# bib

id: cord-000010-prsvv6l9
title: Studying copy number variations using a nanofluidic platform
author: Qin, Jian
date: 2008-08-18
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url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2566873/
doi: 10.1093/nar/gkn518
summary:
  Copy number variations (CNVs) in the human genome are conventionally detected using high-throughput scanning technologies, such as comparative genomic hybridization and high-density single nucleotide polymorphism (SNP) microarrays, or relatively low-throughput techniques, such as quantitative polymerase chain reaction (PCR). We have developed a new technology to study copy numbers using a platform known as the digital array, a nanofluidic biochip capable of accurately quantitating genes of interest in DNA samples. Other existing technologies, such as quantitative polymerase chain reaction (PCR), are limited because of their inability to reliably distinguish less than a twofold difference in copy number of a particular gene in DNA samples (11) (12) (13) . In this study we demonstrate the use of a unique integrated nanofluidic system, the digital array, in the study of CNVs. The digital array (14, 15) is able to accurately quantitate DNA samples based on the fact that single DNA molecules are randomly distributed in more than 9000 reaction chambers and then PCR amplified.

abstract:
  Copy number variations (CNVs) in the human genome are conventionally detected using high-throughput scanning technologies, such as comparative genomic hybridization and high-density single nucleotide polymorphism (SNP) microarrays, or relatively low-throughput techniques, such as quantitative polymerase chain reaction (PCR). All these approaches are limited in resolution and can at best distinguish a twofold (or 50%) difference in copy number. We have developed a new technology to study copy numbers using a platform known as the digital array, a nanofluidic biochip capable of accurately quantitating genes of interest in DNA samples. We have evaluated the digital array's performance using a model system, to show that this technology is exquisitely sensitive, capable of differentiating as little as a 15% difference in gene copy number (or between 6 and 7 copies of a target gene). We have also analyzed commercial DNA samples for their CYP2D6 copy numbers and confirmed that our results were consistent with those obtained independently using conventional techniques. In a screening experiment with breast cancer and normal DNA samples, the ERBB2 gene was found to be amplified in about 35% of breast cancer samples. The use of the digital array enables accurate measurement of gene copy numbers and is of significant value in CNV studies.


id: cord-000125-uvf5qzfd
author: Kenworthy, Rachael
date: 2009-09-03
txt: ./txt/cord-000125-uvf5qzfd.txt
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url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2770676/
doi: 10.1093/nar/gkp714
summary:
  The interaction between a PAMP and a PRR triggers activation of the interferon (IFN) pathway in mammalian cells, which significantly changes the gene-expression profile in the cells and contributes to the well-documented off-target effect of RNAi. IFN induction is especially problematic in antiviral studies employing RNAi, where the antiviral effect of IFN must be distinguished from that of RNAi. Typical IFN-inducing structure patterns include dsRNA of certain length, single-stranded RNA (ssRNA) containing 5 0 -triphosphates (5 0 -ppp), the dsRNA analogue polyinosinic-polycytidylic acid (poly I:C), and certain dsDNA molecules. Mammalian expression plasmids encoding each of these proteins, as well as the dominant negative (DN) mutants of RIG-I and MDA5, were transfected into 293FT cells with shRNAs and an IFN-b promoter reporter construct.

title:
  Short-hairpin RNAs delivered by lentiviral vector transduction trigger RIG-I-mediated IFN activation

abstract:
  Activation of the type I interferon (IFN) pathway by small interfering RNA (siRNA) is a major contributor to the off-target effects of RNA interference in mammalian cells. While IFN induction complicates gene function studies, immunostimulation by siRNAs may be beneficial in certain therapeutic settings. Various forms of siRNA, meeting different compositional and structural requirements, have been reported to trigger IFN activation. The consensus is that intracellularly expressed short-hairpin RNAs (shRNAs) are less prone to IFN activation because they are not detected by the cell-surface receptors. In particular, lentiviral vector-mediated transduction of shRNAs has been reported to avoid IFN response. Here we identify a shRNA that potently activates the IFN pathway in human cells in a sequence- and 5′-triphosphate-dependent manner. In addition to suppressing its intended mRNA target, expression of the shRNA results in dimerization of interferon regulatory factor-3, activation of IFN promoters and secretion of biologically active IFNs into the extracellular medium. Delivery by lentiviral vector transduction did not avoid IFN activation by this and another, unrelated shRNA. We also demonstrated that retinoic-acid-inducible gene I, and not melanoma differentiation associated gene 5 or toll-like receptor 3, is the cytoplasmic sensor for intracellularly expressed shRNAs that trigger IFN activation.


id: cord-000159-8y8ho2x5
title: Recode-2: new design, new search tools, and many more genes
author: Bekaert, Michaël
date: 2009-09-25
txt: ./txt/cord-000159-8y8ho2x5.txt
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url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2808893/
doi: 10.1093/nar/gkp788
summary:
  ''Recoding'' is a term used to describe non-standard read-out of the genetic code, and encompasses such phenomena as programmed ribosomal frameshifting, stop codon readthrough, selenocysteine insertion and translational bypassing. It provides access to detailed information on genes known to utilize translational recoding and allows complex search queries, browsing of recoding data and enhanced visualization of annotated sequence elements. The term ''translational recoding'' describes the utilization of non-standard decoding during protein synthesis and encompasses such processes as ribosomal frameshifting, codon redefinition, translational bypassing and StopGo (1) (2) (3) (4) (5) (6) (7) . To facilitate further development of computational tools for the prediction of recoded genes in the ever faster growing body of sequence data, as well as to provide bench researchers with upto-date information on recoding, an efficient means of Recode database population and annotation are now required. RECODE: a database of frameshifting, bypassing and codon redefinition utilized for gene expression

abstract:
  ‘Recoding’ is a term used to describe non-standard read-out of the genetic code, and encompasses such phenomena as programmed ribosomal frameshifting, stop codon readthrough, selenocysteine insertion and translational bypassing. Although only a small proportion of genes utilize recoding in protein synthesis, accurate annotation of ‘recoded’ genes lags far behind annotation of ‘standard’ genes. In order to address this issue, provide a service to researchers in the field, and offer training data for developers of gene-annotation software, we have gathered together known cases of recoding within the Recode database. Recode-2 is an improved and updated version of the database. It provides access to detailed information on genes known to utilize translational recoding and allows complex search queries, browsing of recoding data and enhanced visualization of annotated sequence elements. At present, the Recode-2 database stores information on approximately 1500 genes that are known to utilize recoding in their expression—a factor of approximately three increase over the previous version of the database. Recode-2 is available at http://recode.ucc.ie


id: cord-000271-812uc4w7
title: Alternative splicing of CD200 is regulated by an exonic splicing enhancer and SF2/ASF
author: Chen, Zhiqi
date: 2010-06-17
txt: ./txt/cord-000271-812uc4w7.txt
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url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2965252/
doi: 10.1093/nar/gkq554
summary:
  Taken together, our data suggest for the first time that SF2/ASF regulates the function of CD200 by controlling CD200 alternative splicing, through direct binding to an ESE located in exon 2 of CD200. Interestingly, in a mouse model of viral infection, we detected for the first time that the normal splicing pattern of CD200 was reversed in the lung tissue of A/J mice infected with mouse hepatitis virus strain I (MHV-1), following an increase in expression of SF2/ASF in this MHV-1 susceptible mouse strain. Two-and-a-half micrograms of siRNA, together with 10 mg of the alternative splicing construct DNA, was transfected to Daudi or SK-N cells by electroporation to detect exogenous expression pattern of CD200 following silencing SF2/ASF. As shown in Figures 3C and D, and 4A and B, expression of the full-length transcript (exon 2 inclusion) was reduced in both Daudi and SK-N cells after mutation or deletion of the ESE in exon 2.

abstract:
  CD200, a type I membrane glycoprotein, plays an important role in prevention of inflammatory disorders, graft rejection, autoimmune diseases and spontaneous fetal loss. It also regulates tumor immunity. A truncated CD200 (CD200(tr)) resulting from alternative splicing has been identified and characterized as a functional antagonist to full-length CD200. Thus, it is important to explore the mechanism(s) controlling alternative splicing of CD200. In this study, we identified an exonic splicing enhancer (ESE) located in exon 2, which is a putative binding site for a splicing regulatory protein SF2/ASF. Deletion or mutation of the ESE site decreased expression of the full-length CD200. Direct binding of SF2/ASF to the ESE site was confirmed by RNA electrophoretic mobility shift assay (EMSA). Knockdown of expression of SF2/ASF resulted in the same splicing pattern as seen after deletion or mutation of the ESE, whereas overexpression of SF2/ASF increased expression of the full-length CD200. In vivo studies showed that viral infection reversed the alternative splicing pattern of CD200 with increased expression of SF2/ASF and the full-length CD200. Taken together, our data suggest for the first time that SF2/ASF regulates the function of CD200 by controlling CD200 alternative splicing, through direct binding to an ESE located in exon 2 of CD200.


id: cord-000293-pc4x5e24
title: Stimulation of ribosomal frameshifting by antisense LNA
author: Yu, Chien-Hung
date: 2010-08-06
txt: ./txt/cord-000293-pc4x5e24.txt
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url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3001050/
doi: 10.1093/nar/gkq650
summary:
  The requirements for À1 ribosomal frameshifting are the presence of a slippery heptanucleotide sequence X XXY YYZ (where X can be A, U, G or C; Y can be A or U; and Z does not equal Y; the spaces indicate the original reading frame) (8) followed by a downstream structural element, such as a pseudoknot, a hairpin or an antisense oligonucleotide duplex [for reviews, see (9) ]. We and others have demonstrated that small RNA oligonucleotides are able to mimic the function of frameshifter pseudoknots or hairpins by redirecting ribosomes into new reading frames (27, 28) . These results demonstrate that LNA modifications indeed enhance the antisense-induced frameshifting efficiency probably due to higher thermodynamic stability and RNA-like structural properties. In addition to RNA oligonucleotides, we demonstrated that LNA/DNA mix-mers are also capable of stimulating efficient À1 ribosomal frameshifting in contrast to DNA oligonucleotides. Triplex structures in an RNA pseudoknot enhance mechanical stability and increase efficiency of À1 ribosomal frameshifting

abstract:
  Programmed ribosomal frameshifting is a translational recoding mechanism commonly used by RNA viruses to express two or more proteins from a single mRNA at a fixed ratio. An essential element in this process is the presence of an RNA secondary structure, such as a pseudoknot or a hairpin, located downstream of the slippery sequence. Here, we have tested the efficiency of RNA oligonucleotides annealing downstream of the slippery sequence to induce frameshifting in vitro. Maximal frameshifting was observed with oligonucleotides of 12–18 nt. Antisense oligonucleotides bearing locked nucleid acid (LNA) modifications also proved to be efficient frameshift-stimulators in contrast to DNA oligonucleotides. The number, sequence and location of LNA bases in an otherwise DNA oligonucleotide have to be carefully manipulated to obtain optimal levels of frameshifting. Our data favor a model in which RNA stability at the entrance of the ribosomal tunnel is the major determinant of stimulating slippage rather than a specific three-dimensional structure of the stimulating RNA element.


id: cord-000294-2g471tb4
title: A flexible loop in yeast ribosomal protein L11 coordinates P-site tRNA binding
author: Rhodin, Michael H. J.
date: 2010-08-12
txt: ./txt/cord-000294-2g471tb4.txt
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url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3001080/
doi: 10.1093/nar/gkq711
summary:
  High-resolution structures reveal that yeast ribosomal protein L11 and its bacterial/archael homologs called L5 contain a highly conserved, basically charged internal loop that interacts with the peptidyl-transfer RNA (tRNA) T-loop. The two mutants also had opposing effects on binding of aa-tRNA to the ribosomal A-site, and downstream functional effects were observed on translational fidelity, drug resistance/hypersensitivity, virus maintenance and overall cell growth. The high degree of similarity across species, from the primary amino acid sequences to their tertiary structures, suggests conserved functional roles beyond serving as mere scaffolding for the rRNAs. Ribosomal protein L11 of Saccharomyces cerevisiae is an essential, highly conserved component of the 60S subunit (in bacteria and archaea, the homologous protein is named L5; the yeast nomenclature is used throughout this text to minimize confusion). Analysis of the recent cryo-EM yeast ribosome structure (4) revealed that these H84 loop bases are located within 3 Å of the stretches of amino acids changed to alanines in both the 51-4A and 54-7A mutants ( Figure 5B ).

abstract:
  High-resolution structures reveal that yeast ribosomal protein L11 and its bacterial/archael homologs called L5 contain a highly conserved, basically charged internal loop that interacts with the peptidyl-transfer RNA (tRNA) T-loop. We call this the L11 ‘P-site loop’. Chemical protection of wild-type ribosome shows that that the P-site loop is inherently flexible, i.e. it is extended into the ribosomal P-site when this is unoccupied by tRNA, while it is retracted into the terminal loop of 25S rRNA Helix 84 when the P-site is occupied. To further analyze the function of this structure, a series of mutants within the P-site loop were created and analyzed. A mutant that favors interaction of the P-site loop with the terminal loop of Helix 84 promoted increased affinity for peptidyl-tRNA, while another that favors its extension into the ribosomal P-site had the opposite effect. The two mutants also had opposing effects on binding of aa-tRNA to the ribosomal A-site, and downstream functional effects were observed on translational fidelity, drug resistance/hypersensitivity, virus maintenance and overall cell growth. These analyses suggest that the L11 P-site loop normally helps to optimize ribosome function by monitoring the occupancy status of the ribosomal P-site.


id: cord-000363-cbzd8ybv
author: Belew, Ashton T.
date: 2010-11-24
txt: ./txt/cord-000363-cbzd8ybv.txt
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url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3074144/
doi: 10.1093/nar/gkq1220
summary:
  The slippery heptamers for these À1 RF signals begin at nucleotides 858, 1653, 279 and 1521 of their respective ORFs. These were cloned into a yeast PGK1 reporter gene so that frameshifted ribosomes are directed to PTCs. All inserts were flanked by sequences derived from Renilla and firefly luciferase genes, providing unique exogenous sequences for specific detection of the reporter mRNAs. Two additional PGK1 reporters without À1 RF signals were used as controls: a readthrough reporter encoded a continuous ORF, while a PTC control contained an in-frame UAA termination codon ( Figure 1 ). Analysis of the Programmed Ribosomal Frameshift Database (http:// prfdb.umd.edu/) reveals that, along with the other four putative À1 RF signals in the EST2 mRNA, the mRNAs encoding Est1p, Stn1p, Cdc13p and Orc5p, all components or regulators of telomerase that are stabilized in NMD À cells, also contain high confidence À1 RF signals (Supplementary Figure S2 ).

title:
  Endogenous ribosomal frameshift signals operate as mRNA destabilizing elements through at least two molecular pathways in yeast

abstract:
  Although first discovered in viruses, previous studies have identified operational −1 ribosomal frameshifting (−1 RF) signals in eukaryotic genomic sequences, and suggested a role in mRNA stability. Here, four yeast −1 RF signals are shown to promote significant mRNA destabilization through the nonsense mediated mRNA decay pathway (NMD), and genetic evidence is presented suggesting that they may also operate through the no-go decay pathway (NGD) as well. Yeast EST2 mRNA is highly unstable and contains up to five −1 RF signals. Ablation of the −1 RF signals or of NMD stabilizes this mRNA, and changes in −1 RF efficiency have opposing effects on the steady-state abundance of the EST2 mRNA. These results demonstrate that endogenous −1 RF signals function as mRNA destabilizing elements through at least two molecular pathways in yeast. Consistent with current evolutionary theory, phylogenetic analyses suggest that −1 RF signals are rapidly evolving cis-acting regulatory elements. Identification of high confidence −1 RF signals in ∼10% of genes in all eukaryotic genomes surveyed suggests that −1 RF is a broadly used post-transcriptional regulator of gene expression.


id: cord-000435-2u49b7xo
title: Stimulation of stop codon readthrough: frequent presence of an extended 3′ RNA structural element
author: Firth, Andrew E.
date: 2011-04-27
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3159437/
doi: 10.1093/nar/gkr224
abstract:
  In Sindbis, Venezuelan equine encephalitis and related alphaviruses, the polymerase is translated as a fusion with other non-structural proteins via readthrough of a UGA stop codon. Surprisingly, earlier work reported that the signal for efficient readthrough comprises a single cytidine residue 3′-adjacent to the UGA. However, analysis of variability at synonymous sites revealed strikingly enhanced conservation within the ∼150 nt 3′-adjacent to the UGA, and RNA folding algorithms revealed the potential for a phylogenetically conserved stem–loop structure in the same region. Mutational analysis of the predicted structure demonstrated that the stem–loop increases readthrough by up to 10-fold. The same computational analysis indicated that similar RNA structures are likely to be relevant to readthrough in certain plant virus genera, notably Furovirus, Pomovirus, Tobravirus, Pecluvirus and Benyvirus, as well as the Drosophilia gene kelch. These results suggest that 3′ RNA stimulatory structures feature in a much larger proportion of readthrough cases than previously anticipated, and provide a new criterion for assessing the large number of cellular readthrough candidates that are currently being revealed by comparative sequence analysis.


id: cord-000482-wifs97yy
title: Stem–loop structures can effectively substitute for an RNA pseudoknot in −1 ribosomal frameshifting
author: Yu, Chien-Hung
date: 2011-07-29
txt: ./txt/cord-000482-wifs97yy.txt
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url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3203594/
doi: 10.1093/nar/gkr579
summary:
  −1 Programmed ribosomal frameshifting (PRF) in synthesizing the gag-pro precursor polyprotein of Simian retrovirus type-1 (SRV-1) is stimulated by a classical H-type pseudoknot which forms an extended triple helix involving base–base and base–sugar interactions between loop and stem nucleotides. In short, pDUAL-HIV(0) was digested by KpnI and BamHI, followed by insertion of complementary oligonucleotides to clone the SRV-1 gag-pro pseudoknot, various hairpins as shown in Figures 2C and 5, and a negative control (NC) which formed no apparent secondary structure downstream of the slippery sequence. These data support the notion that downstream structures serve as barriers to stall translating ribosomes to stimulate frameshifting, and demonstrate that there is a correlation between the thermodynamic stability of a hairpin and its frameshift inducing capacity. In the present study, a 12 bp hairpin derivative of the SRV-1 gag-pro pseudoknot with a calculated stability of À26.9 kcal/mol was capable of inducing 22% of frameshifting, which is only 1.4-fold .

abstract:
  −1 Programmed ribosomal frameshifting (PRF) in synthesizing the gag-pro precursor polyprotein of Simian retrovirus type-1 (SRV-1) is stimulated by a classical H-type pseudoknot which forms an extended triple helix involving base–base and base–sugar interactions between loop and stem nucleotides. Recently, we showed that mutation of bases involved in triple helix formation affected frameshifting, again emphasizing the role of the triple helix in −1 PRF. Here, we investigated the efficiency of hairpins of similar base pair composition as the SRV-1 gag-pro pseudoknot. Although not capable of triple helix formation they proved worthy stimulators of frameshifting. Subsequent investigation of ∼30 different hairpin constructs revealed that next to thermodynamic stability, loop size and composition and stem irregularities can influence frameshifting. Interestingly, hairpins carrying the stable GAAA tetraloop were significantly less shifty than other hairpins, including those with a UUCG motif. The data are discussed in relation to natural shifty hairpins.


id: cord-000532-e18licyc
title: mRNA pseudoknot structures can act as ribosomal roadblocks
author: Tholstrup, Jesper
date: 2011-09-08
txt: ./txt/cord-000532-e18licyc.txt
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url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3245918/
doi: 10.1093/nar/gkr686
summary:
  The results shown in Figure 2 revealed that a 1D SDS-PAGE assay could not firmly identify polypeptides originating from a À1 frameshifted ribosome stalled in the pseudoknot from the non-frameshifted product in a ''Downstream Stop'' construct. In order to quantify the amount of À1 frameshifted ribosomes stalled inside the pseudoknot, we performed a 2D SDS-PAGE separation of the radioactively labelled proteins originating from the ''Upstream Stop'' construct (Supplementary Figures S4 and S5) , which is the type of construct most commonly used throughout literature. In the ''Upstream Stop'' construct the non-frameshifting ribosomes will translate gene10 and terminate at a UAA stop codon in the spacer sequence and produce a 28 kDa polypeptide. In the following subsections ''Identification of transcripts from the T7gene10-PK-lacZ gene fusions'', ''Messenger RNA stability'' and ''Coupling between translation and transcription is required for full-length transcripts'', we will show that the observed proteins did indeed originate from stalled ribosomes and that they were not caused by other effects.

abstract:
  Several viruses utilize programmed ribosomal frameshifting mediated by mRNA pseudoknots in combination with a slippery sequence to produce a well defined stochiometric ratio of the upstream encoded to the downstream-encoded protein. A correlation between the mechanical strength of mRNA pseudoknots and frameshifting efficiency has previously been found; however, the physical mechanism behind frameshifting still remains to be fully understood. In this study, we utilized synthetic sequences predicted to form mRNA pseudoknot-like structures. Surprisingly, the structures predicted to be strongest lead only to limited frameshifting. Two-dimensional gel electrophoresis of pulse labelled proteins revealed that a significant fraction of the ribosomes were frameshifted but unable to pass the pseudoknot-like structures. Hence, pseudoknots can act as ribosomal roadblocks, prohibiting a significant fraction of the frameshifted ribosomes from reaching the downstream stop codon. The stronger the pseudoknot the larger the frameshifting efficiency and the larger its roadblocking effect. The maximal amount of full-length frameshifted product is produced from a structure where those two effects are balanced. Taking ribosomal roadblocking into account is a prerequisite for formulating correct frameshifting hypotheses.


id: cord-000881-s90geszi
author: Lang, Dorothy M.
date: 2012-12-25
txt: ./txt/cord-000881-s90geszi.txt
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url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3561941/
doi: 10.1093/nar/gks1251
summary:
  In contrast to the relatively short lengths of previously described motifs, we found that most homomorphs are long, and each provides a structural connection between the template tunnel or NTP entry tunnel and the exterior of the protein. The structurally aligned sequences that comprised homomorph of Motif F (hmF) for RdRps and HIV are summarized in Figure 3A . Using T7 DNAP as a query (lowest segment of the figure) , only a small portion of the C-terminal edge of Motif D and a few species have similar structures. In the RdRps, the combined regions of structural homology represent $75% of the sequence from the start of homomorph of Motif G (hmG) through the end of hmE in each species ($375 residues). The tertiary position of each of the homomorphs includes at least one residue (and sometimes more) in contact with the exterior surface of the protein and one or more highly conserved functional residues located within or at the wall of the template tunnel.

title:
  Highly similar structural frames link the template tunnel and NTP entry tunnel to the exterior surface in RNA-dependent RNA polymerases

abstract:
  RNA-dependent RNA polymerase (RdRp) is essential to viral replication and is therefore one of the primary targets of countermeasures against these dangerous infectious agents. Development of broad-spectrum therapeutics targeting polymerases has been hampered by the extreme sequence variability of these sequences. RdRps range in length from 400–800 residues, yet contain only ∼20 residues that are conserved in most species. In this study, we made structure-based comparisons that are independent of sequence composition using a recently developed algorithm. We identified residue-to-residue correspondences of multiple protein structures and created (two-dimensional) structure-based alignment maps of 37 polymerase structures that provide both sequence and structure details. Using these maps, we determined that ∼75% of each polymerase species consists of seven protein segments, each of which has high structural similarity to segments in other species, though they are widely divergent in sequence composition and order. We define each of these segments as a ‘homomorph’, and each includes (though most are much larger than) the well-known conserved polymerase motifs. All homomorphs contact the template tunnel or nucleoside triphosphate (NTP) entry tunnel and the exterior of the protein, suggesting they constitute a structural and functional skeleton common among the polymerases.


id: cord-001337-a3y1rfas
author: Sharma, Virag
date: 2014-07-01
txt: ./txt/cord-001337-a3y1rfas.txt
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url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4066793/
doi: 10.1093/nar/gku386
summary:
  The present study combines experimental and bioinformatics approaches to carry out (i) a systematic analysis of the frameshift propensity of all possible motifs (16 Z_ZZN tetramers and 64 X_XXZ_ZZN heptamers) in Escherichia coli and (ii) the identification of genes potentially using this mode of expression amongst 36 Enterobacteriaceae genomes. Prokaryotic signals sometimes possess another type of stimulatory element upstream of the frameshift motif: a Shine-Dalgarno (SD)-like sequence normally involved in translation initiation through pairing with the CCUCC sequence at the 3 end of 16S ribosomal RNA (32) (33) (34) . A preliminary study of 271 IS3 family members (see Supplementary Figure S2 ) led us to choose the following empirical rules: the structure is (i) a simple or branched hairpin of a length ranging from 17 to 140 nt, (ii) that starts 4-10 nt after the last base of the motif , (iii) with a G-C(or C-G) base-pair followed by at least three consecutive Watson-Crick or G-U or U-G base-pairs and (iv) has a G unfold@37 • C ≥ 7.6 kcal.mol −1 ; the G @37 • C value was determined using the default parameters of the RNAfold program from version 1.8.5 of the Vienna RNA package (47) .

title:
  Analysis of tetra- and hepta-nucleotides motifs promoting -1 ribosomal frameshifting in Escherichia coli

abstract:
  Programmed ribosomal -1 frameshifting is a non-standard decoding process occurring when ribosomes encounter a signal embedded in the mRNA of certain eukaryotic and prokaryotic genes. This signal has a mandatory component, the frameshift motif: it is either a Z_ZZN tetramer or a X_XXZ_ZZN heptamer (where ZZZ and XXX are three identical nucleotides) allowing cognate or near-cognate repairing to the -1 frame of the A site or A and P sites tRNAs. Depending on the signal, the frameshifting frequency can vary over a wide range, from less than 1% to more than 50%. The present study combines experimental and bioinformatics approaches to carry out (i) a systematic analysis of the frameshift propensity of all possible motifs (16 Z_ZZN tetramers and 64 X_XXZ_ZZN heptamers) in Escherichia coli and (ii) the identification of genes potentially using this mode of expression amongst 36 Enterobacteriaceae genomes. While motif efficiency varies widely, a major distinctive rule of bacterial -1 frameshifting is that the most efficient motifs are those allowing cognate re-pairing of the A site tRNA from ZZN to ZZZ. The outcome of the genomic search is a set of 69 gene clusters, 59 of which constitute new candidates for functional utilization of -1 frameshifting.


id: cord-001453-l1r416w7
author: Hou, Linlin
date: 2014-11-10
txt: ./txt/cord-001453-l1r416w7.txt
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url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4227792/
doi: 10.1093/nar/gku969
summary:
  title: Archaeal DnaG contains a conserved N-terminal RNA-binding domain and enables tailing of rRNA by the exosome Consistently, a fusion protein containing full-length Csl4 and NTD of DnaG led to enhanced degradation of A-rich RNA by the exosome. The RNase PH-domain containing subunits Rrp41 and Rrp42 are arranged in a catalytically active hexamer, on the top of which a trimeric cap composed of the RNA-binding proteins Rrp4 and Csl4 is bound ( Figure 1B ; 4, 5, [22] [23] [24] . The bacterial primase DnaG is composed of an NTD containing a Zn-finger motif involved in DNA binding, the central, catalytic TOPRIM domain and a CTD neces-sary for the interaction with the replicative helicase DnaB ( Figure 1A , refs. coli cell-free extract was easily detectable by pull-down assays with Strep-Tactin Sepharose beads (for an example see Figure 7A be-low), we conclude that the CTD of DnaG is important for the binding to the archaeal exosome.

title:
  Archaeal DnaG contains a conserved N-terminal RNA-binding domain and enables tailing of rRNA by the exosome

abstract:
  The archaeal exosome is a phosphorolytic 3′–5′ exoribonuclease complex. In a reverse reaction it synthesizes A-rich RNA tails. Its RNA-binding cap comprises the eukaryotic orthologs Rrp4 and Csl4, and an archaea-specific subunit annotated as DnaG. In Sulfolobus solfataricus DnaG and Rrp4 but not Csl4 show preference for poly(rA). Archaeal DnaG contains N- and C-terminal domains (NTD and CTD) of unknown function flanking a TOPRIM domain. We found that the NT and TOPRIM domains have comparable, high conservation in all archaea, while the CTD conservation correlates with the presence of exosome. We show that the NTD is a novel RNA-binding domain with poly(rA)-preference cooperating with the TOPRIM domain in binding of RNA. Consistently, a fusion protein containing full-length Csl4 and NTD of DnaG led to enhanced degradation of A-rich RNA by the exosome. We also found that DnaG strongly binds native and in vitro transcribed rRNA and enables its polynucleotidylation by the exosome. Furthermore, rRNA-derived transcripts with heteropolymeric tails were degraded faster by the exosome than their non-tailed variants. Based on our data, we propose that archaeal DnaG is an RNA-binding protein, which, in the context of the exosome, is involved in targeting of stable RNA for degradation.


id: cord-001502-omq0becw
author: Shabanpoor, Fazel
date: 2015-01-09
txt: ./txt/cord-001502-omq0becw.txt
cache: ./cache/cord-001502-omq0becw.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4288157/
doi: 10.1093/nar/gku1256
summary:
  Studies to date have focused on the delivery of a single SSO conjugated to a CPP, but here we describe the conjugation of two phosphorodiamidate morpholino oligonucleotide (PMO) SSOs to a single CPP for simultaneous delivery and pre-mRNA targeting of two separate genes, exon 23 of the Dmd gene and exon 5 of the Acvr2b gene, in a mouse model of Duchenne muscular dystrophy. Importantly, the cell viability using a bi-specific compound was significantly better than for a mixture of the two individual Pip6a-PMOs. We furthermore assessed the potential of this approach in an in vivo environment through intramuscular administration and demon-Nucleic Acids Research, 2015, Vol. 43, No. 1 31 strated that there were no significant differences in exon skipping activities for both Dmd and Acvr2b targets between bi-specific conjugates (D2 and D3) and a cocktail of the individual P-PMO equivalents.

title:
  Bi-specific splice-switching PMO oligonucleotides conjugated via a single peptide active in a mouse model of Duchenne muscular dystrophy

abstract:
  The potential for therapeutic application of splice-switching oligonucleotides (SSOs) to modulate pre-mRNA splicing is increasingly evident in a number of diseases. However, the primary drawback of this approach is poor cell and in vivo oligonucleotide uptake efficacy. Biological activities can be significantly enhanced through the use of synthetically conjugated cationic cell penetrating peptides (CPPs). Studies to date have focused on the delivery of a single SSO conjugated to a CPP, but here we describe the conjugation of two phosphorodiamidate morpholino oligonucleotide (PMO) SSOs to a single CPP for simultaneous delivery and pre-mRNA targeting of two separate genes, exon 23 of the Dmd gene and exon 5 of the Acvr2b gene, in a mouse model of Duchenne muscular dystrophy. Conjugations of PMOs to a single CPP were carried out through an amide bond in one case and through a triazole linkage (‘click chemistry’) in the other. The most active bi-specific CPP–PMOs demonstrated comparable exon skipping levels for both pre-mRNA targets when compared to individual CPP–PMO conjugates both in cell culture and in vivo in the mdx mouse model. Thus, two SSOs with different target sequences conjugated to a single CPP are biologically effective and potentially suitable for future therapeutic exploitation.


id: cord-001824-7c37elh6
title: The role of wobble uridine modifications in +1 translational frameshifting in eukaryotes
author: Tükenmez, Hasan
date: 2015-10-30
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4627075/
doi: 10.1093/nar/gkv832
abstract:
  In Saccharomyces cerevisiae, 11 out of 42 tRNA species contain 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U), 5-methoxycarbonylmethyluridine (mcm(5)U), 5-carbamoylmethyluridine (ncm(5)U) or 5-carbamoylmethyl-2′-O-methyluridine (ncm(5)Um) nucleosides in the anticodon at the wobble position (U(34)). Earlier we showed that mutants unable to form the side chain at position 5 (ncm(5) or mcm(5)) or lacking sulphur at position 2 (s(2)) of U(34) result in pleiotropic phenotypes, which are all suppressed by overexpression of hypomodified tRNAs. This observation suggests that the observed phenotypes are due to inefficient reading of cognate codons or an increased frameshifting. The latter may be caused by a ternary complex (aminoacyl-tRNA*eEF1A*GTP) with a modification deficient tRNA inefficiently being accepted to the ribosomal A-site and thereby allowing an increased peptidyl-tRNA slippage and thus a frameshift error. In this study, we have investigated the role of wobble uridine modifications in reading frame maintenance, using either the Renilla/Firefly luciferase bicistronic reporter system or a modified Ty1 frameshifting site in a HIS4A::lacZ reporter system. We here show that the presence of mcm(5) and s(2) side groups at wobble uridines are important for reading frame maintenance and thus the aforementioned mutant phenotypes might partly be due to frameshift errors.


id: cord-002366-t94aufs3
title: EuPathDB: the eukaryotic pathogen genomics database resource
author: Aurrecoechea, Cristina
date: 2017-01-04
txt: ./txt/cord-002366-t94aufs3.txt
cache: ./cache/cord-002366-t94aufs3.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5210576/
doi: 10.1093/nar/gkw1105
summary:
  To facilitate the discovery of meaningful biological relationships, the databases couple preconfigured searches with visualization and analysis tools for comprehensive data mining via intuitive graphical interfaces and APIs. All data are analyzed with the same workflows, including creation of gene orthology profiles, so data are easily compared across data sets, data types and organisms. Expanded data content is mostly genomic and functional genomic data while new data types include protein microarray, metabolic pathways, compounds, quantitative proteomics, copy number variation, and polysomal transcriptomics. New features include consistent categorization of searches, data sets and genome browser tracks; redesigned gene pages; effective integration of alternative transcripts; and a EuPathDB Galaxy instance for private analyses of a user''s data. The near-seamless integration of strategy results with tools for functional enrichment analyses and transcript interpretation as well as our new Galaxy workspace and the availability of publicly shared strategies augment the data mining experience in EuPathDB.

abstract:
  The Eukaryotic Pathogen Genomics Database Resource (EuPathDB, http://eupathdb.org) is a collection of databases covering 170+ eukaryotic pathogens (protists & fungi), along with relevant free-living and non-pathogenic species, and select pathogen hosts. To facilitate the discovery of meaningful biological relationships, the databases couple preconfigured searches with visualization and analysis tools for comprehensive data mining via intuitive graphical interfaces and APIs. All data are analyzed with the same workflows, including creation of gene orthology profiles, so data are easily compared across data sets, data types and organisms. EuPathDB is updated with numerous new analysis tools, features, data sets and data types. New tools include GO, metabolic pathway and word enrichment analyses plus an online workspace for analysis of personal, non-public, large-scale data. Expanded data content is mostly genomic and functional genomic data while new data types include protein microarray, metabolic pathways, compounds, quantitative proteomics, copy number variation, and polysomal transcriptomics. New features include consistent categorization of searches, data sets and genome browser tracks; redesigned gene pages; effective integration of alternative transcripts; and a EuPathDB Galaxy instance for private analyses of a user's data. Forthcoming upgrades include user workspaces for private integration of data with existing EuPathDB data and improved integration and presentation of host–pathogen interactions.


id: cord-002494-qm9urt2w
title: SIRT7-dependent deacetylation of CDK9 activates RNA polymerase II transcription
author: Blank, Maximilian F.
date: 2017-03-17
txt: ./txt/cord-002494-qm9urt2w.txt
cache: ./cache/cord-002494-qm9urt2w.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389538/
doi: 10.1093/nar/gkx053
summary:
  In addition to its role in Pol I transcription, we found that SIRT7 also regulates transcription of snoRNAs and mRNAs. Mechanistically, SIRT7 promotes the release of P-TEFb from the inactive 7SK snRNP complex and deacetylates CDK9, a subunit of the elongation factor P-TEFb, which activates transcription by phosphorylating serine 2 within the C-terminal domain (CTD) of Pol II. To investigate whether this region is important for RNA-dependent protein interactions, 5external spacer (5 -ETS) RNA was immobilized on streptavidin beads and incubated with lysates of cells expressing Flag-tagged wildtype SIRT7 or mutants lacking 32 ( N32) or 78 N-terminal amino acids ( N78). To test whether SIRT7 affects Pol II-dependent transcription of snoRNAs, we overexpressed Flag-SIRT7 in HEK293T cells and monitored RNA levels by RT-qPCR. Together with the observation that SIRT7 interacts with elongating Pol II and co-localizes with Pol II phosphorylated at CTD-Ser2 at SIRT7 target genes, these results suggested that reversible acetylation may regulate CDK9 kinase activity and transcription elongation.

abstract:
  SIRT7 is an NAD(+)-dependent protein deacetylase that regulates cell growth and proliferation. Previous studies have shown that SIRT7 is required for RNA polymerase I (Pol I) transcription and pre-rRNA processing. Here, we took a proteomic approach to identify novel molecular targets and characterize the role of SIRT7 in non-nucleolar processes. We show that SIRT7 interacts with numerous proteins involved in transcriptional regulation and RNA metabolism, the majority of interactions requiring ongoing transcription. In addition to its role in Pol I transcription, we found that SIRT7 also regulates transcription of snoRNAs and mRNAs. Mechanistically, SIRT7 promotes the release of P-TEFb from the inactive 7SK snRNP complex and deacetylates CDK9, a subunit of the elongation factor P-TEFb, which activates transcription by phosphorylating serine 2 within the C-terminal domain (CTD) of Pol II. SIRT7 counteracts GCN5-directed acetylation of lysine 48 within the catalytic domain of CDK9, deacetylation promoting CTD phosphorylation and transcription elongation.


id: cord-003305-ya0siivm
author: Liu, Weichi
date: 2018-11-16
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237809/
doi: 10.1093/nar/gky848
title:
  A unique intra-molecular fidelity-modulating mechanism identified in a viral RNA-dependent RNA polymerase

abstract:
  Typically not assisted by proofreading, the RNA-dependent RNA polymerases (RdRPs) encoded by the RNA viruses may need to independently control its fidelity to fulfill virus viability and fitness. However, the precise mechanism by which the RdRP maintains its optimal fidelity level remains largely elusive. By solving 2.1–2.5 Å resolution crystal structures of the classical swine fever virus (CSFV) NS5B, an RdRP with a unique naturally fused N-terminal domain (NTD), we identified high-resolution intra-molecular interactions between the NTD and the RdRP palm domain. In order to dissect possible regulatory functions of NTD, we designed mutations at residues Y471 and E472 to perturb key interactions at the NTD–RdRP interface. When crystallized, some of these NS5B interface mutants maintained the interface, while the others adopted an ‘open’ conformation that no longer retained the intra-molecular interactions. Data from multiple in vitro RdRP assays indicated that the perturbation of the NTD–RdRP interactions clearly reduced the fidelity level of the RNA synthesis, while the processivity of the NS5B elongation complex was not affected. Collectively, our work demonstrates an explicit and unique mode of polymerase fidelity modulation and provides a vivid example of co-evolution in multi-domain enzymes.


id: cord-003711-l3brhmzq
title: Reversible ADP-ribosylation of RNA
author: Munnur, Deeksha
date: 2019-06-20
txt: ./txt/cord-003711-l3brhmzq.txt
cache: ./cache/cord-003711-l3brhmzq.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6582358/
doi: 10.1093/nar/gkz305
summary:
  ADP-ribosylation is a reversible chemical modification catalysed by ADP-ribosyltransferases such as PARPs that utilize nicotinamide adenine dinucleotide (NAD(+)) as a cofactor to transfer monomer or polymers of ADP-ribose nucleotide onto macromolecular targets such as proteins and DNA. We further reveal that ADP-ribosylation of RNA mediated by PARP10 and TRPT1 can be efficiently reversed by several cellular ADP-ribosylhydrolases (PARG, TARG1, MACROD1, MACROD2 and ARH3), as well as by MACROD-like hydrolases from VEEV and SARS viruses. Importantly, PARP3 could only ADP-ribosylate DNA ends and did not have any activity on RNA oligos, while PARP10 specifically modified phosphorylated ssRNA oligo in the conditions tested ( Figure 1A and B). Since PARP10 can ADP-ribosylate both 5 and 3 phosphorylated ends of RNA, we tested both of these modified oligos as substrates for well characterized human ADP-ribosylhydrolases: PARG, TARG1, MACROD1, MACROD2 and ARH1-3.

abstract:
  ADP-ribosylation is a reversible chemical modification catalysed by ADP-ribosyltransferases such as PARPs that utilize nicotinamide adenine dinucleotide (NAD(+)) as a cofactor to transfer monomer or polymers of ADP-ribose nucleotide onto macromolecular targets such as proteins and DNA. ADP-ribosylation plays an important role in several biological processes such as DNA repair, transcription, chromatin remodelling, host-virus interactions, cellular stress response and many more. Using biochemical methods we identify RNA as a novel target of reversible mono-ADP-ribosylation. We demonstrate that the human PARPs - PARP10, PARP11 and PARP15 as well as a highly diverged PARP homologue TRPT1, ADP-ribosylate phosphorylated ends of RNA. We further reveal that ADP-ribosylation of RNA mediated by PARP10 and TRPT1 can be efficiently reversed by several cellular ADP-ribosylhydrolases (PARG, TARG1, MACROD1, MACROD2 and ARH3), as well as by MACROD-like hydrolases from VEEV and SARS viruses. Finally, we show that TRPT1 and MACROD homologues in bacteria possess activities equivalent to the human proteins. Our data suggest that RNA ADP-ribosylation may represent a widespread and physiologically relevant form of reversible ADP-ribosylation signalling.


id: cord-007041-rloey02j
title: Direct sequencing of RNA with MinION Nanopore: detecting mutations based on associations
author: Harel, Noam
date: 2019-12-16
txt: ./txt/cord-007041-rloey02j.txt
cache: ./cache/cord-007041-rloey02j.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7107797/
doi: 10.1093/nar/gkz907
summary:
  We sequenced virus populations in parallel using both MinION and Illumina, allowing us to corroborate the inferences of AssociVar. This then allowed us to directly infer relationships between mutations and to deduce the entire genome sequences of viral strains in the population. We then determined the population frequency of each mutation at passage 1 and passage 15 through whole genome deep sequencing as described below, using Illumina and MinION. After applying AssociVar to the data, we were able to identify five out of the six mutations appearing at a frequency above 10% in the Illumina results in p15A, and all eight positions within the p15B sample (Figure 4 , Supplementary Table S2 ). We applied AssociVar to sequencing data from an evolved population of phages where Illumina sequencing was available, allowing us to corroborate whether mutations we found based on analysis of the MinION data alone were indeed real.

abstract:
  One of the key challenges in the field of genetics is the inference of haplotypes from next generation sequencing data. The MinION Oxford Nanopore sequencer allows sequencing long reads, with the potential of sequencing complete genes, and even complete genomes of viruses, in individual reads. However, MinION suffers from high error rates, rendering the detection of true variants difficult. Here, we propose a new statistical approach named AssociVar, which differentiates between true mutations and sequencing errors from direct RNA/DNA sequencing using MinION. Our strategy relies on the assumption that sequencing errors will be dispersed randomly along sequencing reads, and hence will not be associated with each other, whereas real mutations will display a non-random pattern of association with other mutations. We demonstrate our approach using direct RNA sequencing data from evolved populations of the MS2 bacteriophage, whose small genome makes it ideal for MinION sequencing. AssociVar inferred several mutations in the phage genome, which were corroborated using parallel Illumina sequencing. This allowed us to reconstruct full genome viral haplotypes constituting different strains that were present in the sample. Our approach is applicable to long read sequencing data from any organism for accurate detection of bona fide mutations and inter-strain polymorphisms.


id: cord-009318-zt1o1bcz
author: Rolando, Justin C
date: 2020-04-17
txt: ./txt/cord-009318-zt1o1bcz.txt
cache: ./cache/cord-009318-zt1o1bcz.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7144905/
doi: 10.1093/nar/gkaa099
summary:
  We then develop a real-time digital LAMP (dLAMP) with high-resolution melting temperature (HRM) analysis and use this single-molecule approach to analyze approximately 1.2 million amplification events. By differentiating true and false positives, HRM enables determination of the optimal assay and analysis parameters that leads to the lowest limit of detection (LOD) in a digital isothermal amplification assay. To test this hypothesis, we used a dLAMP assay with CT DNA as the target (combined with sequencing to identify the products of bulk reactions) to analyze both specific and non-specific amplification under conditions that include clinically relevant concentrations of background human DNA. Single digital partition counts were observed at low-T m non-specific amplification in both the presence of template and the NTC and independent of hgDNA concentration ( Figure 9B and C). When using Bst 3.0 (Supplementary Figure S11F) and HRM to remove non-specific amplification, LOD tracks with the number of true-positive events.

title:
  Real-time kinetics and high-resolution melt curves in single-molecule digital LAMP to differentiate and study specific and non-specific amplification

abstract:
  Isothermal amplification assays, such as loop-mediated isothermal amplification (LAMP), show great utility for the development of rapid diagnostics for infectious diseases because they have high sensitivity, pathogen-specificity and potential for implementation at the point of care. However, elimination of non-specific amplification remains a key challenge for the optimization of LAMP assays. Here, using chlamydia DNA as a clinically relevant target and high-throughput sequencing as an analytical tool, we investigate a potential mechanism of non-specific amplification. We then develop a real-time digital LAMP (dLAMP) with high-resolution melting temperature (HRM) analysis and use this single-molecule approach to analyze approximately 1.2 million amplification events. We show that single-molecule HRM provides insight into specific and non-specific amplification in LAMP that are difficult to deduce from bulk measurements. We use real-time dLAMP with HRM to evaluate differences between polymerase enzymes, the impact of assay parameters (e.g. time, rate or florescence intensity), and the effect background human DNA. By differentiating true and false positives, HRM enables determination of the optimal assay and analysis parameters that leads to the lowest limit of detection (LOD) in a digital isothermal amplification assay.


id: cord-011565-8ncgldaq
author: Elworth, R A Leo
date: 2020-06-04
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7261164/
doi: 10.1093/nar/gkaa265
title:
  To Petabytes and beyond: recent advances in probabilistic and signal processing algorithms and their application to metagenomics

abstract:
  As computational biologists continue to be inundated by ever increasing amounts of metagenomic data, the need for data analysis approaches that keep up with the pace of sequence archives has remained a challenge. In recent years, the accelerated pace of genomic data availability has been accompanied by the application of a wide array of highly efficient approaches from other fields to the field of metagenomics. For instance, sketching algorithms such as MinHash have seen a rapid and widespread adoption. These techniques handle increasingly large datasets with minimal sacrifices in quality for tasks such as sequence similarity calculations. Here, we briefly review the fundamentals of the most impactful probabilistic and signal processing algorithms. We also highlight more recent advances to augment previous reviews in these areas that have taken a broader approach. We then explore the application of these techniques to metagenomics, discuss their pros and cons, and speculate on their future directions.


id: cord-048222-1pq6dkl5
author: Imbeaud, Sandrine
date: 2005-03-30
txt: ./txt/cord-048222-1pq6dkl5.txt
cache: ./cache/cord-048222-1pq6dkl5.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1072807/
doi: 10.1093/nar/gni054
summary:
  With that prospect in mind, and with the aim of anticipating future standards by pre-normative research, we identified and tested two software packages recently developed to gauge the integrity of RNA samples with a user-independent strategy: one open source, the degradometer software for calculation of the degradation factor and ''true'' 28S:18S ratio based on peak heights (24) and the freely available RIN algorithm of the Agilent 2100 expert software, based on computation of a ''RNA Integrity Number'' (RIN) (25) . A RIN number is computed for each RNA profile (see Supplementary Table 4 online) resulting in the classification of RNA samples in 10 numerically predefined categories of integrity. The values of the mean fold changes, calculated according to the 2 ÀDDCt quantification method (see Materials and Methods), were found lower than 1.0, corresponding to the expression level (1·) in the sample exhibiting the highest RNA quality (Table 2 and Figure 5 ).

title:
  Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces

abstract:
  While it is universally accepted that intact RNA constitutes the best representation of the steady-state of transcription, there is no gold standard to define RNA quality prior to gene expression analysis. In this report, we evaluated the reliability of conventional methods for RNA quality assessment including UV spectroscopy and 28S:18S area ratios, and demonstrated their inconsistency. We then used two new freely available classifiers, the Degradometer and RIN systems, to produce user-independent RNA quality metrics, based on analysis of microcapillary electrophoresis traces. Both provided highly informative and valuable data and the results were found highly correlated, while the RIN system gave more reliable data. The relevance of the RNA quality metrics for assessment of gene expression differences was tested by Q-PCR, revealing a significant decline of the relative expression of genes in RNA samples of disparate quality, while samples of similar, even poor integrity were found highly comparable. We discuss the consequences of these observations to minimize artifactual detection of false positive and negative differential expression due to RNA integrity differences, and propose a scheme for the development of a standard operational procedure, with optional registration of RNA integrity metrics in public repositories of gene expression data.


id: cord-048327-xgwbl8em
title: Antisense-induced ribosomal frameshifting
author: Henderson, Clark M.
date: 2006-08-18
txt: ./txt/cord-048327-xgwbl8em.txt
cache: ./cache/cord-048327-xgwbl8em.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1616946/
doi: 10.1093/nar/gkl531
summary:
  The ability of cis-acting RNA structures or trans-acting 2 0 -O-Methyl antisense oligonucleotides to induce ribosomal frameshifting was determined by in vitro transcription and translation of a dual luciferase reporter vector, p2Luc. p2Luc contains the Renilla and firefly luciferase genes on either side of a multiple cloning site, and can be transcribed using the T7 promoter located upstream of the Renilla luciferase gene (45) . As the AZ1A antisense oligonucleotide was designed to anneal directly adjacent to the UGA codon of the shift site, it was of interest to determine whether the wild-type antizyme pseudoknot could induce À1 frameshifting when located in the equivalent position. The ability of spermidine to stimulate antisense oligonucleotide induced ribosome frameshifting to the +1 reading frame at the UCC UGA shift site in the absence of the natural 3 0 stimulator demonstrates that this cis-acting element is not required for polyamine responsiveness.

abstract:
  Programmed ribosomal frameshifting provides a mechanism to decode information located in two overlapping reading frames by diverting a proportion of translating ribosomes into a second open reading frame (ORF). The result is the production of two proteins: the product of standard translation from ORF1 and an ORF1–ORF2 fusion protein. Such programmed frameshifting is commonly utilized as a gene expression mechanism in viruses that infect eukaryotic cells and in a subset of cellular genes. RNA secondary structures, consisting of pseudoknots or stem–loops, located downstream of the shift site often act as cis-stimulators of frameshifting. Here, we demonstrate for the first time that antisense oligonucleotides can functionally mimic these RNA structures to induce +1 ribosomal frameshifting when annealed downstream of the frameshift site, UCC UGA. Antisense-induced shifting of the ribosome into the +1 reading frame is highly efficient in both rabbit reticulocyte lysate translation reactions and in cultured mammalian cells. The efficiency of antisense-induced frameshifting at this site is responsive to the sequence context 5′ of the shift site and to polyamine levels.


id: cord-048345-m56agj4z
author: Reddy, Timothy E.
date: 2007-01-03
txt: ./txt/cord-048345-m56agj4z.txt
cache: ./cache/cord-048345-m56agj4z.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1807961/
doi: 10.1093/nar/gkl1062
summary:
  title: Positional clustering improves computational binding site detection and identifies novel cis-regulatory sites in mammalian GABA(A) receptor subunit genes We evaluate the efficacy of our approach using known examples of binding and regulation in yeast and experimentally testing predicted TF-binding sites upstream of the subunit genes coding for the heteromeric mammalian neurotransmitter receptor system, the type A g-aminobutyric acid receptor (GABA A R). In the current study, we test the ability of positional clustering to detect known TF-binding sites in a series of increasingly noisy sets of yeast promoters, and found marked improvement in the percentage of correct predictions over Gibbs sampling alone. We also present de novo predictions of TF-binding sites in promoter regions of GABA A receptor subunit genes (GABRs) whose expression is altered (either up-regulated or down-regulated) in an animal model of temporal lobe epilepsy (35) . Using positional clustering, we predicted 13 TF-binding sites upstream of GABA A receptor subunit genes ( Table 1) .

title:
  Positional clustering improves computational binding site detection and identifies novel cis-regulatory sites in mammalian GABA(A) receptor subunit genes

abstract:
  Understanding transcription factor (TF) mediated control of gene expression remains a major challenge at the interface of computational and experimental biology. Computational techniques predicting TF-binding site specificity are frequently unreliable. On the other hand, comprehensive experimental validation is difficult and time consuming. We introduce a simple strategy that dramatically improves robustness and accuracy of computational binding site prediction. First, we evaluate the rate of recurrence of computational TFBS predictions by commonly used sampling procedures. We find that the vast majority of results are biologically meaningless. However clustering results based on nucleotide position improves predictive power. Additionally, we find that positional clustering increases robustness to long or imperfectly selected input sequences. Positional clustering can also be used as a mechanism to integrate results from multiple sampling approaches for improvements in accuracy over each one alone. Finally, we predict and validate regulatory sequences partially responsible for transcriptional control of the mammalian type A γ-aminobutyric acid receptor (GABA(A)R) subunit genes. Positional clustering is useful for improving computational binding site predictions, with potential application to improving our understanding of mammalian gene expression. In particular, predicted regulatory mechanisms in the mammalian GABA(A)R subunit gene family may open new avenues of research towards understanding this pharmacologically important neurotransmitter receptor system.


id: cord-048359-lz37rh82
author: Li, Jin
date: 2007-06-01
txt: ./txt/cord-048359-lz37rh82.txt
cache: ./cache/cord-048359-lz37rh82.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1919510/
doi: 10.1093/nar/gkm403
summary:
  Subsequently, melting curve analysis, on conventional or nano-technology real-time PCR platforms, detects the samples that contain mutations in a high-throughput and closed-tube manner. Following denaturation and re-annealing of PCR products that leads to formation of cross-hybridized sequences at the positions of mutations ( Figure 1A ) the sample is exposed to Surveyor TM endonuclease that recognizes base pair mismatches or small loops with high specificity (28) and generates a break on both DNA strands 3 0 to the mismatch. Finally, because the amplified mutated sequences contain defined primers at their ends, direct sequencing of enzymatically selected PCR products is readily possible following the real-time melting step, enabling sequencing of low-level mutations identified by Surveyor TM . Here we enabled Surveyor TM , an endonuclease that recognizes selectively mismatches formed by mutations and small deletions following ''cross-hybridized sequence'' formation, to generate mutation-specific DNA fragments that are amplified and screened via differential melting curve analysis.

title:
  s-RT-MELT for rapid mutation scanning using enzymatic selection and real time DNA-melting: new potential for multiplex genetic analysis

abstract:
  The rapidly growing understanding of human genetic pathways, including those that mediate cancer biology and drug response, leads to an increasing need for extensive and reliable mutation screening on a population or on a single patient basis. Here we describe s-RT-MELT, a novel technology that enables highly expanded enzymatic mutation scanning in human samples for germline or low-level somatic mutations, or for SNP discovery. GC-clamp-containing PCR products from interrogated and wild-type samples are hybridized to generate mismatches at the positions of mutations over one or multiple sequences in-parallel. Mismatches are converted to double-strand breaks using a DNA endonuclease (Surveyor™) and oligonucleotide tails are enzymatically attached at the position of mutations. A novel application of PCR enables selective amplification of mutation-containing DNA fragments. Subsequently, melting curve analysis, on conventional or nano-technology real-time PCR platforms, detects the samples that contain mutations in a high-throughput and closed-tube manner. We apply s-RT-MELT in the screening of p53 and EGFR mutations in cell lines and clinical samples and demonstrate its advantages for rapid, multiplexed mutation scanning in cancer and for genetic variation screening in biology and medicine.


id: cord-048370-noscodew
author: Wu, Rebecca P.
date: 2007-08-01
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1976451/
doi: 10.1093/nar/gkm478
title:
  Cell-penetrating peptides as transporters for morpholino oligomers: effects of amino acid composition on intracellular delivery and cytotoxicity

abstract:
  Arginine-rich cell-penetrating peptides (CPPs) are promising transporters for intracellular delivery of antisense morpholino oligomers (PMO). Here, we determined the effect of L-arginine, D-arginine and non-α amino acids on cellular uptake, splice-correction activity, cellular toxicity and serum binding for 24 CPP−PMOs. Insertion of 6-aminohexanoic acid (X) or β-alanine (B) residues into oligoarginine R(8) decreased the cellular uptake but increased the splice-correction activity of the resulting compound, with a greater increase for the sequences containing more X residues. Cellular toxicity was not observed for any of the conjugates up to 10 μM. Up to 60 μM, only the conjugates with ⩾ 5 Xs exhibited time- and concentration-dependent toxicity. Substitution of L-arginine with D-arginine did not increase uptake or splice-correction activity. High concentration of serum significantly decreased the uptake and splice-correction activity of oligoarginine conjugates, but had much less effect on the conjugates containing X or B. In summary, incorporation of X/B into oligoarginine enhanced the antisense activity and serum-binding profile of CPP−PMO. Toxicity of X/B-containing conjugates was affected by the number of Xs, treatment time and concentration. More active, stable and less toxic CPPs can be designed by optimizing the position and number of R, D-R, X and B residues.


id: cord-048478-ftlb5b95
title: Apoptotic signals induce specific degradation of ribosomal RNA in yeast
author: Mroczek, Seweryn
date: 2008-04-01
txt: ./txt/cord-048478-ftlb5b95.txt
cache: ./cache/cord-048478-ftlb5b95.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2396418/
doi: 10.1093/nar/gkm1100
summary:
  One striking characteristic, which accompanies apoptosis in both vertebrates and yeast, is a fragmentation of cellular DNA and mammalian apoptosis is often associated with degradation of different RNAs. We show that in yeast exposed to stimuli known to induce apoptosis, such as hydrogen peroxide, acetic acid, hyperosmotic stress and ageing, two large subunit ribosomal RNAs, 25S and 5.8S, became extensively degraded with accumulation of specific intermediates that differ slightly depending on cell death conditions. For example, in metazoans the programmed cell death (PCD) called apoptosis, in addition to irreversible DNA damage, which is considered an apoptotic hallmark (3) , also involves specific cleavage of several RNA species, including 28S rRNA, U1 snRNA or Ro RNP-associated Y RNAs (4) . Together, this strongly suggests that rRNA degradation observed in apoptotic and oxidative stress conditions is not simply a result of cell death but is produced in the process that requires enzymatic activity and functional cellular machinery.

abstract:
  Organisms exposed to reactive oxygen species, generated endogenously during respiration or by environmental conditions, undergo oxidative stress. Stress response can either repair the damage or activate one of the programmed cell death (PCD) mechanisms, for example apoptosis, and finally end in cell death. One striking characteristic, which accompanies apoptosis in both vertebrates and yeast, is a fragmentation of cellular DNA and mammalian apoptosis is often associated with degradation of different RNAs. We show that in yeast exposed to stimuli known to induce apoptosis, such as hydrogen peroxide, acetic acid, hyperosmotic stress and ageing, two large subunit ribosomal RNAs, 25S and 5.8S, became extensively degraded with accumulation of specific intermediates that differ slightly depending on cell death conditions. This process is most likely endonucleolytic, is correlated with stress response, and depends on the mitochondrial respiratory status: rRNA is less susceptible to degradation in respiring cells with functional defence against oxidative stress. In addition, RNA fragmentation is independent of two yeast apoptotic factors, metacaspase Yca1 and apoptosis-inducing factor Aif1, but it relies on the apoptotic chromatin condensation induced by histone H2B modifications. These data describe a novel phenotype for certain stress- and ageing-related PCD pathways in yeast.


id: cord-262076-b5u5hp2r
title: Inhibition of HIV-1 by multiple siRNAs expressed from a single microRNA polycistron
author: Liu, Ying Poi
date: 2008-03-16
txt: ./txt/cord-262076-b5u5hp2r.txt
cache: ./cache/cord-262076-b5u5hp2r.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/18346971/
doi: 10.1093/nar/gkn109
summary:
  We show that the expression of individual miRNAs is greatly enhanced in multiplex hairpin transcripts that are properly processed into functional miRNAs. HIV-1 replication can be potently inhibited by simultaneous expression of four antiviral miRNAs. These combined results indicate that the multiplex miRNA strategy is a promising therapeutic approach against escape-prone viral pathogens. By repeating this procedure we obtained constructs expressing different combinations of 1, 2, 3, 4 and 6 pri-miRNAs. The RNA structures formed by the transcripts were predicted with the Mfold program (47) at http://frontend.bioinfo.rpi.edu/ applications/mfold/ and found to be similar to the predicted conformation of the wild-type pri-miRNAs. The firefly luciferase (FL) reporters containing HIV-1 target sequences pol47 (Luc-A pol47 ), pol1 (Luc-B pol1 ), gag5 (Luc-C gag5 ), r/t5 (Luc-D r/t5 ), ldr9 (Luc-E ldr9 ) and the anti-HIV shRNAs have been described previously (32) .

abstract:
  RNA interference (RNAi) is a powerful approach to inhibit human immunodeficiency virus type 1 (HIV-1) replication. However, HIV-1 can escape from RNAi-mediated antiviral therapy by selection of mutations in the targeted sequence. To prevent viral escape, multiple small interfering RNAs (siRNAs) against conserved viral sequences should be combined. Ideally, these RNA inhibitors should be expressed simultaneously from a single transgene transcript. In this study, we tested a multiplex microRNA (miRNA) expression strategy by inserting multiple effective anti-HIV siRNA sequences in the miRNA polycistron mir-17-92. Individual anti-HIV miRNAs that resemble the natural miRNA structures were optimized by varying the siRNA position in the hairpin stem to obtain maximal effectiveness against luciferase reporters and HIV-1. We show that an antiviral miRNA construct can have a greater intrinsic inhibitory activity than a conventional short hairpin (shRNA) construct. When combined in a polycistron setting, the silencing activity of an individual miRNA is strongly boosted. We demonstrate that HIV-1 replication can be efficiently inhibited by simultaneous expression of four antiviral siRNAs from the polycistronic miRNA transcript. These combined results indicate that a multiplex miRNA strategy may be a promising therapeutic approach to attack escape-prone viral pathogens.


id: cord-263645-wupre5uj
title: Insights into the development of chemical probes for RNA
author: Morgan, Brittany S
date: 2018-09-19
txt: ./txt/cord-263645-wupre5uj.txt
cache: ./cache/cord-263645-wupre5uj.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1093/nar/gky718
doi: 10.1093/nar/gky718
summary:
  One important example is the development of chemical probes, which has greatly progressed the study of proteins and related diseases (11, 12) but has been challenging for non-ribosomal RNAs. This powerful chemical tool requires small molecules with well-defined biological activity, cell permeability, and selectivity to accurately and reliably probe specific mechanistic and phenotypic questions (11, 12) . While ligands that bind non-ribosomal RNA in vitro have been reported for decades, the development of chemical probes with evidence of specific small molecule:RNA engagement in cell or animal models has dramatically increased in the last four years. Recent studies report several drug-like small molecules that target a range of RNAs in animal models, including riboswitches (15) , miRNAs, (16, 17) splice sites (18) , and mature mRNAs (19) , at least one of which is currently in clinical trials (NCT02268552).

abstract:
  Over the past decade, the RNA revolution has revealed thousands of non-coding RNAs that are essential for cellular regulation and are misregulated in disease. While the development of methods and tools to study these RNAs has been challenging, the power and promise of small molecule chemical probes is increasingly recognized. To harness existing knowledge, we compiled a list of 116 ligands with reported activity against RNA targets in biological systems (R-BIND). In this survey, we examine the RNA targets, design and discovery strategies, and chemical probe characterization techniques of these ligands. We discuss the applicability of current tools to identify and evaluate RNA-targeted chemical probes, suggest criteria to assess the quality of RNA chemical probes and targets, and propose areas where new tools are particularly needed. We anticipate that this knowledge will expedite the discovery of RNA-targeted ligands and the next phase of the RNA revolution.


id: cord-269150-d1sgnxc0
author: Tan, Yong Wah
date: 2012-02-22
txt: ./txt/cord-269150-d1sgnxc0.txt
cache: ./cache/cord-269150-d1sgnxc0.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1093/nar/gks165
doi: 10.1093/nar/gks165
summary:
  In a screen based on a yeast three-hybrid system using the 5′-untranslated region (5′-UTR) of SARS coronavirus (SARS-CoV) RNA as bait against a human cDNA library derived from HeLa cells, we found a positive candidate cellular protein, zinc finger CCHC-type and RNA-binding motif 1 (MADP1), to be able to interact with this region of the SARS-CoV genome. In this study, we describe the interaction of a cellular protein, MADP1 (zinc finger CCHC-type and RNA binding motif 1) with the 5 0 -UTR of IBV and SARS-CoV, using yeast-based three hybrid screen (34) and RNA-binding assays. Using indirect immunofluorescence, we confirmed that MADP1, despite being reported as a nuclear protein (35) , was detected in the cytoplasm of virus-infected cells and partially co-localized with the RTCs. Upon silencing of MADP1 using siRNA, viral RNA synthesis on general has been affected, resulting in a lower replication efficiency and infectivity.

title:
  Binding of the 5′-untranslated region of coronavirus RNA to zinc finger CCHC-type and RNA-binding motif 1 enhances viral replication and transcription

abstract:
  Coronaviruses RNA synthesis occurs in the cytoplasm and is regulated by host cell proteins. In a screen based on a yeast three-hybrid system using the 5′-untranslated region (5′-UTR) of SARS coronavirus (SARS-CoV) RNA as bait against a human cDNA library derived from HeLa cells, we found a positive candidate cellular protein, zinc finger CCHC-type and RNA-binding motif 1 (MADP1), to be able to interact with this region of the SARS-CoV genome. This interaction was subsequently confirmed in coronavirus infectious bronchitis virus (IBV). The specificity of the interaction between MADP1 and the 5′-UTR of IBV was investigated and confirmed by using an RNA pull-down assay. The RNA-binding domain was mapped to the N-terminal region of MADP1 and the protein binding sequence to stem–loop I of IBV 5′-UTR. MADP1 was found to be translocated to the cytoplasm and partially co-localized with the viral replicase/transcriptase complexes (RTCs) in IBV-infected cells, deviating from its usual nuclear localization in a normal cell using indirect immunofluorescence. Using small interfering RNA (siRNA) against MADP1, defective viral RNA synthesis was observed in the knockdown cells, therefore indicating the importance of the protein in coronaviral RNA synthesis.


id: cord-271701-tx0lqgff
author: te Velthuis, Aartjan J.W.
date: 2011-10-29
txt: ./txt/cord-271701-tx0lqgff.txt
cache: ./cache/cord-271701-tx0lqgff.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/22039154/
doi: 10.1093/nar/gkr893
summary:
  Commonly, its core subunit is a single RNA-dependent RNA polymerase (RdRp) that drives the production of template strands for replication, new genome molecules, and-in many RNA virus groupsalso subgenomic (sg) mRNAs. This canonical RdRp is structurally conserved among RNA viruses and widely accepted to drive catalysis of phosphodiester bond formation via a well-established reaction mechanism involving two metal ions that are coordinated by aspartate residues in its motifs A and C (3) (4) (5) . Interestingly, both nsp8 and nsp(7+8) are able to extend the RNA primers beyond template length in the presence of heparin ( Figure 4D and Supplementary Figure S2B ), suggesting that these extensions result from terminal transferase activity and not from template switching, as was previously observed for poliovirus 3D pol (20) . Subsequent alanine substitution of the N-terminal D/ ExD/E motif, composed of D50 and D52 in SARS-CoV, greatly affected primer extension activity on the CU 10 template as shown in Figure 5C .

title:
  The SARS-coronavirus nsp7+nsp8 complex is a unique multimeric RNA polymerase capable of both de novo initiation and primer extension

abstract:
  Uniquely among RNA viruses, replication of the ∼30-kb SARS-coronavirus genome is believed to involve two RNA-dependent RNA polymerase (RdRp) activities. The first is primer-dependent and associated with the 106-kDa non-structural protein 12 (nsp12), whereas the second is catalysed by the 22-kDa nsp8. This latter enzyme is capable of de novo initiation and has been proposed to operate as a primase. Interestingly, this protein has only been crystallized together with the 10-kDa nsp7, forming a hexadecameric, dsRNA-encircling ring structure [i.e. nsp(7+8), consisting of 8 copies of both nsps]. To better understand the implications of these structural characteristics for nsp8-driven RNA synthesis, we studied the prerequisites for the formation of the nsp(7+8) complex and its polymerase activity. We found that in particular the exposure of nsp8's natural N-terminal residue was paramount for both the protein's ability to associate with nsp7 and for boosting its RdRp activity. Moreover, this ‘improved’ recombinant nsp8 was capable of extending primed RNA templates, a property that had gone unnoticed thus far. The latter activity is, however, ∼20-fold weaker than that of the primer-dependent nsp12-RdRp at equal monomer concentrations. Finally, site-directed mutagenesis of conserved D/ExD/E motifs was employed to identify residues crucial for nsp(7+8) RdRp activity.


id: cord-273107-xc61osdx
author: Qureshi, Abid
date: 2014-01-01
txt: ./txt/cord-273107-xc61osdx.txt
cache: ./cache/cord-273107-xc61osdx.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1093/nar/gkt1191
doi: 10.1093/nar/gkt1191
summary:
  title: AVPdb: a database of experimentally validated antiviral peptides targeting medically important viruses Therefore, we have developed AVPdb, available online at http://crdd.osdd.net/servers/avpdb, to provide a dedicated resource of experimentally verified AVPs targeting over 60 medically important viruses including Influenza, HCV, HSV, RSV, HBV, DENV, SARS, etc. Whereas HIPdb is a specific database of experimentally validated HIV inhibiting peptides, which is freely available at http://crdd.osdd.net/servers/hipdb. Further 624 modified peptides were also extracted and have been provided separately in AVPdb. In our database, complete AVP data of almost all human viruses reported in the literature have been included. AVPdb also provides physicochemical properties and predicted structure of AVPs along with more informative tools for data analysis such as BLAST and MAP as well as links to major peptide resources. For modified AVPs also, a separate browse option is provided where the data can be sought by Virus, Modification, Peptide Source, Cell Line, Target and Assay.

title:
  AVPdb: a database of experimentally validated antiviral peptides targeting medically important viruses

abstract:
  Antiviral peptides (AVPs) have exhibited huge potential in inhibiting viruses by targeting various stages of their life cycle. Therefore, we have developed AVPdb, available online at http://crdd.osdd.net/servers/avpdb, to provide a dedicated resource of experimentally verified AVPs targeting over 60 medically important viruses including Influenza, HCV, HSV, RSV, HBV, DENV, SARS, etc. However, we have separately provided HIV inhibiting peptides in ‘HIPdb’. AVPdb contains detailed information of 2683 peptides, including 624 modified peptides experimentally tested for antiviral activity. In modified peptides a chemical moiety is attached for increasing their efficacy and stability. Detailed information include: peptide sequence, length, source, virus targeted, virus family, cell line used, efficacy (qualitative/quantitative), target step/protein, assay used in determining the efficacy and PubMed reference. The database also furnishes physicochemical properties and predicted structure for each peptide. We have provided user-friendly browsing and search facility along with other analysis tools to help the users. Entering of many synthetic peptide-based drugs in various stages of clinical trials reiterate the importance for the AVP resources. AVPdb is anticipated to cater to the needs of scientific community working for the development of antiviral therapeutics.


id: cord-275232-0sg0hv9w
title: A DNA biochip for on-the-spot multiplexed pathogen identification
author: Yeung, Siu-Wai
date: 2006-09-25
txt: ./txt/cord-275232-0sg0hv9w.txt
cache: ./cache/cord-275232-0sg0hv9w.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/17000638/
doi: 10.1093/nar/gkl702
summary:
  The DNA-based identification of the two model pathogens involved a number of steps including a thermal lysis step, magnetic particle-based isolation of the target genomes, asymmetric PCR, and electrochemical sequence-specific detection using silver-enhanced gold nanoparticles. The assay involves the following steps: (i) sample preparation using thermal cell lysis and magnetic particle-based target genome isolation; (ii) target DNA amplification by the PCR; (iii) hybridization of the amplicons to their complementary oligonucleotide capture probes immobilized onto individual detection electrode surfaces and (iv) electrochemical transduction of the recognition event via gold nanoparticles with signal amplification using electrocatalytic silver deposition (10) . The three main steps were (A) sample preparation: thermal cell lysis and magnetic particle-based isolation of specific genomic DNAs; (B) target DNA amplification: generation of single-stranded rich amplicons by asymmetric PCR; (C) product detection: gold nanoparticle labeling, electrocatalytic silver deposition, and electrochemical silver dissolution.

abstract:
  Miniaturized integrated DNA analysis systems have largely been based on a multi-chamber design with microfluidic control to process the sample sequentially from one module to another. This microchip design in connection with optics involved hinders the deployment of this technology for point-of-care applications. In this work, we demonstrate the implementation of sample preparation, DNA amplification, and electrochemical detection in a single silicon and glass-based microchamber and its application for the multiplexed detection of Escherichia coli and Bacillus subtilis cells. The microdevice has a thin-film heater and temperature sensor patterned on the silicon substrate. An array of indium tin oxide (ITO) electrodes was constructed within the microchamber as the transduction element. Oligonucleotide probes specific to the target amplicons are individually positioned at each ITO surface by electrochemical copolymerization of pyrrole and pyrrole−probe conjugate. These immobilized probes were stable to the thermal cycling process and were highly selective. The DNA-based identification of the two model pathogens involved a number of steps including a thermal lysis step, magnetic particle-based isolation of the target genomes, asymmetric PCR, and electrochemical sequence-specific detection using silver-enhanced gold nanoparticles. The microchamber platform described here offers a cost-effective and sample-to-answer technology for on-site monitoring of multiple pathogens.


id: cord-275519-98qxf6xo
author: Chun, Jong-Yoon
date: 2007-02-07
txt: ./txt/cord-275519-98qxf6xo.txt
cache: ./cache/cord-275519-98qxf6xo.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/17287288/
doi: 10.1093/nar/gkm051
summary:
  This structure results in two primer segments with distinct annealing properties: a longer 5′-segment that initiates stable priming, and a short 3′-segment that determines target-specific extension. This DPO-based system is a fundamental tool for blocking extension of non-specifically primed templates, and thereby generates consistently high PCR specificity even under less than optimal PCR conditions. Since the development of the polymerase chain reaction (PCR), a variety of modifications in primer design and reaction conditions have been proposed to enhance and optimize specificity (1-3), but a fundamental solution for eliminating non-specific priming still remains a challenge and limits the versatility of PCR in nucleic-acid-based tests (NATs). In this article, we describe and demonstrate how effectively DPO eliminates extension of non-specifically primed templates and generates high PCR specificity under a range of sub-optimal or stringent reaction conditions. We further evaluated the DPO-based multiplex PCR system for the detection of a single nucleotide polymorphism (SNP) in CYP2C19.

title:
  Dual priming oligonucleotide system for the multiplex detection of respiratory viruses and SNP genotyping of CYP2C19 gene

abstract:
  Successful PCR starts with proper priming between an oligonucleotide primer and the template DNA. However, the inevitable risk of mismatched priming cannot be avoided in the currently used primer system, even though considerable time and effort are devoted to primer design and optimization of reaction conditions. Here, we report a novel dual priming oligonucleotide (DPO) which contains two separate priming regions joined by a polydeoxyinosine linker. The linker assumes a bubble-like structure which itself is not involved in priming, but rather delineates the boundary between the two parts of the primer. This structure results in two primer segments with distinct annealing properties: a longer 5′-segment that initiates stable priming, and a short 3′-segment that determines target-specific extension. This DPO-based system is a fundamental tool for blocking extension of non-specifically primed templates, and thereby generates consistently high PCR specificity even under less than optimal PCR conditions. The strength and utility of the DPO system are demonstrated here using multiplex PCR and SNP genotyping PCR.


id: cord-275859-ix8du1er
author: Mouzakis, Kathryn D.
date: 2012-12-15
txt: ./txt/cord-275859-ix8du1er.txt
cache: ./cache/cord-275859-ix8du1er.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/23248007/
doi: 10.1093/nar/gks1254
summary:
  In contrast, there is a strong correlation between frameshift efficiency and the local thermodynamic stability of the first 3–4 bp in the stem–loop, which are predicted to reside at the opening of the mRNA entrance channel when the ribosome is paused at the slippery site. Here, we investigate the role of the HIV-1 RNA structure in frameshifting, focusing on elucidating the relationships between frameshift efficiency and (i) the downstream RNA stem-loop thermodynamic stability, (ii) spacer length and (iii) surrounding genomic secondary structure. Our data further indicate that the base pairs important for frameshifting are located at a distance of 8 nt from the slippery site, which corresponds to the length of the spacer and is consistent with a structural model of the ribosome paused at the frameshift site. Instead, we observe a strong correlation (R 2 = 0.88) between frameshift efficiency and local stability of the first 3 bp at the base of the stem-loop using a one-phase exponential decay function ( Figure 3C and Supplementary Table S3 ).

title:
  HIV-1 frameshift efficiency is primarily determined by the stability of base pairs positioned at the mRNA entrance channel of the ribosome

abstract:
  The human immunodeficiency virus (HIV) requires a programmed −1 ribosomal frameshift for Pol gene expression. The HIV frameshift site consists of a heptanucleotide slippery sequence (UUUUUUA) followed by a spacer region and a downstream RNA stem–loop structure. Here we investigate the role of the RNA structure in promoting the −1 frameshift. The stem–loop was systematically altered to decouple the contributions of local and overall thermodynamic stability towards frameshift efficiency. No correlation between overall stability and frameshift efficiency is observed. In contrast, there is a strong correlation between frameshift efficiency and the local thermodynamic stability of the first 3–4 bp in the stem–loop, which are predicted to reside at the opening of the mRNA entrance channel when the ribosome is paused at the slippery site. Insertion or deletions in the spacer region appear to correspondingly change the identity of the base pairs encountered 8 nt downstream of the slippery site. Finally, the role of the surrounding genomic secondary structure was investigated and found to have a modest impact on frameshift efficiency, consistent with the hypothesis that the genomic secondary structure attenuates frameshifting by affecting the overall rate of translation.


id: cord-284990-klsl1nzn
author: Zhang, Dapeng
date: 2011-02-08
txt: ./txt/cord-284990-klsl1nzn.txt
cache: ./cache/cord-284990-klsl1nzn.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1093/nar/gkr036
doi: 10.1093/nar/gkr036
summary:
  By analyzing the toxin proteins encoded in the neighborhood of the SUKH superfamily we predict that they possess domains belonging to diverse nuclease and nucleic acid deaminase families. Our above observations indicate that outside of CDI systems, the SUKH superfamily genes are linked to genes encoding the HNH and NucA nucleases; hence, it is likely that even these nucleases function as distinct but analogous toxins that cleave nucleic acids in target cells. Together, the above observations raised the possibility that the SUKH superfamily protein might serve as immunity proteins, not just in certain proteobacterial CDI systems, but also more generally function, across all major bacterial lineages, to protect against linked genes, which are predicted to act as toxins. In bacteria the SUKH superfamily domains are one of the most widespread immunity proteins that appear to function in conjunction with a repertoire of nuclease toxins that are extremely diverse in sequence and structure (Figures 3 and 4) .

title:
  A novel immunity system for bacterial nucleic acid degrading toxins and its recruitment in various eukaryotic and DNA viral systems

abstract:
  The use of nucleases as toxins for defense, offense or addiction of selfish elements is widely encountered across all life forms. Using sensitive sequence profile analysis methods, we characterize a novel superfamily (the SUKH superfamily) that unites a diverse group of proteins including Smi1/Knr4, PGs2, FBXO3, SKIP16, Syd, herpesviral US22, IRS1 and TRS1, and their bacterial homologs. Using contextual analysis we present evidence that the bacterial members of this superfamily are potential immunity proteins for a variety of toxin systems that also include the recently characterized contact-dependent inhibition (CDI) systems of proteobacteria. By analyzing the toxin proteins encoded in the neighborhood of the SUKH superfamily we predict that they possess domains belonging to diverse nuclease and nucleic acid deaminase families. These include at least eight distinct types of DNases belonging to HNH/EndoVII- and restriction endonuclease-fold, and RNases of the EndoU-like and colicin E3-like cytotoxic RNases-folds. The N-terminal domains of these toxins indicate that they are extruded by several distinct secretory mechanisms such as the two-partner system (shared with the CDI systems) in proteobacteria, ESAT-6/WXG-like ATP-dependent secretory systems in Gram-positive bacteria and the conventional Sec-dependent system in several bacterial lineages. The hedgehog-intein domain might also release a subset of toxic nuclease domains through auto-proteolytic action. Unlike classical colicin-like nuclease toxins, the overwhelming majority of toxin systems with the SUKH superfamily is chromosomally encoded and appears to have diversified through a recombination process combining different C-terminal nuclease domains to N-terminal secretion-related domains. Across the bacterial superkingdom these systems might participate in discriminating `self’ or kin from `non-self’ or non-kin strains. Using structural analysis we demonstrate that the SUKH domain possesses a versatile scaffold that can be used to bind a wide range of protein partners. In eukaryotes it appears to have been recruited as an adaptor to regulate modification of proteins by ubiquitination or polyglutamylation. Similarly, another widespread immunity protein from these toxin systems, namely the suppressor of fused (SuFu) superfamily has been recruited for comparable roles in eukaryotes. In animal DNA viruses, such as herpesviruses, poxviruses, iridoviruses and adenoviruses, the ability of the SUKH domain to bind diverse targets has been deployed to counter diverse anti-viral responses by interacting with specific host proteins.


id: cord-289274-3g67f8sw
author: Tosoni, Elena
date: 2015-10-15
txt: ./txt/cord-289274-3g67f8sw.txt
cache: ./cache/cord-289274-3g67f8sw.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/26354862/
doi: 10.1093/nar/gkv897
summary:
  The implementation of electrophorethic mobility shift assay and pull-down experiments coupled with mass spectrometric analysis revealed that the cellular protein nucleolin is able to specifically recognize G-quadruplex structures present in the LTR promoter. In this direction, the significance of these structures as focal points of interactions with host and viral factors is supported also by the observation that G4-folded sequences are specifically recognized by various viral proteins, such as the Epstein Barr Virus Nuclear Antigen 1 (34, 35) and the SARS coronavirus unique domain (SUD), which occurs exclusively in highly pathogenic strains (36) . The LTR-II+III+IV oligonucleotide was incubated with extracts of HIV-1 producing and non-producing 293T cells to test whether the presence of viral proteins affected in any detectable way the observed EMSA profiles ( Figure 1C ). Positive identification was also confirmed by performing EMSA analysis of samples that included the G4-folded wt and mutant LTR-II+III+IV sequences with either nuclear extracts or purified human NCL. (B) EMSA analysis of the binding of nuclear extract (NE) proteins and purified NCL to the wt and mutant LTR sequences.

title:
  Nucleolin stabilizes G-quadruplex structures folded by the LTR promoter and silences HIV-1 viral transcription

abstract:
  Folding of the LTR promoter into dynamic G-quadruplex conformations has been shown to suppress its transcriptional activity in HIV-1. Here we sought to identify the proteins that control the folding of this region of proviral genome by inducing/stabilizing G-quadruplex structures. The implementation of electrophorethic mobility shift assay and pull-down experiments coupled with mass spectrometric analysis revealed that the cellular protein nucleolin is able to specifically recognize G-quadruplex structures present in the LTR promoter. Nucleolin recognized with high affinity and specificity the majority, but not all the possible G-quadruplexes folded by this sequence. In addition, it displayed greater binding preference towards DNA than RNA G-quadruplexes, thus indicating two levels of selectivity based on the sequence and nature of the target. The interaction translated into stabilization of the LTR G-quadruplexes and increased promoter silencing activity; in contrast, disruption of nucleolin binding in cells by both siRNAs and a nucleolin binding aptamer greatly increased LTR promoter activity. These data indicate that nucleolin possesses a specific and regulated activity toward the HIV-1 LTR promoter, which is mediated by G-quadruplexes. These observations provide new essential insights into viral transcription and a possible low mutagenic target for antiretroviral therapy.


id: cord-291070-y0wf456f
author: Zhang, Guang Lan
date: 2005-07-01
txt: ./txt/cord-291070-y0wf456f.txt
cache: ./cache/cord-291070-y0wf456f.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/15980450/
doi: 10.1093/nar/gki479
summary:
  PRED(BALB/c) is a computational system that predicts peptides binding to the major histocompatibility complex-2 (H2(d)) of the BALB/c mouse, an important laboratory model organism. To our knowledge, this is the first online server for the prediction of peptides binding to a complete set of major histocompatibility complex molecules in a model organism (H2(d) haplotype). PRED BALB/c is a computational system for the prediction of peptides binding to all five MHC molecules in BALB/c mice (H2 d ) class I (H2-K d , H2-L d and H2-D d ) and class II (I-A d and I-E d ) that allows analysis of proteins for the presence of binding motifs to all five H2 d molecules in parallel. We derived the initial quantitative matrices for PRED BALB/c using logarithmic equations based on the frequency of amino acids at specific positions within the training set of 9mer peptides as described previously (16) . To our knowledge, PRED BALB/c is the first online server for the prediction of peptides binding to a complete set of MHC molecules in a model organism (H2 d haplotype).

title:
  PRED(BALB/c): a system for the prediction of peptide binding to H2(d) molecules, a haplotype of the BALB/c mouse

abstract:
  PRED(BALB/c) is a computational system that predicts peptides binding to the major histocompatibility complex-2 (H2(d)) of the BALB/c mouse, an important laboratory model organism. The predictions include the complete set of H2(d) class I (H2-K(d), H2-L(d) and H2-D(d)) and class II (I-E(d) and I-A(d)) molecules. The prediction system utilizes quantitative matrices, which were rigorously validated using experimentally determined binders and non-binders and also by in vivo studies using viral proteins. The prediction performance of PRED(BALB/c) is of very high accuracy. To our knowledge, this is the first online server for the prediction of peptides binding to a complete set of major histocompatibility complex molecules in a model organism (H2(d) haplotype). PRED(BALB/c) is available at .


id: cord-297760-uzzuoy9v
title: siVirus: web-based antiviral siRNA design software for highly divergent viral sequences
author: Naito, Yuki
date: 2006-07-01
txt: ./txt/cord-297760-uzzuoy9v.txt
cache: ./cache/cord-297760-uzzuoy9v.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/16845046/
doi: 10.1093/nar/gkl214
summary:
  title: siVirus: web-based antiviral siRNA design software for highly divergent viral sequences siVirus () is a web-based online software system that provides efficient short interfering RNA (siRNA) design for antiviral RNA interference (RNAi). siVirus searches for functional, off-target minimized siRNAs targeting highly conserved regions of divergent viral sequences. siVirus will be a useful tool for designing optimal siRNAs targeting highly divergent pathogens, including human immunodeficiency virus (HIV), hepatitis C virus (HCV), influenza virus and SARS coronavirus, all of which pose enormous threats to global human health. Consequently, only a limited fraction of 21mers is suitable for use as antiviral siRNAs. In this study, we developed a novel web-based online software system, siVirus, which provides functional, off-target minimized siRNAs targeting highly conserved regions of divergent viral sequences. Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference siDirect: highly effective, target-specific siRNA design software for mammalian RNA interference

abstract:
  siVirus () is a web-based online software system that provides efficient short interfering RNA (siRNA) design for antiviral RNA interference (RNAi). siVirus searches for functional, off-target minimized siRNAs targeting highly conserved regions of divergent viral sequences. These siRNAs are expected to resist viral mutational escape, since their highly conserved targets likely contain structurally/functionally constrained elements. siVirus will be a useful tool for designing optimal siRNAs targeting highly divergent pathogens, including human immunodeficiency virus (HIV), hepatitis C virus (HCV), influenza virus and SARS coronavirus, all of which pose enormous threats to global human health.


id: cord-302368-uhhtvdif
author: Longhini, Andrew P.
date: 2016-04-07
txt: ./txt/cord-302368-uhhtvdif.txt
cache: ./cache/cord-302368-uhhtvdif.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1093/nar/gkv1333
doi: 10.1093/nar/gkv1333
summary:
  Finally, we showcase the improvement in spectral quality arising from reduced crowding and narrowed linewidths, and accurate analysis of NMR relaxation dispersion (CPMG) and TROSY-based CEST experiments to measure μs-ms time scale motions, and an improved NOESY strategy for resonance assignment. Additionally, we show that the measurements of relaxation parameters using CPMG, R 1 , and CEST are possible for both small and large RNAs. Furthermore, we demonstrate substantial improvements in signalto-noise and line width for relaxation optimized spectroscopy (TROSY) experiments compared to the traditional heteronuclear single quantum coherence (HSQC) exNucleic Acids Research, 2016, Vol. 44, No. 6 e52 periments for isolated two-spin systems approximated by our purine and pyrimidine labeling schemes (30) (31) (66) (67) . Thus, RNAs synthesized with our selective site-specifically labeled NTPs should benefit from TROSY based NMR experiments that reduce the problems of crowding, fast signal decay, low resolution, and decreased S/N ratios (12, 34, 31, (66) (67) (80) (81) .

title:
  Chemo-enzymatic synthesis of site-specific isotopically labeled nucleotides for use in NMR resonance assignment, dynamics and structural characterizations

abstract:
  Stable isotope labeling is central to NMR studies of nucleic acids. Development of methods that incorporate labels at specific atomic positions within each nucleotide promises to expand the size range of RNAs that can be studied by NMR. Using recombinantly expressed enzymes and chemically synthesized ribose and nucleobase, we have developed an inexpensive, rapid chemo-enzymatic method to label ATP and GTP site specifically and in high yields of up to 90%. We incorporated these nucleotides into RNAs with sizes ranging from 27 to 59 nucleotides using in vitro transcription: A-Site (27 nt), the iron responsive elements (29 nt), a fluoride riboswitch from Bacillus anthracis (48 nt), and a frame-shifting element from a human corona virus (59 nt). Finally, we showcase the improvement in spectral quality arising from reduced crowding and narrowed linewidths, and accurate analysis of NMR relaxation dispersion (CPMG) and TROSY-based CEST experiments to measure μs-ms time scale motions, and an improved NOESY strategy for resonance assignment. Applications of this selective labeling technology promises to reduce difficulties associated with chemical shift overlap and rapid signal decay that have made it challenging to study the structure and dynamics of large RNAs beyond the 50 nt median size found in the PDB.


id: cord-302895-471zei5o
author: Deng, Zengqin
date: 2013-12-24
txt: ./txt/cord-302895-471zei5o.txt
cache: ./cache/cord-302895-471zei5o.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/24369429/
doi: 10.1093/nar/gkt1310
summary:
  Biochemical studies using recombinant arterivirus and coronavirus helicases revealed similar enzymatic properties, including nucleic acid-stimulated ATPase and 5 0 -3 0 duplex unwinding activities on both RNA and DNA substrates containing 5 0 single-stranded regions (34, 35) . Amino acid substitutions in ZBD or the adjacent ''spacer'' that connects it to the downstream domain can profoundly affect EAV helicase activity and RNA synthesis, with most replacements of conserved Cys or His residues yielding replicationnegative virus phenotypes (36, 37) . Thus, our study not only highlights how nidovirus helicase activity depends on the extensive relay of interactions between the ZBD, accessory and HEL1 domains but also provides a framework to propose and explore a role for the enzyme in the posttranscriptional quality control of nidovirus RNAs. Nsp10 of the EAV-Bucyrus isolate (NCBI Reference Sequence NC_002532) is composed of amino acids 2371-2837 of replicase pp1ab, which will throughout this study be referred to as nsp10 residues 1-467.

title:
  Structural basis for the regulatory function of a complex zinc-binding domain in a replicative arterivirus helicase resembling a nonsense-mediated mRNA decay helicase

abstract:
  All positive-stranded RNA viruses with genomes >∼7 kb encode helicases, which generally are poorly characterized. The core of the nidovirus superfamily 1 helicase (HEL1) is associated with a unique N-terminal zinc-binding domain (ZBD) that was previously implicated in helicase regulation, genome replication and subgenomic mRNA synthesis. The high-resolution structure of the arterivirus helicase (nsp10), alone and in complex with a polynucleotide substrate, now provides first insights into the structural basis for nidovirus helicase function. A previously uncharacterized domain 1B connects HEL1 domains 1A and 2A to a long linker of ZBD, which further consists of a novel RING-like module and treble-clef zinc finger, together coordinating three Zn atoms. On substrate binding, major conformational changes were evident outside the HEL1 domains, notably in domain 1B. Structural characterization, mutagenesis and biochemistry revealed that helicase activity depends on the extensive relay of interactions between the ZBD and HEL1 domains. The arterivirus helicase structurally resembles the cellular Upf1 helicase, suggesting that nidoviruses may also use their helicases for post-transcriptional quality control of their large RNA genomes.


id: cord-304794-z2kx314h
title: G-quadruplexes in viruses: function and potential therapeutic applications
author: Métifiot, Mathieu
date: 2014-11-10
txt: ./txt/cord-304794-z2kx314h.txt
cache: ./cache/cord-304794-z2kx314h.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1093/nar/gku999
doi: 10.1093/nar/gku999
summary:
  Conversely, a G-quadruplex or G4 is formed by nucleic acid sequences (DNA or RNA) containing G-tracts or Gblocks (adjacent runs of guanines) and composed of various numbers of guanines. Short RNA templates from the central region of the HIV-1 genome contain G-rich sequences near the central polypurine tract (cPPT) at the 3 end of the pol gene (IN coding sequence); this is a region where one of the two primers used for synthesizing the (−) strand DNA is produced during reverse transcription. In addition, one could imagine alternative therapeutic strategies focused on targeting RNA structures within viral ORFs to interfere with the virus cycle as well as to promote antigen presentation and to stimulate the host immune response. Topology of a DNA G-quadruplex structure formed in the HIV-1 promoter: a potential target for anti-HIV drug development U3 Region in the HIV-1 genome adopts a G-quadruplex structure in its RNA and DNA sequence

abstract:
  G-rich nucleic acids can form non-canonical G-quadruplex structures (G4s) in which four guanines fold in a planar arrangement through Hoogsteen hydrogen bonds. Although many biochemical and structural studies have focused on DNA sequences containing successive, adjacent guanines that spontaneously fold into G4s, evidence for their in vivo relevance has recently begun to accumulate. Complete sequencing of the human genome highlighted the presence of ∼300 000 sequences that can potentially form G4s. Likewise, the presence of putative G4-sequences has been reported in various viruses genomes [e.g., Human immunodeficiency virus (HIV-1), Epstein–Barr virus (EBV), papillomavirus (HPV)]. Many studies have focused on telomeric G4s and how their dynamics are regulated to enable telomere synthesis. Moreover, a role for G4s has been proposed in cellular and viral replication, recombination and gene expression control. In parallel, DNA aptamers that form G4s have been described as inhibitors and diagnostic tools to detect viruses [e.g., hepatitis A virus (HAV), EBV, cauliflower mosaic virus (CaMV), severe acute respiratory syndrome virus (SARS), simian virus 40 (SV40)]. Here, special emphasis will be given to the possible role of these structures in a virus life cycle as well as the use of G4-forming oligonucleotides as potential antiviral agents and innovative tools.


id: cord-308331-55ge7kmr
author: Routh, Andrew
date: 2013-10-09
txt: ./txt/cord-308331-55ge7kmr.txt
cache: ./cache/cord-308331-55ge7kmr.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1093/nar/gkt916
doi: 10.1093/nar/gkt916
summary:
  Using ViReMa, we demonstrate that by mapping the distribution and frequency of recombination events in the genome of flock house virus (FHV), we can discover de novo functional genomic motifs required for viral replication and encapsidation. Here, segments at the 5 0 and the 3 0 end of a complex recombination event have been mapped to nt 500-550 and nt 1040-1080 of FHV RNA 1, but there remain a small number of trimmed nucleotides in the middle. We generated 5 043 791 synthetic reads containing 99 033 unique recombination events and aligned these reads to the FHV genome with ViReMa using a seed length of 20 nt. Our analysis of FHV demonstrates that by isolating a small number of virus particles, deep sequencing the encapsidated RNA and mapping the positions of recombination events, functional RNA motifs can be discovered.

title:
  Discovery of functional genomic motifs in viruses with ViReMa–a Virus Recombination Mapper–for analysis of next-generation sequencing data

abstract:
  We developed an algorithm named ViReMa (Viral-Recombination-Mapper) to provide a versatile platform for rapid, sensitive and nucleotide-resolution detection of recombination junctions in viral genomes using next-generation sequencing data. Rather than mapping read segments of pre-defined lengths and positions, ViReMa dynamically generates moving read segments. ViReMa initially attempts to align the 5′ end of a read to the reference genome(s) with the Bowtie seed-based alignment. A new read segment is then made by either extracting any unaligned nucleotides at the 3′ end of the read or by trimming the first nucleotide from the read. This continues iteratively until all portions of the read are either mapped or trimmed. With multiple reference genomes, it is possible to detect virus-to-host or inter-virus recombination. ViReMa is also capable of detecting insertion and substitution events and multiple recombination junctions within a single read. By mapping the distribution of recombination events in the genome of flock house virus, we demonstrate that this information can be used to discover de novo functional motifs located in conserved regions of the viral genome.


id: cord-314572-1pou702r
author: Lin, Ya-Hui
date: 2016-10-14
txt: ./txt/cord-314572-1pou702r.txt
cache: ./cache/cord-314572-1pou702r.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/27521370/
doi: 10.1093/nar/gkw718
summary:
  Conformational and functional analyses indicate that the engineered theophylline-responsive RNA functions as a mammalian riboswitch with robust theophylline-dependent −1 PRF stimulation activity in a stable human 293T cell-line. In a first step to constructing a ligand-responsive −1 PRF stimulator, we designed Switch-0 RNA with a theophylline aptamer replacing the stem 3 of SARS-PK ( Figure 1A and C). We rationalized that such an engineered switch hairpin of reasonable stability (predicted free energy of −12.7 kcal/mole (37)) would be the dominant conformation that could interfere with the formation of pseudoknot stem 2 in the absence of theophylline (Supplementary Figure S2A) . To improve the dynamic range of ligand response and to see if theophylline aptamers can be functional while existing in both positive and negative regulators of −1 PRF, we fused previously designed theophylline-dependent upstream attenuator, theoOFF2 (24) with Switch-1 ( Figure 5A ) and examined theophylline-dependent −1 PRF activity in vitro.

title:
  Rational design of a synthetic mammalian riboswitch as a ligand-responsive -1 ribosomal frame-shifting stimulator

abstract:
  Metabolite-responsive RNA pseudoknots derived from prokaryotic riboswitches have been shown to stimulate −1 programmed ribosomal frameshifting (PRF), suggesting −1 PRF as a promising gene expression platform to extend riboswitch applications in higher eukaryotes. However, its general application has been hampered by difficulty in identifying a specific ligand-responsive pseudoknot that also functions as a ligand-dependent -1 PRF stimulator. We addressed this problem by using the −1 PRF stimulation pseudoknot of SARS-CoV (SARS-PK) to build a ligand-dependent −1 PRF stimulator. In particular, the extra stem of SARS-PK was replaced by an RNA aptamer of theophylline and designed to couple theophylline binding with the stimulation of −1 PRF. Conformational and functional analyses indicate that the engineered theophylline-responsive RNA functions as a mammalian riboswitch with robust theophylline-dependent −1 PRF stimulation activity in a stable human 293T cell-line. Thus, RNA–ligand interaction repertoire provided by in vitro selection becomes accessible to ligand-specific −1 PRF stimulator engineering using SARS-PK as the scaffold for synthetic biology application.


id: cord-314877-db7tze8j
title: Activation of viral transcription by stepwise largescale folding of an RNA virus genome
author: Chkuaseli, Tamari
date: 2020-08-12
txt: ./txt/cord-314877-db7tze8j.txt
cache: ./cache/cord-314877-db7tze8j.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/32785642/
doi: 10.1093/nar/gkaa675
summary:
  When viewed in the context of the RNA secondary structure model for the TBSV genome (48) , the DE/CE interaction corresponds to the closing stem of a sizable RNA domain, termed large domain 3 (LD3), which, along with formation of the adjacent LD2, acts to unite the AS2 and RS2 sequences ( Figure 1B) . Translational readthrough for the CIRV genome requires a long-distance RNA-RNA interaction (LDRI) between RTSL and the 3 UTR, involving the PRTE and DRTE partner sequences, respectively ( Figure 1A , B) (39) . The binding of RTSL-TL to SL59-5 was investigated functionally by introducing compensatory nucleotide substitutions into the candidate partner sequences and assessing the effects on sg mRNA1 accumulation following transfection of mutant viral RNA genomes into protoplasts pairing potential in mutants TC-6 and TC-7 diminished sg mRNA1 plus-and minus-strand levels below ∼10% of wt, while regenerating pairing capacity with alternate nucleotides in mutant TC-8 restored levels up to ∼50-62% of wt ( Figure 3B, C) .

abstract:
  The genomes of RNA viruses contain regulatory elements of varying complexity. Many plus-strand RNA viruses employ largescale intra-genomic RNA-RNA interactions as a means to control viral processes. Here, we describe an elaborate RNA structure formed by multiple distant regions in a tombusvirus genome that activates transcription of a viral subgenomic mRNA. The initial step in assembly of this intramolecular RNA complex involves the folding of a large viral RNA domain, which generates a discontinuous binding pocket. Next, a distally-located protracted stem-loop RNA structure docks, via base-pairing, into the binding site and acts as a linchpin that stabilizes the RNA complex and activates transcription. A multi-step RNA folding pathway is proposed in which rate-limiting steps contribute to a delay in transcription of the capsid protein-encoding viral subgenomic mRNA. This study provides an exceptional example of the complexity of genome-scale viral regulation and offers new insights into the assembly schemes utilized by large intra-genomic RNA structures.


id: cord-319116-2ts6zpdb
title: G-quadruplexes and G-quadruplex ligands: targets and tools in antiviral therapy
author: Ruggiero, Emanuela
date: 2018-04-20
txt: ./txt/cord-319116-2ts6zpdb.txt
cache: ./cache/cord-319116-2ts6zpdb.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/29554280/
doi: 10.1093/nar/gky187
summary:
  Since the number of reports describing the presence of G4s in virus genomes has boomed in the past 2 years and treatment with several G4 ligands has shown potentially interesting therapeutic activity, we here aim at presenting, organizing and discussing an up-to-date close-up of the literature on G4s in viruses and the classes of molecules that have shown antiviral activity by viral G4 targeting. The research of G4s in the HIV-1 genome has been quite productive, concerning not only the two RNA viral genome copies, but also the integrated proviral genome, specifically For each virus the following information is shown: virion structure and dimension, genome size and organization; schematic representation of the G4 (red dots) location in the viral genomes or in the mRNA and G4 binding proteins; number of G4s assessed through bioinformatics analysis, according to the corresponding references; G4 ligands reported to date to display antiviral effect and corresponding references.

abstract:
  G-quadruplexes (G4s) are non-canonical nucleic acids secondary structures that form within guanine-rich strands of regulatory genomic regions. G4s have been extensively described in the human genome, especially in telomeres and oncogene promoters; in recent years the presence of G4s in viruses has attracted increasing interest. Indeed, G4s have been reported in several viruses, including those involved in recent epidemics, such as the Zika and Ebola viruses. Viral G4s are usually located in regulatory regions of the genome and implicated in the control of key viral processes; in some cases, they have been involved also in viral latency. In this context, G4 ligands have been developed and tested both as tools to study the complexity of G4-mediated mechanisms in the viral life cycle, and as therapeutic agents. In general, G4 ligands showed promising antiviral activity, with G4-mediated mechanisms of action both at the genome and transcript level. This review aims to provide an updated close-up of the literature on G4s in viruses. The current state of the art of G4 ligands in antiviral research is also reported, with particular focus on the structural and physicochemical requirements for optimal biological activity. The achievements and the to-dos in the field are discussed.


id: cord-319649-d6dqr03e
author: Yang, Jie
date: 2013-12-05
txt: ./txt/cord-319649-d6dqr03e.txt
cache: ./cache/cord-319649-d6dqr03e.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/24319147/
doi: 10.1093/nar/gkt1256
summary:
  Here, we expressed VP5 from type 5 Helicoverpa armigera cypovirus (HaCPV-5) in a eukaryotic system and determined that this VP5 possesses RNA chaperone-like activity, which destabilizes RNA helices and accelerates strand annealing independent of ATP. In this study, we expressed HaCPV-5 VP5 in a eukaryotic expression system and determined that this CPV VP5 possesses an RNA chaperone-like activity to ATP-independently destabilize RNA helices and accelerate strand annealing. Moreover, we found that HaCPV-5 VP5 could facilitate the transcription initiation of an alternative polymerase (i.e. reverse transcriptase) through a CPV panhandle-structured RNA template, thereby strongly suggesting a direct role of the RNA chaperone activity of VP5 in the initiation of cypoviral dsRNA replication. In the family Reoviridae, CPV VP5 may not be the only RNA chaperone, as rotavirus nonstructural protein 2 (NSP2), which is a multifunctional enzyme involved in rotaviral dsRNA replication, was previously shown to contain ATP-independent nucleic acid helix-destabilizing activity (45) .

title:
  A cypovirus VP5 displays the RNA chaperone-like activity that destabilizes RNA helices and accelerates strand annealing

abstract:
  For double-stranded RNA (dsRNA) viruses in the family Reoviridae, their inner capsids function as the machinery for viral RNA (vRNA) replication. Unlike other multishelled reoviruses, cypovirus has a single-layered capsid, thereby representing a simplified model for studying vRNA replication of reoviruses. VP5 is one of the three major cypovirus capsid proteins and functions as a clamp protein to stabilize cypovirus capsid. Here, we expressed VP5 from type 5 Helicoverpa armigera cypovirus (HaCPV-5) in a eukaryotic system and determined that this VP5 possesses RNA chaperone-like activity, which destabilizes RNA helices and accelerates strand annealing independent of ATP. Our further characterization of VP5 revealed that its helix-destabilizing activity is RNA specific, lacks directionality and could be inhibited by divalent ions, such as Mg(2+), Mn(2+), Ca(2+) or Zn(2+), to varying degrees. Furthermore, we found that HaCPV-5 VP5 facilitates the replication initiation of an alternative polymerase (i.e. reverse transcriptase) through a panhandle-structured RNA template, which mimics the 5′-3′ cyclization of cypoviral positive-stranded RNA. Given that the replication of negative-stranded vRNA on the positive-stranded vRNA template necessitates the dissociation of the 5′-3′ panhandle, the RNA chaperone activity of VP5 may play a direct role in the initiation of reoviral dsRNA synthesis.


id: cord-319681-kjet3e50
author: Lin, Zhaoru
date: 2012-06-28
txt: ./txt/cord-319681-kjet3e50.txt
cache: ./cache/cord-319681-kjet3e50.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/22743270/
doi: 10.1093/nar/gks629
summary:
  The mRNA signal for À1 FS is composed of two elements, a slippery sequence with consensus X_XXY_ YYZ (underlines denote zero frame; X can be any base, Y is A or U, Z is not G in eukaryotic systems) where the ribosome changes frame, and a downstream stimulatory RNA structure, a stem-loop or pseudoknot (reviewed in 3, 4) . A version of the À1 FS reporter plasmid with the IBV slippery sequence (p2lucIBV-AON) was also Spacer-length dependence of programmed À1 or À2 ribosomal frameshifting on a U 6 A heptamer supports a role for mRNA tension in frameshifting Based on the published literature, including our own studies of 80S ribosomes stalled at the IBV frameshift-stimulatory pseudoknot (22, 37) , we proposed a mechanical model of frameshifting in which a failure of intrinsic ribosomal helicase activity (15, 28) to unwind efficiently the stimulatory RNA during the translocation step leads to the build up of tension in the mRNA and subsequently, breakage of codon:anticodon contacts and realignment of the tRNAs in the À1 reading frame.

title:
  Spacer-length dependence of programmed −1 or −2 ribosomal frameshifting on a U(6)A heptamer supports a role for messenger RNA (mRNA) tension in frameshifting

abstract:
  Programmed −1 ribosomal frameshifting is employed in the expression of a number of viral and cellular genes. In this process, the ribosome slips backwards by a single nucleotide and continues translation of an overlapping reading frame, generating a fusion protein. Frameshifting signals comprise a heptanucleotide slippery sequence, where the ribosome changes frame, and a stimulatory RNA structure, a stem–loop or RNA pseudoknot. Antisense oligonucleotides annealed appropriately 3′ of a slippery sequence have also shown activity in frameshifting, at least in vitro. Here we examined frameshifting at the U(6)A slippery sequence of the HIV gag/pol signal and found high levels of both −1 and −2 frameshifting with stem–loop, pseudoknot or antisense oligonucleotide stimulators. By examining −1 and −2 frameshifting outcomes on mRNAs with varying slippery sequence-stimulatory RNA spacing distances, we found that −2 frameshifting was optimal at a spacer length 1–2 nucleotides shorter than that optimal for −1 frameshifting with all stimulatory RNAs tested. We propose that the shorter spacer increases the tension on the mRNA such that when the tRNA detaches, it more readily enters the −2 frame on the U(6)A heptamer. We propose that mRNA tension is central to frameshifting, whether promoted by stem–loop, pseudoknot or antisense oligonucleotide stimulator.


id: cord-320325-sjab8zsk
author: Mendez, Aaron S
date: 2018-12-14
txt: ./txt/cord-320325-sjab8zsk.txt
cache: ./cache/cord-320325-sjab8zsk.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1093/nar/gky932
doi: 10.1093/nar/gky932
summary:
  Using purified KSHV SOX protein, we reconstituted the cleavage reaction in vitro and reveal that SOX displays robust, sequence-specific RNA binding to residues proximal to the cleavage site, which must be presented in a particular structural context. Using an RNA substrate that is efficiently cleaved by SOX in cells, we revealed that specific RNA sequences within and outside of the cleavage site significantly contribute to SOX binding efficiency and target processing. Given that both substrates contain the requisite unpaired bulge at the predicted cleavage site (see Figure 2A and Supplementary Figure S2 ), these observations suggest that additional sequence or structural features impact SOX targeting efficiency on individual RNAs. Two SOX point mutants, P176S and F179A, located in an unstructured region of the protein that bridges domains I and II have been shown to be selectively required for its endonucleolytic processing of RNA substrates (Supplementary Figure S3A and S3B) (8, 21) .

title:
  Site specific target binding controls RNA cleavage efficiency by the Kaposi''s sarcoma-associated herpesvirus endonuclease SOX

abstract:
  A number of viruses remodel the cellular gene expression landscape by globally accelerating messenger RNA (mRNA) degradation. Unlike the mammalian basal mRNA decay enzymes, which largely target mRNA from the 5′ and 3′ end, viruses instead use endonucleases that cleave their targets internally. This is hypothesized to more rapidly inactivate mRNA while maintaining selective power, potentially though the use of a targeting motif(s). Yet, how mRNA endonuclease specificity is achieved in mammalian cells remains largely unresolved. Here, we reveal key features underlying the biochemical mechanism of target recognition and cleavage by the SOX endonuclease encoded by Kaposi's sarcoma-associated herpesvirus (KSHV). Using purified KSHV SOX protein, we reconstituted the cleavage reaction in vitro and reveal that SOX displays robust, sequence-specific RNA binding to residues proximal to the cleavage site, which must be presented in a particular structural context. The strength of SOX binding dictates cleavage efficiency, providing an explanation for the breadth of mRNA susceptibility observed in cells. Importantly, we establish that cleavage site specificity does not require additional cellular cofactors, as had been previously proposed. Thus, viral endonucleases may use a combination of RNA sequence and structure to capture a broad set of mRNA targets while still preserving selectivity.


id: cord-320627-7vi6skvh
title: A molecular beacon, bead-based assay for the detection of nucleic acids by flow cytometry
author: Horejsh, Douglas
date: 2005-01-19
txt: ./txt/cord-320627-7vi6skvh.txt
cache: ./cache/cord-320627-7vi6skvh.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/15659574/
doi: 10.1093/nar/gni015
summary:
  We have developed a fluid array system using microsphere-conjugated molecular beacons and the flow cytometer for the specific, multiplexed detection of unlabelled nucleic acids in solution. Using beads of different sizes and molecular beacons in two fluorophore colours, synthetic nucleic acid control sequences were specifically detected for three respiratory pathogens, including the SARS coronavirus in proof-of-concept experiments. In this report, we describe the construction of molecular beacon-conjugated beads that we have called ''BeadCons'', whose specific hybridization with complementary target sequences can be resolved by flow cytometry (see Figure 1 ). In the multiplex detection experiment, the test sample contained 0.5 ml of the positive oligo DNA (100 mM stock) diluted in 9.5 ml of a complex mixture of oligonucleotides (equimolar levels of 10 mM each, equalling a 100 mM total concentration; sequences listed in Supplementary Table 1 ).

abstract:
  Molecular beacons are dual-labelled probes that are typically used in real-time PCR assays, but have also been conjugated with solid matrices for use in microarrays or biosensors. We have developed a fluid array system using microsphere-conjugated molecular beacons and the flow cytometer for the specific, multiplexed detection of unlabelled nucleic acids in solution. For this array system, molecular beacons were conjugated with microspheres using a biotin-streptavidin linkage. A bridged conjugation method using streptavidin increased the signal-to-noise ratio, allowing for further discrimination of target quantitation. Using beads of different sizes and molecular beacons in two fluorophore colours, synthetic nucleic acid control sequences were specifically detected for three respiratory pathogens, including the SARS coronavirus in proof-of-concept experiments. Considering that routine flow cytometers are able to detect up to four fluorescent channels, this novel assay may allow for the specific multiplex detection of a nucleic acid panel in a single tube.


id: cord-321352-174q2pjw
title: PCAF interacts with XBP-1S and mediates XBP-1S-dependent transcription
author: Lew, Qiao Jing
date: 2010-09-04
txt: ./txt/cord-321352-174q2pjw.txt
cache: ./cache/cord-321352-174q2pjw.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/20817929/
doi: 10.1093/nar/gkq785
summary:
  Further induction (more than 2-fold) of the XBP-1S-mediated activation of HTLV-1 and BiP promoters was detected in the PCAF-expressing cells ( Figure 3A and B) . Co-transfection of the PCAF shRNA in the XBP-1S-expressing cells led to 35, 74 and 52% inhibition of BiP, CHOP, and EDEM transcription, respectively ( Figure 6B ), demonstrating the involvement of PCAF in the XBP-1S-dependent transcription. In the XBP-1S/PCAF co-transfected cells, more XBP-1S proteins were found to bind to the promoter region of BiP and CHOP genes ( Figure 7A and B). This observation could explain why CREB1 and other CREB/ATF family proteins fail to up-regulate HTLV-1 transcription in the absence of Tax. In contrast, the requirement for PCAF in the XBP-1S-dependent HTLV-1 basal transcription was clearly demonstrated in the cell-based reporter assays ( Figures 3A and 4A) .

abstract:
  X-box binding protein 1 (XBP-1) is a key regulator required for cellular unfolded protein response (UPR) and plasma cell differentiation. In addition, involvement of XBP-1 in host cell–virus interaction and transcriptional regulation of viruses, such as human T-lymphotropic virus type 1 (HTLV-1), has been revealed recently. Two XBP-1 isoforms, XBP-1U and XBP-1S, which share an identical N-terminal domain, are present in cells. XBP-1S is a transcription activator while XBP-1U is the inactive isoform. Although the transactivation domain of XBP-1S has been identified within the XBP-1S-specific C-terminus, molecular mechanism of the transcriptional activation by XBP-1S still remains unknown. Here we report the interaction between p300/CBP-associated factor (PCAF) and XBP-1S through the C-terminal domain of XBP-1S. No binding between XBP-1U and PCAF is detected. In a cell-based reporter assay, overexpression of PCAF further stimulates the XBP-1S-mediated cellular and HTLV-1 transcription while knockdown of PCAF exhibits the opposite effect. Expression of endogenous XBP-1S cellular target genes, such as BiP and CHOP, is significantly inhibited when PCAF is knocked down. Furthermore, PCAF is recruited to the promoters of XBP-1S target genes in vivo, in a XBP-1S-dependent manner. Collectively, our results demonstrate that PCAF mediates the XBP-1S-dependent transcription through the interaction with XBP-1S.


id: cord-324367-il9mz5na
title: Translational recoding: canonical translation mechanisms reinterpreted
author: Rodnina, Marina V
date: 2020-02-20
txt: ./txt/cord-324367-il9mz5na.txt
cache: ./cache/cord-324367-il9mz5na.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/31511883/
doi: 10.1093/nar/gkz783
summary:
  This review summarizes the recent advances in understanding the mechanisms of three types of recoding events: stop-codon readthrough, –1 ribosome frameshifting and translational bypassing. In this review, we will focus on three types of recod-ing: (i) stop-codon readthrough; (ii) ribosome frameshifting and (iii) translational bypassing ( Figure 1 ). The key element for recruitment of the SelB-GTP-Sec-tRNA Sec to the stop codon on bacterial ribosomes is a selenocysteine insertion sequence (SECIS) in the mRNA, a SL structure located immediately downstream of the in-frame UGA codon at which Sec is incorporated (67) . A recent crystal structure of a translocation intermediate formed in the absence of EF-G indeed shows that the interactions of the ribosome with the codon-anticodon complex are disrupted and the A-site tRNA in the complex is shifted by one nucleotide toward the -1-frame of the mRNA (78) (Figure 4) .

abstract:
  During canonical translation, the ribosome moves along an mRNA from the start to the stop codon in exact steps of one codon at a time. The collinearity of the mRNA and the protein sequence is essential for the quality of the cellular proteome. Spontaneous errors in decoding or translocation are rare and result in a deficient protein. However, dedicated recoding signals in the mRNA can reprogram the ribosome to read the message in alternative ways. This review summarizes the recent advances in understanding the mechanisms of three types of recoding events: stop-codon readthrough, –1 ribosome frameshifting and translational bypassing. Recoding events provide insights into alternative modes of ribosome dynamics that are potentially applicable to other non-canonical modes of prokaryotic and eukaryotic translation.


id: cord-325985-xfzhn1n1
title: Comprehensive viral oligonucleotide probe design using conserved protein regions
author: Jabado, Omar J.
date: 2007-12-13
txt: ./txt/cord-325985-xfzhn1n1.txt
cache: ./cache/cord-325985-xfzhn1n1.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/18079152/
doi: 10.1093/nar/gkm1106
summary:
  The method uses the Protein Families database (Pfam) and motif finding algorithms to identify oligonucleotide probes in conserved amino acid regions and untranslated sequences. Our method for probe design employs protein alignment information, discovered protein motifs, nucleic acid motifs and finally, sliding windows to ensure near complete coverage of the database. The EMBL nucleotide sequence database [July 2007, Release 91; 461,353 nucleic acid sequences (31) ] was chosen as the reference for this study because it is tightly integrated with the Pfam protein family database (23, 32 Taxon growth was estimated using a standard least squares method, with the SPSS statistical package. We have described a method that capitalizes on the Pfam protein alignment database and a motif finding algorithm to automate the extraction of nucleic acid sequence for probes from conserved protein regions.

abstract:
  Oligonucleotide microarrays have been applied to microbial surveillance and discovery where highly multiplexed assays are required to address a wide range of genetic targets. Although printing density continues to increase, the design of comprehensive microbial probe sets remains a daunting challenge, particularly in virology where rapid sequence evolution and database expansion confound static solutions. Here, we present a strategy for probe design based on protein sequences that is responsive to the unique problems posed in virus detection and discovery. The method uses the Protein Families database (Pfam) and motif finding algorithms to identify oligonucleotide probes in conserved amino acid regions and untranslated sequences. In silico testing using an experimentally derived thermodynamic model indicated near complete coverage of the viral sequence database.


id: cord-330067-ujhgb3b0
title: CoVDB: a comprehensive database for comparative analysis of coronavirus genes and genomes
author: Huang, Yi
date: 2007-10-02
txt: ./txt/cord-330067-ujhgb3b0.txt
cache: ./cache/cord-330067-ujhgb3b0.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/17913743/
doi: 10.1093/nar/gkm754
summary:
  To overcome the problems we encountered in the existing databases during comparative sequence analysis, we built a comprehensive database, CoVDB (http://covdb.microbiology.hku.hk), of annotated coronavirus genes and genomes. CoVDB provides a convenient platform for rapid and accurate batch sequence retrieval, the cornerstone and bottleneck for comparative gene or genome analysis. In CoVDB, with the aim of facilitating gene retrieval, we tried to unify the naming of these non-structural proteins from different groups of coronaviruses. When we compared their putative amino acid sequences to the corresponding ones in other group 1 coronavirus genomes using BLAST, as well as searching for conserved domains using motifscan, results showed that the putative proteins encoded by these ORFs belonged to a protein family in Pfam originally assigned as ''Corona_NS3b'' (accession number PF03053). database, CoVDB, of annotated coronavirus genes and genomes, which offers efficient batch sequence retrieval and analysis.

abstract:
  The recent SARS epidemic has boosted interest in the discovery of novel human and animal coronaviruses. By July 2007, more than 3000 coronavirus sequence records, including 264 complete genomes, are available in GenBank. The number of coronavirus species with complete genomes available has increased from 9 in 2003 to 25 in 2007, of which six, including coronavirus HKU1, bat SARS coronavirus, group 1 bat coronavirus HKU2, groups 2c and 2d coronaviruses, were sequenced by our laboratory. To overcome the problems we encountered in the existing databases during comparative sequence analysis, we built a comprehensive database, CoVDB (http://covdb.microbiology.hku.hk), of annotated coronavirus genes and genomes. CoVDB provides a convenient platform for rapid and accurate batch sequence retrieval, the cornerstone and bottleneck for comparative gene or genome analysis. Sequences can be directly downloaded from the website in FASTA format. CoVDB also provides detailed annotation of all coronavirus sequences using a standardized nomenclature system, and overcomes the problems of duplicated and identical sequences in other databases. For complete genomes, a single representative sequence for each species is available for comparative analysis such as phylogenetic studies. With the annotated sequences in CoVDB, more specific blast search results can be generated for efficient downstream analysis.


id: cord-333502-3ulketgy
title: PATRIC: The VBI PathoSystems Resource Integration Center
author: Snyder, E. E.
date: 2006-11-16
txt: ./txt/cord-333502-3ulketgy.txt
cache: ./cache/cord-333502-3ulketgy.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/17142235/
doi: 10.1093/nar/gkl858
summary:
  The PathoSystems Resource Integration Center (PATRIC) is one of eight Bioinformatics Resource Centers (BRCs) funded by the National Institute of Allergy and Infection Diseases (NIAID) to create a data and analysis resource for selected NIAID priority pathogens, specifically proteobacteria of the genera Brucella, Rickettsia and Coxiella, and corona-, caliciand lyssaviruses and viruses associated with hepatitis A and E. (i) collection and organization of existing genomic data for the eight pathosystems under a single, unified framework (ii) genome annotation and curation following standardized procedures (iii) visualization of raw data from analytical programs, as well as curated data (iv) creation of orthologous gene groups within each organism category allowing comparative analysis of gene content (v) prediction and visualization of bacterial metabolic pathways to complement functional analysis of proteins (vi) integration of online literature reviews from PathInfo (14) for selected organisms.

abstract:
  The PathoSystems Resource Integration Center (PATRIC) is one of eight Bioinformatics Resource Centers (BRCs) funded by the National Institute of Allergy and Infection Diseases (NIAID) to create a data and analysis resource for selected NIAID priority pathogens, specifically proteobacteria of the genera Brucella, Rickettsia and Coxiella, and corona-, calici- and lyssaviruses and viruses associated with hepatitis A and E. The goal of the project is to provide a comprehensive bioinformatics resource for these pathogens, including consistently annotated genome, proteome and metabolic pathway data to facilitate research into counter-measures, including drugs, vaccines and diagnostics. The project's curation strategy has three prongs: ‘breadth first’ beginning with whole-genome and proteome curation using standardized protocols, a ‘targeted’ approach addressing the specific needs of researchers and an integrative strategy to leverage high-throughput experimental data (e.g. microarrays, proteomics) and literature. The PATRIC infrastructure consists of a relational database, analytical pipelines and a website which supports browsing, querying, data visualization and the ability to download raw and curated data in standard formats. At present, the site warehouses complete sequences for 17 bacterial and 332 viral genomes. The PATRIC website () will continually grow with the addition of data, analysis and functionality over the course of the project.


id: cord-334127-wjf8t8vp
title: NCBI Viral Genomes Resource
author: Brister, J. Rodney
date: 2015-01-28
txt: ./txt/cord-334127-wjf8t8vp.txt
cache: ./cache/cord-334127-wjf8t8vp.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/25428358/
doi: 10.1093/nar/gku1207
summary:
  This, in turn, has placed increased emphasis on leveraging the knowledge of individual scientific communities to identify important viral sequences and develop well annotated reference virus genome sets. Whereas primary databases are archival repositories of sequence data, reference databases provide curated datasets that enable a number of activities, among them are transfer annotation to related genomes (11) (12) (13) , sequence assembly and virus discovery (14) (15) (16) (17) , viral dynamics and evolution (18) (19) (20) and pathogen detection (14, (21) (22) (23) . The second model captures and standardizes host information for all viruses, and whenever a new RefSeq record is created, a manually curated ''viral host'' property is assigned to the relevant species within the NCBI Taxonomy database. The link to the Retrovirus Resource (http://www.ncbi.nlm.nih.gov/genome/viruses/retroviruses) provides access to the Retrovirus Genotyping Tool and HIV-1, Human Interaction Database (50, 51) .

abstract:
  Recent technological innovations have ignited an explosion in virus genome sequencing that promises to fundamentally alter our understanding of viral biology and profoundly impact public health policy. Yet, any potential benefits from the billowing cloud of next generation sequence data hinge upon well implemented reference resources that facilitate the identification of sequences, aid in the assembly of sequence reads and provide reference annotation sources. The NCBI Viral Genomes Resource is a reference resource designed to bring order to this sequence shockwave and improve usability of viral sequence data. The resource can be accessed at http://www.ncbi.nlm.nih.gov/genome/viruses/ and catalogs all publicly available virus genome sequences and curates reference genome sequences. As the number of genome sequences has grown, so too have the difficulties in annotating and maintaining reference sequences. The rapid expansion of the viral sequence universe has forced a recalibration of the data model to better provide extant sequence representation and enhanced reference sequence products to serve the needs of the various viral communities. This, in turn, has placed increased emphasis on leveraging the knowledge of individual scientific communities to identify important viral sequences and develop well annotated reference virus genome sets.


id: cord-335377-zrbn637z
title: RNA dimerization plays a role in ribosomal frameshifting of the SARS coronavirus
author: Ishimaru, Daniella
date: 2012-12-26
txt: ./txt/cord-335377-zrbn637z.txt
cache: ./cache/cord-335377-zrbn637z.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/23275571/
doi: 10.1093/nar/gks1361
summary:
  Furthermore, the inability to dimerize caused by the silent codon change in Stem 3 of SARS-CoV changed the viral growth kinetics and affected the levels of genomic and subgenomic RNA in infected cells. We further show that kissing dimer formation plays a role in frameshift-stimulation and modulates the relative abundance of full-length and subgenomic viral RNAs. Plasmids containing wild-type pseudoknot as well as the ÁS3 pk mutant were described in Plant et al (1) . Our previous NMR analysis of exchangeable imino protons of the SARS-CoV pseudoknot ( Figure 1A , wild-type pk) provided unequivocal evidence for the existence of Stem 3 (1). Surprisingly, in the context of the SARS-CoV Stem 3 sequence, 5 0 -cuug-3 0 tetraloop-capped mutants readily formed extended duplex structures as revealed by native gel and NMR analysis.

abstract:
  Messenger RNA encoded signals that are involved in programmed -1 ribosomal frameshifting (-1 PRF) are typically two-stemmed hairpin (H)-type pseudoknots (pks). We previously described an unusual three-stemmed pseudoknot from the severe acute respiratory syndrome (SARS) coronavirus (CoV) that stimulated -1 PRF. The conserved existence of a third stem–loop suggested an important hitherto unknown function. Here we present new information describing structure and function of the third stem of the SARS pseudoknot. We uncovered RNA dimerization through a palindromic sequence embedded in the SARS-CoV Stem 3. Further in vitro analysis revealed that SARS-CoV RNA dimers assemble through ‘kissing’ loop–loop interactions. We also show that loop–loop kissing complex formation becomes more efficient at physiological temperature and in the presence of magnesium. When the palindromic sequence was mutated, in vitro RNA dimerization was abolished, and frameshifting was reduced from 15 to 5.7%. Furthermore, the inability to dimerize caused by the silent codon change in Stem 3 of SARS-CoV changed the viral growth kinetics and affected the levels of genomic and subgenomic RNA in infected cells. These results suggest that the homodimeric RNA complex formed by the SARS pseudoknot occurs in the cellular environment and that loop–loop kissing interactions involving Stem 3 modulate -1 PRF and play a role in subgenomic and full-length RNA synthesis.


id: cord-337998-08tknscm
author: Sztuba-Solinska, Joanna
date: 2016-11-16
txt: ./txt/cord-337998-08tknscm.txt
cache: ./cache/cord-337998-08tknscm.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/27651462/
doi: 10.1093/nar/gkw825
summary:
  Selective 2′-hydroxyl acylation analyzed by primer extension analysis of the secondary structure of the EBOV minigenomic RNA indicates formation of a small stem-loop composed of the HSPA8 motif, a 3′ stem-loop (nucleotides 1868–1890) that is similar to a previously identified structure in the replicative intermediate (RI) RNA and a panhandle domain involving a trailer-to-leader interaction. 3E-5E-GFP minigenome RNA secondary structure and host protein interactions were examined using selective 2 -hydroxyl acylation analyzed by primer extension (SHAPE) (38, 39) , antisense-interfered SHAPE (aiSHAPE) (40) , electrophoretic mobility shift assays (EMSA), siRNA, and mutational analysis, using both the 3E-5E-GFP minigenome system and EBOV reverse genetics. The secondary structure of the EBOV 3E-5E-GFP minigenome RNA predicted by RNAstructure software version 5.7 (48) and chemical probing data from SHAPE were used to generate 10 three-dimensional (3D) models for the trailer-to-leader panhandle interaction in the wt-EBOV genome and variants, A30U and A26U/A30U, using open-source RNAComposer, version 1.0 (http://rnacomposer.cs.put.poznan.pl/).

title:
  A small stem-loop structure of the Ebola virus trailer is essential for replication and interacts with heat-shock protein A8

abstract:
  Ebola virus (EBOV) is a single-stranded negative-sense RNA virus belonging to the Filoviridae family. The leader and trailer non-coding regions of the EBOV genome likely regulate its transcription, replication, and progeny genome packaging. We investigated the cis-acting RNA signals involved in RNA–RNA and RNA–protein interactions that regulate replication of eGFP-encoding EBOV minigenomic RNA and identified heat shock cognate protein family A (HSC70) member 8 (HSPA8) as an EBOV trailer-interacting host protein. Mutational analysis of the trailer HSPA8 binding motif revealed that this interaction is essential for EBOV minigenome replication. Selective 2′-hydroxyl acylation analyzed by primer extension analysis of the secondary structure of the EBOV minigenomic RNA indicates formation of a small stem-loop composed of the HSPA8 motif, a 3′ stem-loop (nucleotides 1868–1890) that is similar to a previously identified structure in the replicative intermediate (RI) RNA and a panhandle domain involving a trailer-to-leader interaction. Results of minigenome assays and an EBOV reverse genetic system rescue support a role for both the panhandle domain and HSPA8 motif 1 in virus replication.


id: cord-341154-wwq0sd2r
author: Liao, Pei-Yu
date: 2010-09-07
txt: ./txt/cord-341154-wwq0sd2r.txt
cache: ./cache/cord-341154-wwq0sd2r.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/20823091/
doi: 10.1093/nar/gkq761
summary:
  The model reveals three kinetic pathways to −1 PRF that yield two possible frameshift products: those incorporating zero frame encoded A-site tRNAs in the recoding site, and products incorporating −1 frame encoded A-site tRNAs. Using known kinetic rate constants, the individual contributions of different steps of the translation elongation cycle to −1 PRF and the ratio between two types of frameshift products were evaluated. Protein sequencing was originally employed to generate the simultaneous slippage model, and to confirm that the À1 PRF site for HIV-1 is U UUU UUA located within the gag/ pol overlap (where the P-site of the ribosome during frameshifting is underlined) (1). In agreement with the model predictions, experimental perturbation of different translation steps resulted in different levels of À1 PRF efficiency as well as in the relative ratios of two types of frameshift proteins. Our model suggests that in both mechanisms, incomplete translocation and slippage of P-and A-site tRNAs participate in synthesizing frameshift proteins to varying extents for different À1 PRF signals.

title:
  The many paths to frameshifting: kinetic modelling and analysis of the effects of different elongation steps on programmed –1 ribosomal frameshifting

abstract:
  Several important viruses including the human immunodeficiency virus type 1 (HIV-1) and the SARS-associated Coronavirus (SARS-CoV) employ programmed −1 ribosomal frameshifting (PRF) for their protein expression. Here, a kinetic framework is developed to describe −1 PRF. The model reveals three kinetic pathways to −1 PRF that yield two possible frameshift products: those incorporating zero frame encoded A-site tRNAs in the recoding site, and products incorporating −1 frame encoded A-site tRNAs. Using known kinetic rate constants, the individual contributions of different steps of the translation elongation cycle to −1 PRF and the ratio between two types of frameshift products were evaluated. A dual fluorescence reporter was employed in Escherichia coli to empirically test the model. Additionally, the study applied a novel mass spectrometry approach to quantify the ratios of the two frameshift products. A more detailed understanding of the mechanisms underlying −1 PRF may provide insight into developing antiviral therapeutics.


id: cord-348427-worgd0xu
title: Virus Variation Resource – improved response to emergent viral outbreaks
author: Hatcher, Eneida L.
date: 2017-01-04
txt: ./txt/cord-348427-worgd0xu.txt
cache: ./cache/cord-348427-worgd0xu.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1093/nar/gkw1065
doi: 10.1093/nar/gkw1065
summary:
  The resource now includes expanded data processing pipelines and analysis tools, and supports selection and retrieval of nucleotide and protein sequences from four new viral groups: Ebolaviruses, MERS coronavirus, rotavirus, and Zika virus ( Table 2 ). New processes have been added to parse source descriptor terms from Gen-Bank records and map these to controlled vocabulary, and the resource now supports retrieval of sequences based on standardized isolation source and host terms in addition to standardized gene and protein names. The resource includes data processing pipelines that retrieve sequences from GenBank, provide standardized gene and protein an-notation, and map sequence source descriptors (i.e. metadata) to uniform vocabularies. To resolve this issue, the Virus Variation database loading pipeline parses Gen-Bank records, identifies important metadata terms, such as sample isolation host, date, country and source, and maps these to a standardized vocabulary using a hierarchical approach.

abstract:
  The Virus Variation Resource is a value-added viral sequence data resource hosted by the National Center for Biotechnology Information. The resource is located at http://www.ncbi.nlm.nih.gov/genome/viruses/variation/ and includes modules for seven viral groups: influenza virus, Dengue virus, West Nile virus, Ebolavirus, MERS coronavirus, Rotavirus A and Zika virus. Each module is supported by pipelines that scan newly released GenBank records, annotate genes and proteins and parse sample descriptors and then map them to controlled vocabulary. These processes in turn support a purpose-built search interface where users can select sequences based on standardized gene, protein and metadata terms. Once sequences are selected, a suite of tools for downloading data, multi-sequence alignment and tree building supports a variety of user directed activities. This manuscript describes a series of features and functionalities recently added to the Virus Variation Resource.


id: cord-350189-2su7oqbz
title: Locked nucleic acid (LNA) mediated improvements in siRNA stability and functionality
author: Elmén, Joacim
date: 2005-01-14
txt: ./txt/cord-350189-2su7oqbz.txt
cache: ./cache/cord-350189-2su7oqbz.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/15653644/
doi: 10.1093/nar/gki193
summary:
  A priori, this suggests that LNA may be used to increase the functional half-life of siRNA in vivo by two different mechanisms, e.g. by enhancing the resistance of the constituent RNA strands against degradation by single-stranded RNases and by stabilizing the siRNA duplex structure that is critical for activity. Next, we examined the effect of making single RNA to LNA exchanges at base-paired positions in the antisense strand of the firefly luciferase siLNA1. Although we cannot exclude that these modifications somehow prevent loading of the antisense strand into RISC, we believe this to be unlikely given the functionality of many significantly more modified siLNAs. Rather, as these positions are all close to the site where RNA target cleavage occurs [between pos. The SARS siRNA (Table 1) has identical closing base-pairs at both ends (A:U) making it likely that enough of both the antisense and sense strand would be incorporated into RISC to observe activity on the respective targets.

abstract:
  Therapeutic application of the recently discovered small interfering RNA (siRNA) gene silencing phenomenon will be dependent on improvements in molecule bio-stability, specificity and delivery. To address these issues, we have systematically modified siRNA with the synthetic RNA-like high affinity nucleotide analogue, Locked Nucleic Acid (LNA). Here, we show that incorporation of LNA substantially enhances serum half-life of siRNA's, which is a key requirement for therapeutic use. Moreover, we provide evidence that LNA is compatible with the intracellular siRNA machinery and can be used to reduce undesired, sequence-related off-target effects. LNA-modified siRNAs targeting the emerging disease SARS, show improved efficiency over unmodified siRNA on certain RNA motifs. The results from this study emphasize LNA's promise in converting siRNA from a functional genomics technology to a therapeutic platform.


