key: cord-1003651-yf8fvx7k authors: Bekaert, Michaël; Rousset, Jean-Pierre title: An Extended Signal Involved in Eukaryotic −1 Frameshifting Operates through Modification of the E Site tRNA date: 2005-01-07 journal: Mol Cell DOI: 10.1016/j.molcel.2004.12.009 sha: 2e55acf52fad6034e63d74551c6bbf3425ec153b doc_id: 1003651 cord_uid: yf8fvx7k By using a sensitive search program based on hidden Markov models (HMM), we identified 74 viruses carrying frameshift sites among 1500 fully sequenced virus genomes. These viruses are clustered in specific families or genera. Sequence analysis of the frameshift sites identified here, along with previously characterized sites, identified a strong bias toward the two nucleotides 5′ of the shifty heptamer signal. Functional analysis in the yeast Saccharomyces cerevisiae demonstrated that high frameshifting efficiency is correlated with the presence of a Ψ39 modification in the tRNA present in the E site of the ribosome at the time of frameshifting. These results demonstrate that an extended signal is involved in eukaryotic frameshifting and suggest additional interactions between tRNAs and the ribosome during decoding. lated by the modification status of the E site tRNA. Over-gions from the 20 viruses that we had functionally characterized (see Experimental Procedures) and used it to all, our results propose an extended model for Ϫ1 frameshift sites. search the GenBank viral genome database (release 02/ 10/2004). 285 motifs were identified and subsequently manually inspected to eliminate false positives. We Results and Discussion checked (1) that the first nucleotide of the heptamer is in frame with the AUG of the upstream coding region, Characterization of Viral Ϫ1 Frameshift Sites (2) for a protein motif associated to the upstream and The RECODE database resource (http://recode.genetics. downstream coding regions, and (3) for the presence utah.edu, Baranov et al., 2001) describes 35 viruses susof a potential secondary structure downstream of the pected or demonstrated to carry a frameshift site. Few heptamer. By this procedure, we identified 74 frameshift Ϫ1 frameshift signals are fully documented. The gesites in viral genomes. Most false positives exhibited no nomes of these 35 viruses are entirely sequenced, and secondary structure after the shifty site and were found only five sites are precisely characterized, including the in the large and highly complex herpesvirus, papillostructure of the stimulatory pseudoknot (BWYV, HIV-1, mavirus, and nucleopolyhedrovirus genomes. We con-MMTV, PEMV-1, and SRV-1). For the remaining sites, 13 sider this assessment to be accurate, because it dehave been analyzed by extensive directed mutagenesis pends not only on in silico methods but also on the coupled with quantification of frameshifting efficiency, biological assay of the HMM learning set. It is noteworbut the others are only partially characterized. Most of thy that this method is very efficient, even though we the sites are therefore putative; i.e., they carry the typical did not take into account the stimulatory secondary heptamer and secondary structure but have never been structures when we defined the profile. RNA folding alproven to be functional. gorithms are time consuming and cannot be restrained Initially, we functionally characterized a larger number to a defined window in the vicinity of the heptamer. of viruses containing a putative frameshifting site. To Moreover, the theoretical evaluation of thermodynamic explore the widest viral diversity possible (order, family, stability of secondary structures is not accurate for and genus), we deduced a neighbor-joining tree from pseudoknots (Walter et al., 1994) . the 35 viruses, based on the multiple alignment of the With the HMM profile based on only 20 sites, we were polymerase protein sequence (Figure 1 ). From this tree, able to find all known frameshifting viruses and 39 that we selected a subset of 20 viruses representative of the are new or uncharacterized ( Table 2 ). The list of the global viral diversity. To assay the frameshift compeviruses with the position of their frameshift signals is in tence of each putative site, we cloned the entire viral Supplemental Table S1 available online at http://www. Ϫ1 frameshift region of the 20 representative viruses in a molecule.org/cgi/content/full/17/1/61/DC1/. Ten putadual-reporter vector and estimated in vivo frameshifting tive frameshift sites were never previously annotated efficiency in yeast (see Experimental Procedures). and are associated to an upstream and a downstream Frameshift sites from different eukaryotic species have been shown to function in yeast (Bekaert et al., 2003; coding region. 12 frameshifting structures were already Stahl et al., 1995) . The existence of a functional annotated as such in the RECODE database, and eight frameshift signal was demonstrated for all candidates were only annotated in the sequence field of GenBank (Table 1) or in relation to a publication that did not mention any It is unlikely that the low level of expression of ScYLV evidence of frameshift. For the remaining 44 sequences, is due to the use of a heterologous host cell, because ten a site was suspected, but it was not precisely localized frameshift sites from other plant viruses are functional in between two coding regions. For those, we were able our assay. This site might be nonfunctional or carry to propose a precise position for the frameshift event polymorphic variations. The Ϫ1 frameshifting frequenand in some cases, a more accurate annotation. For cies varied between 8% and 31%, compatible with those example, for the Ovine astrovirus (ssRNA ϩ , Astroviridae previously obtained with in vitro or in vivo assays (e.g., family), a putative Ϫ1 Luteoviridae, Retroviridae, Tombusviridae, and Totiviri-We then aligned the newly characterized sites with dae. Within each family, only a few subfamilies/genera sites already identified. Strikingly, we observed an imwere capable of Ϫ1 frameshifting (see Supplemental portant bias not only at the slippery heptamer but also Table S1 for details). However, in this latter case, all in the spacer region and just upstream of the heptamer. members of the genus submitted to HMM analyses ap-The upstream bias was never before observed, and its pear capable of Ϫ1 frameshifting: they carry not only detailed analysis is presented below. the HMM profile but also secondary structures as a canonical frameshift signal (Supplemental Table S1 ). For example, manual checking of the Poleroviruses found by Sensitive Search of Viral Frameshift Sites We established a HMM profile of efficient viral Ϫ1 using HMM successfully identifies a pseudoknot three to nine nucleotides downstream from the heptamer site. frameshift signals with the alignment of the slippery re- an order 1 HMM search where the probability of a given nucleotide is dependent on the identity of the previous pression concluded that translation of the second ORF is initiated on its own internal AUG codon (Huang and nucleotide. Accordingly, Figure 2 shows the bias of dinucleotide distribution. The 2 score for the last dinucleo-Ghabrial, 1996). However, this does not exclude the possibility that both mechanisms are at play to express tide position before the heptamer is 80 with 15 degrees (Table 3 ): all con-16 possible sequences within the context of the wildtype (wt) frameshift signal of the Avian infectious bron-structs that exhibited a high-frameshifting level use a cognate (or near-cognate) tRNA carrying the ⌿39 modi-chitis virus (IBV), because it has been extensively used as a model virus for Ϫ1 frameshifting studies (Brierley fication. Conversely, the sequences that do not involve a codon decoded by a tRNA with the ⌿39 modification et al., 1991, 1992). Table 3 shows that a 3.3-fold variation was found between the frameshifting efficiencies di-direct low-frameshifting efficiency. This observation prompted us to investigate the effect of the mutation of rected by these 16 IBV variant sites. Compared to the wt sequence, the frameshifting level is significantly reduced (p value Ͻ 10 Ϫ4 ) in ten of the mutants. The dinucleotide situated 5Ј of the heptamer corresponds to the first two nucleotides of the preceding codon; its impact can thus be interpreted as an effect either of the amino acid, the codon, or the decoding tRNA. Because it was previously shown that tRNA modi- Two low-and two high-frameshift rate constructs which the UUUAAAC heptamer was mutated to UUU AUAC. In this case, tandem slippage should be ineffi-were tested in modification mutants (Table 4 ). With the low-frameshifting rate subset (frameshift efficiency cient due to the presence of two mismatches after repairing of the A site tRNA in the Ϫ1 frame, but single lower than 10%), pus3⌬ mutants show no significant effect (Table 4 ). In contrast, with the high-frameshifting slippage would not be affected. The frameshifting efficiency obtained with this construct was Ͻ0.1%, similar rate subset (frameshift efficiency higher than 18%), which involves decoding by a ⌿39 modified tRNA, a to the background level. This result demonstrates that in these experiments, frameshifting actually occurred reduced frameshifting frequency was observed in pus3⌬ mutants. This frequency was similar to that directed by through a tandem tRNA slippage mechanism. This implies that the three sites are involved in ribosomal frame-the low-frameshifting rate subset, indicating that most of the effect was reversed in the mutant. We verified shifting (see below). The ⌿39 modification is conserved over the tree of that the effect is actually due to the modifying activity of Pus3p and not to a possible chaperone-like activity life; its role on Ϫ1 frameshifting could thus be similar in a broad spectrum of organisms. This is consistent with by using the pus3[D151A] mutant, which harbors a mutation in the active site of the PUS3 protein. In this mutant, the fact that the bias at the two positions upstream of the heptamer was deduced from a wide variety of viruses the high-frameshifting constructs yield lower frameshifting efficiency, as in a pus3⌬ mutant context (Table 4) . of different origins. However, each host cell, like the yeast strains used here, carries a specific tRNA pool The effect of the dinucleotide upstream of the heptamer suggests that the three ribosomal site tRNAs are that differs from one organism to another. This could explain the different dinucleotide usage observed be-involved in the mechanism of Ϫ1 frameshifting. However, although the mechanism of frameshifting in eu-tween viruses; however, not enough sequence data are available to assess this point. In any case, the existence karyotes is thought to involve mostly tandem slippage of the tRNAs occupying the A and P sites, single slippage of a bias indicates an important role of tRNA modification on Ϫ1 frameshifting in eukaryotes. A role of tRNA at the P site has been reported to occur (Jacks et al., Brierley et al., 1997; Licznar et al., 2003) , during the accommodation step of the A-tRNA and not during the preceding decoding reaction (Nierhaus, 1990 ; but not in eukaryotes. In these cases, the tRNAs involved were acting at the A or P site. Noller et al., 2002) . However, in the case of a Ϫ1 frameshift event, E-tRNA release at the decoding step Overall, these results demonstrate that the effect of the upstream context of the heptamer is directed by the would facilitate the slippery event of A and P site tRNAs, and this precisely might be the effect of ⌿39. Biochemi-modification status of the tRNA decoding the Ϫ1 codon. cal experiments will be required to clarify this point. with several partners. The closest distance between the sequences give rise to patterns inconsistent with acanticodon stem backbones of the P-and E-tRNAs is cepted trees (data not shown). Inconsistency of frameabout 6 Å , which is closer than the distance separating shifting patterns with accepted phylogenetic trees is not the A-and P-tRNAs. The two tRNAs are not in direct surprising taking into account the recombinant nature of contact but are linked by the 16S rRNA helices H24, many viruses; functional requirements probably account H28, and H29, and loops 690 and 790, both of which for both this complete conservation and the variability they directly interact with through their anticodon loops of the frameshifting site sequences. Indeed, in the Retro- (Yusupov et al., 2001) . Another link between E and P viridae family, the Alpharetrovirus genus is exceptional sites is through the mRNA. A single possible contact because some members exhibit frameshift signals but was noted between the mRNA and E-tRNA in the crystal others do not. In fact, this genus is subdivided in two structure, but the latter was noncognate. Even this noncategories: replication-competent viruses, which poscognate E site anticodon was close enough to the cosess the pol gene, and defective viruses, which do not. don, such that cognate interaction would be structurally Logically, frameshift signals are found only in the latter plausible; moreover, there is biochemical evidence for category. It is even more interesting that despite their codon-anticodon specificity in the E site (Lill and Winposition among the Totiviridae, the Leishmaniavirus getermeyer, 1987; Rheinberger et al., 1986). E site tRNA nus members do not carry Ϫ1 frameshift sites but, is thus sufficiently connected to the P site to suggest rather, use ϩ1 frameshifting to express their polymerase that it very likely plays a role in promoting the stability domain. This suggests that strong biological constraints of P site codon-anticodon pairing. ⌿39 modification can are at play in the selection of a recoding event in the be expected to improperly fill the E site during the sliplife cycle of these viruses, possibly related to the incorpage-prone state, probably resulting in an unstable P poration of the polymerase as a fusion protein in the site codon-anticon interaction and enhanced Ϫ1 frameviral particle. shifting. This is reminiscent of the role played by a partic- ular context of a bacterial tmRNA resume codon. In this An interesting feature of the results presented here is case, an unusual E site conformation destabilizes the P the involvement of an extended nonanucleotide signal site codon-anticodon interaction and induces framein ribosomal frameshifting. As demonstrated above, no shifting (Trimble et al., 2004) . single slippage is observed in the experimental system The results presented here demonstrate that the slipused here; this nonanucleotide-directed frameshifting pery component of Ϫ1 frameshift signals, at least in thus involves classical tandem slippage where both A yeast, is more complex than previously anticipated. and P site tRNAs slip by one nucleotide upstream. This Compared to the initial model of Jacks et al. (1988) , implies that the three ribosomal sites are involved in sequence elements of both the 3Ј and 5Ј heptamer ele-Ϫ1 frameshifting. Two hypotheses can be proposed to ments are now shown to participate in frameshiting effiaccount for the role of the E site tRNA in frameshifting. ciency through interactions between tRNA, mRNA, and Firstly, frameshifting might be enhanced by the absence the ribosome. Similarly, downstream secondary strucof a tRNA in the E site. In this case, the ⌿39 modification tures can directly or indirectly influence frameshifting. would destabilize the tRNA:E site interaction. Secondly, A combinatorial use of upstream codons, heptamer se-⌿39 might interfere directly or indirectly with the interacquences, downstream codons, and stimulatory secondtion of the P site tRNA with the mRNA, decreasing pairary structures permit a given frameshifting efficiency for ing stability. a given virus in a given host. Whether or not these differ-The first hypothesis is supported by recent results in ent sequence elements act independently remains to which premature release of the E site tRNA from the be established. ribosome has been shown to be coupled with highlevel ϩ1 frameshifting at the prfB gene, encoding the 1.83 (Thompson et al., 1994) alignment of viral polymerase amino acid sequences retrieved from GenBank was used. It was predict that the ⌿39 modification induces a higher fre-employed to deduce a neighbor-joining tree with 1000 bootstrap ratory and the "frameshift team" for numerous stimulating discussions. We are especially grateful to Anne-Lise Haenni for critically replications (Saitou and Nei, 1987) his3⌬200, leu2-3, 112, and ura3-52) , and its derivative pus3⌬ (Mat a, of the "slippery-sequence" component of a coronavirus ribosomal ade1-14, trp1-289, his3⌬200, leu2-3, 112, ura3-52, and pus3⌬::KAN) . virus type 1 RNA dimerization and viral infectivity Identification and characterisation of Signals for ribosomal frameshifting in the Rous sarcoma virus gagpol region Structure of the 80S ribosome from 715-718. Saccharomyces cerevisiae-tRNA-ribosome and subunit-subunit in-Kim viral RNA pseudoknot drastically change ribosomal frameshifting efficiency. Proc. Natl. Acad. Sci. 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