key: cord-010120-mqvm9zn4 authors: Dinman, Jonathan D. title: Ribosomal frameshifting in yeast viruses date: 2004-01-29 journal: Yeast DOI: 10.1002/yea.320111202 sha: doc_id: 10120 cord_uid: mqvm9zn4 Proper maintenance of translational reading frame by ribosomes is essential for cell growth and viability. In the last 10 years it has been shown that a number of viruses induce ribosomes to shift reading frame in order to regulate the expression of gene products having enzymatic functions. Studies on ribosomal frameshifting in viruses of yeast have been particularly enlightening. The roles of viral mRNA sequences and secondary structures have been elucidated and a picture of how these interact with host chromosomal gene products is beginning to emerge. The efficiency of ribosomal frameshifting is important for viral particle assembly, and has identified ribosomal frameshifting as a potential target for antiviral agents. The availability of mutants of host chromosomal gene products involved in maintaining the efficiency of ribosomal frameshifting bodes well for the use of yeast in future studies of ribosomal frameshifting. How not frameshifting can be instructive: Ty5 and Tfl 1125 Summary 1125 Maintenance of correct reading frame is fundamental to the integrity of the translation process and, ultimately, to cell growth and viability. However, a number of cases of directed frameshifting have been identified. Frameshifting events produce fusion proteins, in which the Nand C-terminal domains are encoded by two distinct, overlapping open reading frames (ORFs). These are, for the most part, seen in viruses, e.g. retroviruses, coronaviruses, the yeast L-A dsRNA virus, the dsRNA virus of Giardia lamblia, the Ty family of viruses in yeast, (+) ssRNA viruses of plants, bacteriophage T7, and a number of bacterial transposons, as well as in a few bacterial cellular genes and the ornithine decarboxylase antizyme gene in mammals (for reviews, see references 1, 13, 29, 30, 35) . Ribosomal frameshifting is different from frameshift suppression in that these events are directed by specific mRNA sequences and structures, rather than being a consequence of mutations in host gene products, e.g. tRNAs containing four base anticodon loops. The study of these ribosomal frameshifts is important both because of their critical role in animal and plant pathogens, and because of the information they provide about the mechanisms by which the reading frame is normally maintained. Most of the cases of ribosomal frameshifting seen to date are found in viruses which use their (+) strands as (1) mRNAs encoding multiple protein products, (2) the species of RNA that is packaged into nascent viral particles and (3) the template for replication of the viral genetic material. Production of multiple protein products could be achieved by mRNA splicing or editing. These mechanisms could lead to the production of altered (+) strands, resulting in the production of mutant viral genomes, unless splicing or editing removed an RNA site required for packaging or replication of the genomic RNA. Perhaps for this reason, (+) ssRNA and dsRNA viruses are not known to use splicing or mRNA editing and retroviruses remove the ackaging site (Y) when of viruses use ribosomal frameshifting and/or readthrough of termination codons to make fusion proteins. Neither of these mechanisms alters the template and so neither packages mutant viral genomes. " Ribosomal frameshifting in the -1, or 5' direction in retroviruses, (+) ssRNA viruses and dsRNA viruses results in the production of Gagpol fusion proteins. It requires a special sequence, X XXY YYZ (the 0-frame is indicated by spaces) called the 'slippery site'.'4 The simultaneous slippage of ribosome-bound A-and P-site tRNAs by one base in the 5' direction still leaves their nonwobble bases correctly paired in the new reading frame. A second promoting element,34 usually an mRNA pseudoknot, is located immediately 3' to the slippery It is thought that the role of the mRNA pseudoknot is to induce elongating ribosomes to pause over the slippery site. Both of these elements have been found to be required for the promotion of efficient -1 ribosomal frameshifting in the L-A virus of yeast. In eukaryotes, frameshifting in the +1, or 3' direction has been observed in the Ty retrotransposable elements in yeast (for review, see reference 25), and in the ornithine decarboxylase antizyme gene in mammalian ~e l l s .~~,~~ In Tyl and Ty3, +1 they splice their RNA.4 I: s6 All of these classes ribosomal frameshifting between the T Y A and T Y B genes in Tyl and the GAG3 and POL3 genes in Ty3 also results in the production of Gag-Pol fusion proteins. Although heptameric sequences and the induction of a ribosomal pause are required to promote efficient frameshifting, the actual mechanisms involved are very different from those used in -1 ribosomal frameshifting. Here, 1 will present a review of ribosomal frameshifting research in yeast. The elements involved in promoting -1 and + I ribosomal frameshifting will be discussed. The importance of the efficiency of ribosomal frameshifting for the production of the correct ratios of viral proteins, and the consequences for viral propagation when these ratios are perturbed, will be considered. Research in yeast focused on the generation and genetic characterization of yeast strains in which mutations in unique chromosomal genes result in cells having ribosomal frameshifting efficiencies significantly greater than normal cells will bc examined, and a prospectus considering the implications of these classes of mutants upon the fields of translational control and virology will be presented. The 5' g c r g gene encodes the major coat protein and the 3' pol gene encodes a multifunctional protein domain which includes the RNA-dependent RNA polymerase and a domain required for viral RNA packaging,27.32.'3.4X A -1 ribosomal frameshift event is responsible for the production of the Gag-Pol fusion protein. 'x.26.33 M , , a satellite dsRNA virus of L-A which encodes a secreted killer toxin (reviewed in reference 12), is encapsidated and is replicated in L-A encoded particles. According to the 'simultaneous slippage' (Figure 2 ), -1 ribosomal frameshifting occurs at a heptameric 'slippery site', X XXY YYZ (0-frame indicated by spaces). This can occur when ribosome bound A-and P-site tRNAs unpair from the 0-frame on the mRNA, and then re-pair their non-wobble bases in the -1 frame of the mRNA. Oligonucleotide site-directed in vitro mutagenesis has been extensively used to dissect the slippery site in L-A, retroviruses and (+) ssRNA viruses. Experimmts altering the sequence of the first triplet (X XX), which corresponds to tRNAs slipping between the 0and -1 frames at the ribosomal P-site, show that disruption of the identity of the three bases significantly reduces the ability of ribosomes to shift reading frame. Substitution of any three identical nucleotides in this position is sufficient to direct efficient -1 ribosomal frameshifting. Different combinations yield different iefficiencies of frameshifting, with pyrimi-dines promoting the most efficient frameshifting, followed by purines, such that UUU>CCC> AAA>GGG. l 8 In the second triplet (corresponding to the ribosomal A-site), only triplets of A and U promote efficient levels of -1 ribosomal frameshifting. The seventh base can be A, U or C, but not G. 'R,'9 The simultaneous slippage model stresses the ability of the non-wobble bases of ribosome-bound tRNAs to be able to re-pair to the -1 frame. However, ribosome-bound tRNAs must first unpair from the 0-frame before they can re-pair to Elongating ribosome pauses at pseudoknot. New incoming -1 frame Wdon Figure 2 . The simultaneous slippage as applied to the L-A slippery site. Peptidyl-tRNA and aminoacyl tRNA occupy the ribosomal P-and A-sites respectively as the ribosome pauses at the mRNA pseudoknot. Both tRNAs can simultaneously slip one nucleotide in the 3' ( -1 ) direction, in such a way that their non-wobble bases can repair to the codons in the -1 reading frame. After peptidyl transfer and translocation, incoming tRNA recognizes the AGG codon in the new ( -1) reading frame. the -1 frame. The A and U restriction in the second triplet suggests that tRNA-mRNA pairing is stronger at the ribosomal A-site than at the P-site because the A.U base pair contains one less hydrogen bond than the G C base pair.I8 This was tested by examining the efficiencies of -1 ribosomal frameshifting using constructs having A-site triplets with fewer 0-frame hydrogen bonds (i.e. A.U rich and better able to un-pair), but capable of forming fewer hydrogen bonds in the -1 frame (i.e. less able to re-pair), as opposed to those having more 0-frame hydrogen bonds (i.e. G C rich, less capable of un-pairing), but having a greater potential of re-pairing to the -1 frame. The A.U rich 0-frame triplets were more capable of promoting efficient -I ribosomal frameshifting than those having more 0-frame hydrogen bonds (i.e. G C rich), even when they were less well suited to base pairing in the -1 frame.'' These data reinforce the notion that tRNA-mRNA pairing is stronger at the ribosomal A-site than at the P-site, although it is also possible that there are specific tRNAs that are particularly good at frameshifting in the context of the slippery site. A second element, an mRNA pseudoknot, is required to romote efficient -1 ribosomal f r a m e~h i f t i n g .~~~'~ An mRNA pseudoknot is a stem-loop whose loop can base pair to a sequence 3' to the stem. These elements are commonly referred to as stem 1 (Sl) -loop 1 (L1) -stem 2 (S2) ~ loop 2 (L2) ( Figure 3B ). The mRNA pseudoknot of L-A is particularly interesting in that it can potentially assume a variety of conformers, ranging from a long stem l/short stem 2 to a short stem I/long stem 2. Oligonucleotide site-directed mutagenesis was used to determine the biologically active mRNA pseudoknot conformation." In all cases that allowed for maximization of base pairing in stem 1, frameshifting was at or near wild-type levels, whereas changes which maximized stem 2 showed decreased frameshifting efficiencies comparable to mutants in which base pairing was entirely disrupted. Changes which strengthened stem 1 tended to increase the ability of the pseudoknot to promote efficient frameshifting, a finding which was also noted in coronavir~ses.~ The mRNA pseudoknot induces elongating ribosomes to pause over the slippery site,51.54 and this is thought to increase the probability of 5' ribosomal movement. Remarkably, energetically equivalent stem-loop structures will not substi-t~t e .~.~~ The spacing between slippery sites and pseudoknots is also ~r i t i c a l ,~,~,~~,~~ For efficient frameshifting, the ribosome must pause with the slippery site precisely positioned in its A and P sites. How are mRNA pseudoknots particularly able to promote efficient -1 ribosomal frameshifting? One idea invokes a 'pseudoknot recognizing factor'. However, evidence for the existence of such a factor has not been forthcoming, either by The ribosome can relatively easily unwind a simple stem-loop because there is no restriction on the rotation of the loop, even with a long stem. There is no unique pause site. (B) The ribosome meets added resistance to unwinding stem 1 of an mRNA pseudoknot, because loop 1 cannot easily rotate. If the mRNA pseudoknot is properly placed, the ribosome pauses over the slippery site and frameshifting occurs more often. S1, Stem 1; S2, Stem 2; L1, Loop I ; L2. Loop 2 compel ition assays in in vitvo translation systems (ten Dem, cited in reference 24) or by gel retardation assays (J. Dinman, unpublished data). Another suggestion is that, since the 5' and 3' ends of the pseudoknot are not contiguous, a ribosomeassociated helicase has greater difficulty unwinding a pseudoknot than a simple stem or perhaps some unique structural feature is what makes mRNA pseudoknots less resistant to unwinaing. Here we propose a 'Torsional Resistance Model' for how RNA pseudoknots promote efficient -1 ribosornal frameshifting (Figure 3) . Addition or deletion of only three nucleotides in the spacer between the slippery site and stem 1 prevents efficien.. frame~hifting.'.~~'~.~' Th us, the five to eight nucleotide spacing between slippery sites and pseudoknots is critical. If elongating ribosomes were able. before pausing, to unwind just one extra codon, i.e. one third of a helical turn of stem 1, the ribosomal A-and P-sites would not be correctly positioned over the slippery site, and -1 ribosomal frameshifting would not proceed efficiently. A simple stem-loop structure, no matter how long, does not force the ribosomes to stop at one special point, and so cannot efficiently promote -1 ribosomal frameshifting ( Figure 3A) . How then do RNA ,2seudoknots make elongating ribosomes pause in the right place? As the ribosome unwinds a stem loop 1 to rotate. If this loop is loop it forces anchored or restrained, as in a pseudoknot b; stem 2, then stem 1 cannot be unwound. The ribosome is thus forced to pause at a special point in stem 1 ( Figure 3B ). A simple stem-loop is not restrained, and can rotate freely; only the base pairs at the bottom of the stem resist ribosome movement. The pseudoknot has both these base pairs and those of stem 2 resisting ribosome motion. Thus, as the ribosome tries to unwind stem 1, stem 2 forces the supercoiling of stem 1, providing extra resistance to ribosome movement. Several predictions of this model are borne out by the experimental data: .Disrupting the first three base pairs of stem 1 would allow the ribosome to elongate be ond the .Destabilizing stem 2 would allow it to be unwound more readily, decreasing the efficiency of f r a m e~h i f t i n g .~.~~ .Replacing bulges in stem 1 with base pairs would increase the energy required to unwind its first three base pairs. A longer ribosomal pause over the slippery site would follow, yielding increased efficiencies of -1 ribosomal frame~hifting.~.'~ A weak point of this model is that, in some mRNA pseudoknots, stem 1 is only five or six base pairs in slippery site, eliminating frameshifting. 4Y length, i.e. less than a full helical turn. In these mRNA pseudoknots, unwinding of just one extra codon, i.e. one third of a helical turn of stem 1, would be sufficient to completely open up stem 1, rendering useless the contribution of stem 2. At this juncture, no definitive experiments have been designed to test these theories. Perhaps the identification and characterization of yeast chromosomal mutants capable of either efficient -1 ribosomal frameshifting in response to a simple stem loop, or conversely, incapable of efficient -1 ribosomal frameshifting through an RNA pseudoknot, will provide the necessary tools to definitively address the question of how RNA pseudoknots promote efficient -1 ribosomal frameshifting. + 1 RIBOSOMAL FRAMESHIFTING: The Ty retrotransposable elements of 5'. cevevisiue are all approximately 5 kb in length and are flanked by direct repeats (Figure 4 ; for reviews, see references 7, 50). They have the same general genomic organization as do viruses that use -1 ribosomal frameshifting, i.e. a 5' gag ORF, followed by apol ORF. The 5' ORF in Tyl, Ty2 and Ty4 is called TYA and in Ty3 it is called GAG3. The 3' ORFs are called TYB and POL3, respectively. TYB and POL3 are expressed as protein fusions to the product of the upstream genes in these elements. 15, 38344 In the Ty elements, however, pol is in the +1 reading frame relative to gag, and +1 ribosomal frameshifting is used to form Gag-pol fusion protein^.^,*^ Ty5 is different, in that it has a single long O R F (D. Votyas, cited in reference 25). +1 Ribosomal frameshifting in Tyl is directed by a heptanucleotide sequence CUU AGG C (0-frame indicated by space^).^ In Ty3, it is promoted by the heptameric sequence GCG AGU U." A ribosomal pause is required in each case, and sequence downstream helps to promote efficient + 1 ribosomal frameshifting. At this point, however, the similarities between + I and -1 ribosomal frameshifting end. Although both +1 and -1 ribosomal frameshifting occur at heptameric 'slippery sites', the nature of these sites are completely different. Unlike -1 ribosomal frameshifting, the simultaneous slippage of ribosome-bound A-and P-site tRNAs from the 0-frame to the +1 frame would not allow their non-wobble bases to repair. Also, in -1 ribosomal frameshifting, the downstream sequence that is required to promote efficient frameshifting is the mRNA pseudoknot. Although a potential pseudoknot structure can be inferred in Tyl, the structure is not required, and no such structure can be inferred from the Ty3 sequence.25 The purpose of the downstream sequences in the Ty elements is not understood, but they do not involve pseudoknots. In -1 ribosomal frameshifting, the RNA pseudoknot promotes a ribosomal pause. In +1 ribosomal frameshifting in the Ty elements, the ribosomal pause is promoted by 'hungry codons', in the 0-frame A-site (i.e. nucleotides 4-6 of the slippery site). Hungry codons correspond to tRNAs that are not abundant in the cell. Elongating ribosomes pause at the slippery site with their P-sites occupied by peptidyl-tRNAs, awaiting the arrival of the cognate tRNA that should base pair to the hungry codon at the ribosomal A-site. It is during this pause that the shift in reading frame occurs. Figure 5 shows how +1 ribosomal frameshifting in Tyl, Ty2, Ty3 and Ty4 is thought to occur. In the Tyl, Ty2 and Ty4 elements, the slippery Leu tRNA,,, occupying the P-site of the +1 ribosomal frameshift signal recognizes its cognate codon CUU by two out of three decoding.55 The normal decoding of the in-frame A-site AGG is slow because of the low availability of the cognate Arg tRNA,,,, causing a translational pause, during which + 1 ribosomal frameshifting occurs. Two Val tRNqAC "Hungry" 0-frame I codon New +1 frame d o n Figure 5 . +1 Ribosomal frameshifting in Tyl, 2 and 4, and in Ty3: two distinct mechanisms. In Tyl, Ty2 and Ty4, the slippery Leu tRNA,,, recognizes its cognate codon CUU by two out of three decoding. The ribosome pauses at the AGG 'hungry codon' due to the low availability of its cognate Arg tRNAccV. During this translational pause, Gly tRNA,,, likely binds transiently to the + I frame codon GGC, followed by slippage of the Leu tRNA,,, to the UUA codon. In Ty3, after recognition of the GCG codon by Ala tRNA,,,, AGU serves as the 'hungry' codon, corresponding to the low abundance Ser tRNA,,,. This allows for the recognition of the + I frame codon GUU by Val tRNA,,,. The body of the peptidyl-Ala tRNA,,, (shown as 'bent to the right') allows the outof-frame tRNA to be accepted by the ribosome, allowing peptide transfer to occur, shifting the ribosome into the +1 reading frame. slightly different models have been proposed. In the first, the P-site Leu tRNAUAI; first slips in the +1 direction, followed by binding of Gly tRNA,,,. to the + I frame A -~i t e .~ In the second mechanism, the Gly tRNAGCc transiently binds to the +1 frame codon GGC in the ribosomal A-site, followed by the slippage of Leu tRNAUAG in the +1 direction to the UUA ~o d o n .~~ The second model is currently favored because, since the amount of the Gly tRNA,,, is important,46 the + 1 tRNA must bind before slippage, assuming that the elongating ribosome is not in equilibrium. Further, this model would unify the +1 ribosomal frameshifting mechanisms used by Tyl, TyZ and Ty4 with that of Ty3 (see below). Thus, this is the model depicted in Figure 5A . In Ty3 ( Figure 5B ), Ala tRNAcGc is bound to the GCG codon in the 0-frame P-site and the 0-frame A-site AGU codon, corresponding to the rare Ser tRNAGcU, provides the pause. However, in this case, the Ala tRNA,,, cannot slip onto the +1 CGA codon but, rather, it is thought to force a Val tRNA,,, into the +1 GUU codon at the ribosomal A-site. Saturation mutagenesis of the frameshift site of Ty3 has demonstrated that there is no correlation between the ability of a peptidyl-tRNA to slip and its ability to promote efficient + 1 ribosomal frame~hifting.~~ Eight different tRNAs were shown to be capable of promoting frameshifting, four of which cannot slip. Some other aspects of these tRNAs must allow them to promote frameshifting by directing out-of-frame binding of the incoming aminoacyl-tRNA. Substitution of tRNA bodies with different anticodons showed that it is the body of the P-site tRNA that promotes forcing of the incoming A-site tRNA into the + I frame.46 These data imply that interactions between the P-site tRNA and the incoming ternary complex mispositions the aminoacyl tRNA onto the +1 codon. Thus, Ty3 frameshifting occurs without tRNA slippage: a special tRNA in the P-site is able to promote A-site tRNA binding to the + 1 codon, provided that there is a translational pause provided by a hungry ~o d o n .~~,~~ We have determined that the efficiency of -1 ribosomal frameshifting in the naturally occurring L-A slippery site is 1.8-2.0%.'8,'9 The 39 nm L-A viral particle contains 120 Gag proteins,",22 and the 1.9% efficiency of frameshifting can be interpreted as providing 1 Gag-pol molecule for every 59 Gag proteins made, i.e. each viral particle contains two Gag-pol molecules. Genetic and geometric considerations from the frameshifting data led us to hypothesize that Gag-pol functions as a dimer in the viral particle.'' Reconstructions of the L-A virus particle from cryoelectron microscopic observations show that Gag is also a dimer.I4 Changing the efficiency of -1 ribosomal frameshifting would change the ratio of Gag to Gag-pol. This might in turn affect viral particle assembly, and therefore the ability of the cell to propagate the virus. Changing the slippery site sequence affects the efficiency of -1 ribosomal frarne~hifting.'~,'' The efficiency of -1 ribosomal frameshifting can also be affected by mutations in the cellular gene products that presumably interact with these tRNA and mRNA factors."320 Using both molecular and genetic methods, we demonstrated that the 1.9% efficiency of ribosomal frameshifting yields the optimum ratio of structural Gag to enzymatic Gag-pol proteins. Changing frameshifting efficiencies more than two-to three-fold greater (or 70% less) than wild-type levels results in the loss of the M, satellite virus, whether the virus is supported by L-A cDNA clones containing altered slippery sites,'' or by the wild-type L-A virus in host cells containing chromosomal mutations which result in cells having higher efficiencies of -1 ribosomal frameshifting in res onse to the L-A frameshift signal (see below)."32o Even slight changes in -1 ribosomal frameshifting efficiencies significantly lower M I copy numbers. A +1 frameshifting signal derived from Tyl can substitute for a -1 signal in maintaining MI as long as frameshifting efficiencies fall within this acceptable 'frameshift window '. l9 Analogously, the importance of the efficiency of + 1 ribosomal frameshifting in determining the relative ratios of Gag to Gag-pol have been tested in Tyl. Ty elements transpose through an RNA intermediate using the same replication and integration strategy employed by the metazoan v i r~s e s .~,~~,~~ Thus, in order to transpose they must go through a viral intermediate. Increasing the abundance of the Arg tRNAcuu critical for inducing the translational pause by providing it in trans on a high copy vector nearly abolishes + I ribosomal frame~hifting,~~ and dramatically reduces Tyl transposition frequencies. Similarly, deleting the sin le copy gene for this tRNA gene, called HSX13' promotes extremely high levels of frameshifting and also results in loss of Tyl transposition. 60 We have found that starvation for the polyamine spermidine and the consequent elevation of intracellular concentrations of putrecine also increases the efficiency of +1 frameshifting in T Y Z .~.~ Loss of the ability of a HIS3-marked TyZ cDNA clone to transpose into the yeast genome paralleled the increase in +1 ribosomal frameshifting in polyamine-starved spe2 cells.3 Taken together, these findings support the hypothesis that the efficiency of ribosomal frameshifting is critical for viral propagation, and that agents which affect ribosomal frameshifting efficiency may have antiviral a c t i v i t i e~.~'~"~'~~ CHROMOSOMAL MUTATIONS WHICH Recently a number of host chromosomal mutants which affect the efficiency of -1 ribosomal frameshifting have been described. 19-21. 39 The mutants isolated in our laboratory are called mof (Maintenance Of Frame). To date, nine such mof mutants have been characterized. These mutations show differential effects on various frameshifting signals and have numerous secondary phenotypes (Table 1) . These mutants appear to be affecting the elongation phase of protein synthesis. AFFECT THE EFFICIENCY OF -1 The fold increase in frameshifting efficiency is from reference 20. M, dsRNA denotes the ability of the mutant cells to maintain the M, satellite virus of L-A. ts, temperature sensitive. pet, ability to grow on glycerol. a non-fermentable carbon source. Upf phenotype denotes the amount of endogenous un-spliced CYH2 precursor mRNA in these cells. The mof4-1 mutation is an allele of the UPFl gene In normal cells, nonsense mRNAs, e.g. unspliced mRNAs which escape into the cytoplasm, or mis-transcribed mRNAs which contain premature termination signals, are rapidly degraded. A class of genes, called UPF ( U P Frameshift) and NMD (Nonsense Mediated Decay), are involved in the degradation of nonsense mRNAs. The halflives of nonsense mRNAs are increased in this class of mutants, and they also have frameshift suppressor phenotypes (reviewed in reference 47). The constructs that we originally used for the detection of rnof mutants have the lacZ gene downstream of the L-A -1 ribosomal frameshift signal in the -1 reading frame relative to a translational start site (see Figure 1B ). To elongating ribosomes, this would present a long mRNA containing a short 5' ORF that is quickly interrupted by a termination codon, i.e. a nonsense mRNA. The assay that was designed to detect mof mutants relied upon finding cells expressing increased amounts of P-galactosidase (P-gal) as a result of an increase in the efficiency of -1 ribosomal frameshifting. However, the same result could also be observed if the cells were upf or nmd mutants, because a longer half-life of the 'nonsense' reporter mRNA would result in the greater accumulation of the frameshifted P-gal gene product. Thus, mutants in the nonsense-mediated mRNA decay pathway could be mistaken for mof mutants on the basis of their higher P-gal activities. We have identified mof4-1 as an allele of the UPFl gene. The UPFl protein contains a putative zinc finger domain and a predicted helicase domain.40 The original upfl-2 mutation has a tryptophan-to-termination nonsense mutation at amino acid residue 205. We have sequenced the mof4-1 mutation and determined that it consists of a cystine (Cys)-to-tyrosine missense mutation at amino acid 62, the first Cys residue in the putative zinc finger. ' Although the half-lives of the -1 ribosomal frameshifting reporter mRNAs are increased in upf and nmd mutants, the ribosomes translating them would continue to frameshift with the same efficiency. Thus, although upflnmd mutants should be indistinguishable from mof mutants by the P-gal assay, upflnmd mutants should be able to maintain the M, virus because the ratio of Gag to Gag-pol would remain unaffected. True mof mutants, by virtue of their effect upon -1 ribosomal frameshifting efficiency, should not be capable of propagating M,, i.e. they should have Mak-phenotypes (MAK = MAintenance of Killer; for review, see reference 58). m o f l -I , mof2-1, mof4-I, mof5-1 and mof6-1 have the Mak phenotype and are thus true mof mutants. mof4-1 has a strong nonsense mRNA decay mutant phenotype, and mof2-1, mof5-1 and mof8-1 also have weak Upfnonsense mRNA decay mutant phenotypes (Table upf3 -1 and upf4-1 are all capable of maintaining MI, indicating that mof4-I is a unique mof allele of 1). ~p f l -2 , UPFI:: URA3, ~p f 2 -I , UPF2:: URA3, UPFl .I7 Recent studies examining UPFl show that mutations in the zinc finger domain affect frameshift suppression, whereas mutations in the helicase domain affect the nonsense-mediated mRNA decay phenotype." Our results with rnof4-1 complement these findings, in that the mof4-1 mutation, which is in the zinc finger domain, has a specific translational defect that results in an increased efficiency of -1 ribosomal frameshifting. Recently, Lee et ~1 .~' have identified two Zncreased FrameShift (ifs) mutants in yeast. Using a construct containing the yeast CUP1 gene downstream of a -1 ribosomal frameshift signal from the mouse mammary tumor virus Gag-Pol junction, ifs mutants were identified by loss of copper sensitivity in cupA cells. Both of these had -1 ribosomal frameshifting efficiencies approximately two-fold greater than wild-type cells, as measured by P-gal activities. They cloned and sequenced one of these, $1. Com arison of IFSl to UPF2 cal. We have also determined that $2 and mof4-1 fall into the same complementation group and that the Ifs phenotype of ifs2 can be corrected with a clone of UPFl. Both ifsl and ifs2 mutants are able to propagate M,. This could be due to the fact that a two-fold increase in -1 ribosomal frameshifting efficiency is not large enough to affect the propagation of M I in these cells. Alternatively, there could be no change in the efficiency of -1 ribosomal frameshifting in these mutants per se, but rather the ability of these mutants to grow in the presence of copper might be due to the increased half-lives of the nonsense reporter mRNAs. These data demonstrate that the mof4-1 is a unique maintenance of frame allele of UPFl. It has both the Mak and Upfphenotypes, and promotes a greater efficiency of -1 ribosomal frameshifting in response to a specific viral signal. As such, it represents the first time that a single protein has been linked to both the processes of translation and mRNA decay. This demonstrates that there is a connection between the phenomena defined by the mof and upflnmd mutants, illuminating the continuity of the translational process, from mRNA stability through the translation of the complete protein product. mof9: 5s rRNA is involved in fidelity of translarional reading frame Yeast ribosomes are composed of at least 77 ribosomal proteins and four ribosomal RNAs ( N M D 2 ) sequenceI6, P shows that they are identi-(rRNAs; for a review, see reference 59). Slightly more than half of the cloned ribosomal protein genes are represented by two isoforms in yeast, whereas there are over 100 copies of the rRNA genes in the genome. One would expect that the mof mutants would be associated with ribosomal protein genes rather than rRNAs, because a mutation in only one copy of an rRNA gene would be expected to be masked by the presence of over 100 copies of the wild-type gene. Surprisingly, mOf9-1, which increases the efficiency of ribosomal frameshifting 2.5to 3-fold, is complemented by a clone of 5s rRNA on either a single or high copy vector.2' The mof9-1 mutation maps to the yeast rDNA locus, and two other independent mutations of 5s rRNA at that locus also have the MOB-phenotype and can be complemented by wild-type 5s rRNA. Mutant 5s rRNAs expressed from episomal vectors as 20-50% of total cellular 5s rRNA also have the Mof9-phenotype. The mof9 mutants also increase the efficiency of +1 ribosomal frameshifting directed by a Tyl frameshift signal, but have no effect upon readthrough of UAG or UUA termination codons, indicating that not all translational specificity is affected. There is no detectable increase in the amount of steady-state l a c 2 mRNA transcribed from the -1 ribosomal frameshift test plasmid. Therefore the increased amount of P-gal activity is the direct result of an increase in the efficiency of ribosomal frameshifting, and is not due to a defect in the nonsense-mediated mRNA degradation pathway. Prior to these studies, no specific role had been assigned to 5s rRNA, aside from a vague 'scaffold' function. The mof9 data suggest a role for 5s rRNA in maintaining reading frame in translation. CHROMOSOMAL MUTATIONS AFFECTING + 1 RIBOSOMAL FRAMESHIFTING As noted above, + 1 ribosomal frameshifting in Tyl depends upon the low abundance Arg tRNAAGG, encoded by the single-copy H S X l gene. Deletion of this gene results in extremely high levels of +1 ribosomal frameshifting as directed by a Tyl +1 frameshifting signal, and the loss of the ability of Tyl cDNA clones to transpose. Conversely, overexpression of this tRNA gene on high copy plasmids decreases + 1 ribosomal frameshifting efficiency, and results in the loss of the ability of TyZ cDNA clones to transpose. A second class of mutants which affect the efficiency of +1 ribosomal frameshifting are involved in the biosynthesis of p~l y a m i n e s .~.~ Upon starvation for spermidine, the efficiency of +1 ribosomal frameshifting directed by a Tyl frameshift signal increases dramatically in deletion mutants of SPE2 (S-adenosyl decarboxylase). Paralleling the increase in + 1 frameshifting efficiencq is a decrease in transposition frequency of Tyl. Interestingly, deletion of SPEl (orinithine decarboxylase, which produces putrescine, a biosynthetic precursor of spermidine) can reverse the increase in + 1 ribosomal frameshifting efficiency in spe2A cells depleted of spermidine. The high level of +I ribosomal frameshifting efficiency in spe2A cells is the result of the combined effects of both sperniidine deprivation and the large increase in the level of intracellular putrescine resulting from the derepression of the SPEI gene in spermidinedeficient strains. Since the overexpression of Arg tRNA,,, suppressed the increase of +1 ribosomal frameshifting in spermidine-depleted spe2A cells, the results from these studies suggest that spermidine may be required for selection and/or insertion of cognate tRNA at the ribosomal A-site. As noted above, Ty5 consists of a single ORF and ribosomal frameshifting does not appear to be involved. Likewise, all of the proteins in the retrotransposable element of the fission yeast Schizosucchuromyces pombe, Tfl, are derived from a single primary translation product.41 How do these elements regulate the relative ratios of structural Gag proteins to those having enzymatic functions, i.e. integrase (Int), reverse transcriptase (RT), protease (Pro) and RNAse H? The ratio of structural (Gag) proteins to those having enzymatic functions in Tfl particles is 30:l. Although there has been no demonstration that Tfl particles require this 30:l ratio to transpose, DNA blot results indicate that the bulk of mature cDNA is produced by particles having this ratio of structural to enzymatic proteins (H. Levin, personal communication) . The implications for viral particle assembly and replication in these 'non-frameshifting' retrotransposable elements are very exciting. The studies on -I and +l ribosomal frameshifting in the L-A and Ty viruses of yeast serve as an example of how such pairings can further our understanding of translational elongation, nonsense-mediated mRNA decay, the control of viral and cellular gene expression and the dynamics of viral capsid assembly and RNA packaging. The molecular and biochemical characterization of the host chromosomal mutants affecting the efficiency of ribosomal frameshifting will provide a unique set of tools for these investigations. That these mutants affect the ability of cells to propagate viruses which use ribosomal frameshifting suggests that the characterization of the mof mutations and Mof gene products may serve to identify targets for the rational design of antiviral agents. The yeast cellular host constitutes an ideal system for drug screening along these lines. Toward a genetic dissection of the basis of triplet decoding, and its natural subversion: programmed reading frame shifts and hops Spermidine deficiency increases + 1 ribosomal frameshifting efficiency and inhibits TyI retrotransposition in Saccharomyces cerevisiue SPEI and SPEZ: Two essential genes in the biosynthesis of polyamines that modulate + 1 ribosomal frameshifting in Suecharomyces cerevisiae Ribosomal frameshifting in the yeast retrotransposon Ty: tRNAs induce slippage on a 7 nucleotide minimal site The coat protein of the yeast double-stranded RNA virus L-A attaches covalently to the cap structure of eukaryotic mRNA Ty elements transpose through an RNA intermediate Yeast transposable elements Characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an RNA pseudoknot Mutational analysis of the R N A pseudoknot component of a coronavirus ribosomal frameshifting signal Mutational analysis of the 'slippery-sequence' component of a coronavirus ribosomal frameshfiting signal Virus-like particle of yeast K1 killer toxin, a poreforming protein from yeast Translational frameshifting in the control of transposition in bacteria Fungal virus capsids, cytoplasmic compartments for the replication of double-stranded RNA, formed as icosahedral shells of asymmetric gag dimers Nucleotide sequence of a yeast Ty element: evidence for an unusual mechanism of gene expression Identification and characterization o f genes that are required for the accelerated degradation of mRNAs containing a premature translational termination codon A -1 ribosomal frameshift in a double-stranded RNA virus forms a gag-pol fusion protein Ribosomal frameshifting eficiency and gaglgag-pol ratio are critical for yeast M, double-stranded RNA virus propagation Translational maintenance of frame: mutants of Saccharomyces cerevisiae with altered -1 ribosomal frameshifting efficiencies rRNA is involved in fidelity of translational reading frame Three different M 1 RNA-containing viruslike particle types in Saccharomyces cerevisiae: in vitro M1 double-stranded RNA synthesis A novel programmed frameshift expresses the POW gene of retrotransposon Ty3 of yeast: frameshifting without tRNA slippage Post-transcriptional regulation of transposition by Ty retrotransposons of Saccharomyces cerevisiae Gene overlap results in a viral protein having an RNA binding domain and a major coat protein domain Interaction of two cis sites with the R N A replicase of the yeast L-A virus Ty element transposition: reverse transcriptase and virus-like particles Translational suppression in retroviral gene expression Rapid and regulated degradation of ornithine decarboxylase Identification of a novel component of the nonsense-mediated mRNA decay pathway by use of an interacting protein screen Translation of the L-species dsRNA genome of the killer-associated virus-like particles of Saccharomyces cerevisiae The doublestranded R N A genome of yeast virus L-A encodes its own putative RNA polymerase by fusing two open reading frames Signals for ribosomal frameshifting in the Rous sarcoma virus gag-pol region Translational suppression in gene expression in retroviruses and retrotransposons Ty element-induced temperature-sensitive mutations of Sacchuromyces cerevisiue A rare tRNA-Arg(CUU) that regulates Tyl element ribosomal frameshifting is essential for Tyl retrotransposition in Succharomyces cerevisiae Transposition of a Ty3 GAG3-POL3 fusion mutant is limited by availability of capsid protein A genetic screen identifies cellular factors involved in retroviral ~ 1 ribosomal frameshifting Novel gene expression mechanism in a fission yeast retroelement: Tfl proteins are derived from a single primary translation product Autoregulatory frameshifting in decoding mammalian ornithine decarboxylase antizyme Identification and analysis of the gug-pol ribosomal frameshift site of feline immunodeficiency virus Pulling the ribosome out of frame by + 1 at a programmed frameshift site by cognate binding of aminoacyl-tRNA Nonsense-mediated mRNA decay in yeast RNAdependent RNA polymerase consensus sequence of the L-A double-stranded RNA virus: definition of essential domains Polyamines regulate the expression of ornithine decarboxylase antizyme in vitro by inducing ribosomal frameshifting Yeast retrotransposons RNA pseudoknots: Translational frameshifting and readthrough on viral RNAs Ribosomal movement impeded at a pseudoknot required for ribosomal frameshifting Special peptidyl-tRNA molecules can promote translational frameshifting without slippage Encapsidation sequences for spleen necrosis virus, an avian retrovirus, are between the 5' long terminal repeat and the start of the gag gene Linking mRNA turnover and translational termination by the Upfl protein Double-stranded and singlestranded RNA viruses of Saccharomyces cerevisiae Host genes that influence transposition in yeast: the abundance of a rare tRNA regulates Tyl transposition frequency ACKNOWLEDGEMENTS 1 would like to acknowledge Phil Farabaugh, Henry Levin, Jim Umen, Susanna Lee, Ying Cui and Stuart Peltz for sharing strains, manuscripts and information prior to publication. I also want to thank Reed Wickner for all that he has done to make my postdoctoral days rich, rewarding and fun.