An mRNA sequence derived from a programmed frameshifting signal decreases codon discrimination during translation initiation

Sequences and structures in the mRNA can alter the accuracy of translation. In some cases, mRNA secondary structures like hairpin loops or pseudoknots can cause frequent errors of translational reading frame (programmed frameshifting) or misreading of termination codons as sense (nonsense readthrough). In other cases, the primary mRNA sequence stimulates the error probably by interacting with an element of the ribosome to interfere with error correction. One such primary mRNA sequence, the Ty3 stimulator, increases programmed +1 frameshifting 7.5 times in the yeast Saccharomyces cerevisiae. Here we show that this stimulator also increases the usage of non-AUG initiation codons in the bacterium Escherichia coli but not in S. cerevisiae. These data suggest that in E. coli, though not in yeast, an element of the ribosome's elongation accuracy mechanism ensures initiation accuracy.     

Translational suppressors and antisuppressors alter the efficiency of the Ty1 programmed translational frameshift

Certain viruses, transposons, and cellular genes have evolved specific sequences that induce high levels of specific translational errors. Such "programmed misreading" can result in levels of frameshifting or nonsense codon readthrough that are up to 1,000-fold higher than normal. Here we determine how a number of mutations in yeast affect the programmed misreading used by the yeast Ty retrotransposons. These mutations have previously been shown to affect the general accuracy of translational termination. We find that among four nonsense suppressor ribosomal mutations tested, one (a ribosomal protein mutation) enhanced the efficiency of the Tyl frameshifting, another (an rRNA mutation) reduced frameshifting, and two others (another ribosomal protein mutation and another rRNA mutation) had no effect. Three antisuppressor rRNA mutations all reduced Tyl frameshifting; however the antisuppressor mutation in the ribosomal protein did not show any effect. Among nonribosomal mutations, the allosuppressor protein phosphatase mutation enhanced Tyl frameshifting, whereas the partially inactive prion form of the release factor eRF3 caused a slight decrease, if any effect. A mutant form of the other release factor, eRF1, also had no effect on frameshifting. Our data suggest that Ty frameshifting is under the control of the cellular translational machinery. Surprisingly we find that translational suppressors can affect Ty frameshifting in either direction, whereas antisuppressors have either no effect or cause a decrease.     

Characterization of RNA elements that regulate gag-pol ribosomal frameshifting in equine infectious anemia virus

Synthesis of Gag-Pol polyproteins of retroviruses requires ribosomes to shift translational reading frame once or twice in a -1 direction to read through the stop codon in the gag reading frame. It is generally believed that a slippery sequence and a downstream RNA structure are required for the programmed -1 ribosomal frameshifting. However, the mechanism regulating the Gag-Pol frameshifting remains poorly understood. In this report, we have defined specific mRNA elements required for sufficient ribosomal frameshifting in equine anemia infectious virus (EIAV) by using full-length provirus replication and Gag/Gag-Pol expression systems. The results of these studies revealed that frameshifting efficiency and viral replication were dependent on a characteristic slippery sequence, a five-base-paired GC stretch, and a pseudoknot structure. Heterologous slippery sequences from human immunodeficiency virus type 1 and visna virus were able to substitute for the EIAV slippery sequence in supporting EIAV replication. Disruption of the GC-paired stretch abolished the frameshifting required for viral replication, and disruption of the pseudoknot reduced the frameshifting efficiency by 60%. Our data indicated that maintenance of the essential RNA signals (slippery sequences and structural elements) in this region of the genomic mRNA was critical for sufficient ribosomal frameshifting and EIAV replication, while concomitant alterations in the amino acids translated from the same region of the mRNA could be tolerated during replication. The data further indicated that proviral mutations that reduced frameshifting efficiency by as much as 50% continued to sustain viral replication and that greater reductions in frameshifting efficiency lead to replication defects. These studies define for the first time the RNA sequence and structural determinants of Gag-Pol frameshifting necessary for EIAV replication, reveal novel aspects relative to frameshifting elements described for other retroviruses, and provide new genetic determinants that can be evaluated as potential antiviral targets.     

Programmed frameshifting in the synthesis of mammalian antizyme is +1 in mammals, predominantly +1 in fission yeast, but -2 in budding yeast.

The coding sequence for mammalian ornithine decarboxylase antizyme is in two different partially overlapping reading frames with no independent ribosome entry to the second ORF. Immediately before the stop codon of the first ORF, a proportion of ribosomes undergo a quadruplet translocation event to shift to the +1 reading frame of the second and main ORF. The proportion that frameshifts is dependent on the polyamine level and, because the product antizyme is a negative regulator of intracellular polyamine levels, the frameshifting acts to complete an autoregulatory circuit by sensing polyamine levels. An mRNA element just 5' of the shift site and a 3' pseudoknot are important for efficient frameshifting. Previous work has shown that a cassette with the mammalian shift site and associated signals directs efficient shifting in the budding yeast Saccharomyces cerevisiae at the same codon to the correct frame, but that the shift is -2 instead of +1. The product contains an extra amino acid corresponding to the shift site. The present work shows efficient frameshifting also occurs in the fission yeast, Schizosaccharomyces pombe. This frameshifting is 80% +1 and 20% -2. The response of S. pombe translation apparatus to the mammalian antizyme recoding signals is more similar to that of the mammalian system than to that of S. cerevisiae. S. pombe provides a good model system for genetic studies on the mechanism of at least this type of programmed mammalian frameshifting.     

Evidence against a direct role for the Upf proteins in frameshifting or nonsense codon readthrough

The Upf proteins are essential for nonsense-mediated mRNA decay (NMD). They have also been implicated in the modulation of translational fidelity at viral frameshift signals and premature termination codons. How these factors function in both mRNA turnover and translational control remains unclear. In this study, mono- and bicistronic reporter systems were used in the yeast Saccharomyces cerevisae to differentiate between effects at the levels of mRNA turnover and those at the level of translation. We confirm that upfDelta mutants do not affect programmed frameshifting, and show that this is also true for mutant forms of eIF1/Sui1p. Further, bicistronic reporters did not detect defects in translational readthrough due to deletion of the UPF genes, suggesting that their function in termination is not as general a phenomenon as was previously believed. The demonstration that upf sui1 double mutants are synthetically lethal demonstrates an important functional interaction between the NMD and translation initiation pathway.     

How translational accuracy influences reading frame maintenance

Most missense errors have little effect on protein function, since they only exchange one amino acid for another. However, processivity errors, frameshifting or premature termination result in a synthesis of an incomplete peptide. There may be a connection between missense and processivity errors, since processivity errors now appear to result from a second error occurring after recruitment of an errant aminoacyl-tRNA, either spontaneous dissociation causing premature termination or translational frameshifting. This is clearest in programmed translational frameshifting where the mRNA programs errant reading by a near-cognate tRNA; this error promotes a second frameshifting error (a dual-error model of frameshifting). The same mechanism can explain frameshifting by suppressor tRNAs, even those with expanded anticodon loops. The previous model that suppressor tRNAs induce quadruplet translocation now appears incorrect for most, and perhaps for all of them. We suggest that the 'spontaneous' tRNA-induced frameshifting and 'programmed' mRNA-induced frameshifting use the same mechanism, although the frequency of frameshifting is very different. This new model of frameshifting suggests that the tRNA is not acting as the yardstick to measure out the length of the translocation step. Rather, the translocation of 3 nucleotides may be an inherent feature of the ribosome.     

Connection between stop codon reassignment and frequent use of shifty stop frameshifting

Ciliated protozoa of the genus Euplotes have undergone genetic code reassignment, redefining the termination codon UGA to encode cysteine. In addition, Euplotes spp. genes very frequently employ shifty stop frameshifting. Both of these phenomena involve noncanonical events at a termination codon, suggesting they might have a common cause. We recently demonstrated that Euplotes octocarinatus peptide release factor eRF1 ignores UGA termination codons while continuing to recognize UAA and UAG. Here we show that both the Tetrahymena thermophila and E. octocarinatus eRF1 factors allow efficient frameshifting at all three termination codons, suggesting that UGA redefinition also impaired UAA/UAG recognition. Mutations of the Euplotes factor restoring a phylogenetically conserved motif in eRF1 (TASNIKS) reduced programmed frameshifting at all three termination codons. Mutation of another conserved residue, Cys124, strongly reduces frameshifting at UGA while actually increasing frameshifting at UAA/UAG. We will discuss these results in light of recent biochemical characterization of these mutations.     

Recoding in archaea

Standard decoding of the genetic information into polypeptides is performed by one of the most sophisticated cell machineries, the translating ribosome, which, by following the genetic code, ensures the correspondence between the mature mRNA and the protein sequence. However, the expression of a minority of genes requires programmed deviations from the standard decoding rules, globally named recoding. This includes ribosome programmed -/+1 frameshifting, ribosome hopping, and stop codon readthrough. Recoding in Archaea was unequivocally demonstrated only for the translation of the UGA stop codon into the amino acid selenocysteine. However, a new recoding event leading to the 22nd amino acid pyrrolysine and the preliminary reports on a gene regulated by programmed -1 frameshifting have been recently described in Archaea. Therefore, it appears that the study of this phenomenon in Archaea is still at its dawn and that most of the genes whose expression is regulated by recoding are still uncharacterized.     

Prion: disease or relief?

The self-perpetuating amyloid isoform, or prion, of the yeast translation termination factor eRF3 modulates programmed translational frameshifting that controls a regulatory circuit determining the polyamine levels in a yeast cell. But it is still unclear whether this effect is adaptive or pathological.     

Suppression of frameshift mutation as a result of partial inactivation of translation termination factors in Saccharomyces cerevisiae yeast]

Special search for frameshift mutations, which are suppressed by the cytoplasmic [PSI] factor and by omnipotent nonsense suppressors (recessive mutations in the SUP35 and SUP45 genes), partially inactivating a translation termination complex, was initiated in the LYS2 gene in the yeast Saccharomyces cerevisiae. Mutations were obtained after exposure to UV light and treatment with a mixture consisting of 1.6- and 1.8-dinitropyrene (DNP). This mixture was shown to induce mutations of the frameshift type with a high frequency. The majority of these mutations were insertions of one A or T, which is in good agreement with the data obtained in studies of DNP-induced mutagenesis in other eukaryotes. Frameshift suppression in yeast was first shown on the example of the mutation obtained in this work (lys2-90), which carried the insertion of an extra T in the sequence of five T. This frameshift suppression was shown to occur in the presence of the [PSI] factor (i.e., due to the prion form of the translation release factor eRF3) and as a result of mutations in genes SUP35 or SUP45, which partially inactivate translation termination factors eRF3 and eRF1, respectively. Alternative mechanisms of programmed translational frameshifting in the course of translation and the possibility of enhancing the effectiveness of such frameshifting in the presence of the [PSI] factor are considered.     

Pseudogene recoding revealed from proteomic analysis of salmonella serovars

Recoding refers to the reprogramming of mRNA translation by nonstandard read-out rules. In this study, we used stable isotope labeling with amino acids in cell culture (SILAC) technology to investigate the proteome of host-adapted Salmonella serovars, which are characteristic of accumulation of pseudogenes. Interestingly, a few annotated pseudogenes were indeed able to express peptides downstream of the inactivation site, suggesting the occurrence of recoding. Two mechanisms of recoding, namely, programmed frameshifting and codon redefinition, were both found. We believe that the phenomena of recoding are not rare in bacteria. More studies are required for a better understanding of bacterial translation and the implication of pseudogene recoding in Salmonella serovars.     

Binary specification of nonsense codons by splicing and cytoplasmic translation.

Premature translation termination codons resulting from nonsense or frameshift mutations are common causes of genetic disorders. Complications arising from the synthesis of C-terminally truncated polypeptides can be avoided by 'nonsense-mediated decay' of the mutant mRNAs. Premature termination codons in the beta-globin mRNA cause the common recessive form of beta-thalassemia when the affected mRNA is degraded, but the more severe dominant form when the mRNA escapes nonsense-mediated decay. We demonstrate that cells distinguish a premature termination codon within the beta-globin mRNA from the physiological translation termination codon by a two-step specification mechanism. According to the binary specification model proposed here, the positions of splice junctions are first tagged during splicing in the nucleus, defining a stop codon operationally as a premature termination codon by the presence of a 3' splicing tag. In the second step, cytoplasmic translation is required to validate the 3' splicing tag for decay of the mRNA. This model explains nonsense-mediated decay on the basis of conventional molecular mechanisms and allows us to propose a common principle for nonsense-mediated decay from yeast to man.     

Conventional 3' end formation is not required for NMD substrate recognition in Saccharomyces cerevisiae

The recognition and rapid degradation of mRNAs with premature translation termination codons by the nonsense-mediated pathway of mRNA decay is an important RNA quality control system in eukaryotes. In mammals, the efficient recognition of these mRNAs is dependent upon exon junction complex proteins deposited on the RNA during pre-mRNA splicing. In yeast, splicing does not play a role in recognition of mRNAs that terminate translation prematurely, raising the possibility that proteins deposited during alternative pre-mRNA processing events such as 3' end formation might contribute to the distinction between normal and premature translation termination. We have utilized mRNAs with a 3' poly(A) tail generated by ribozyme cleavage to demonstrate that the normal process of 3' end cleavage and polyadenylation is not required for mRNA stability or the detection of a premature stop codon. Thus, in yeast, the distinction between normal and premature translation termination events is independent of both splicing and conventional 3' end formation.     

Lack of pseudouridine 38/39 in the anticodon arm of yeast cytoplasmic tRNA decreases in vivo recoding efficiency

Many different modified nucleotides are found in naturally occurring tRNA, especially in the anticodon region. Their importance for the efficiency of the translational process begins to be well documented. Here we have analyzed the in vivo effect of deleting genes coding for yeast tRNA-modifying enzymes, namely Pus1p, Pus3p, Pus4p, or Trm4p, on termination readthrough and +1 frameshift events. To this end, we have transformed each of the yeast deletion strains with a lacZ-luc dual-reporter vector harboring selected programmed recoding sites. We have found that only deletion of the PUS3 gene, encoding the enzyme that introduces pseudouridines at position 38 or 39 in tRNA, has an effect on the efficiency of the translation process. In this mutant, we have observed a reduced readthrough efficiency of each stop codon by natural nonsense suppressor tRNAs. This effect is solely due to the absence of pseudouridine 38 or 39 in tRNA because the inactive mutant protein Pus3[D151A]p did not restore the level of natural readthrough. Our results also show that absence of pseudouridine 39 in the slippery tRNA(UAG)(Leu) reduces +1 frameshift efficiency. Therefore, the presence of pseudouridine 38 or 39 in the tRNA anticodon arm enhances misreading of certain codons by natural nonsense tRNAs as well as promotes frameshifting on slippery sequences in yeast.     

Frameshifting at the internal stop codon within the mRNA for bacterial release factor-2 on eukaryotic ribosomes

A translational frameshift is necessary in the synthesis of Escherichia coli release factor 2 (RF-2) to bypass an in-frame termination codon within the coding sequence. High-efficiency frameshifting around this codon can occur on eukaryotic ribosomes as well as prokaryotic ribosomes. This was determined from the relative efficiency of translation of RF-2 RNA compared with that for the other release factor RF-1, which lacks the in-frame premature stop codon. Since the termination product is unstable an absolute measure of the efficiency of frameshifting has not been possible. A gene fusion between trpE and RF-2 was carried out to give a stable termination product as well as the frameshift product, thereby allowing a direct determination of frameshifting efficiency. The extension of RF-2 RNA near its start codon with a fragment of the trpE gene, while still allowing high efficiency frameshifting on prokaryotic ribosomes, surprisingly gives a different estimate of frameshifting on the eukaryotic ribosomes than that obtained with RF-2 RNA alone. This paradox may be explained by long distance context effects on translation rates in the frameshift region created by the trpE sequences in the gene fusion, and may reflect that pausing and translation rate are fundamental factors in determining the efficiency of frameshifting.     

Reprogrammed genetic decoding in cellular gene expression

Reprogrammed genetic decoding signals in mRNAs productively overwrite the normal decoding rules of translation. These "recoding" signals are associated with sites of programmed ribosomal frameshifting, hopping, termination codon suppression, and the incorporation of the unusual amino acids selenocysteine and pyrrolysine. This review summarizes current knowledge of the structure and function of recoding signals in cellular genes, the biological importance of recoding in gene regulation, and ways to identify new recoded genes.     

The Pokeweed Antiviral Protein Specifically Inhibits Ty1-Directed +1 Ribosomal Frameshifting and Retrotransposition in Saccharomyces cerevisiae

Programmed ribosomal frameshifting is a molecular mechanism that is used by many RNA viruses to produce Gag-Pol fusion proteins. The efficiency of these frameshift events determines the ratio of viral Gag to Gag-Pol proteins available for viral particle morphogenesis, and changes in ribosomal frameshift efficiencies can severely inhibit virus propagation. Since ribosomal frameshifting occurs during the elongation phase of protein translation, it is reasonable to hypothesize that agents that affect the different steps in this process may also have an impact on programmed ribosomal frameshifting. We examined the molecular mechanisms governing programmed ribosomal frameshifting by using two viruses of the yeast Saccharomyces cerevisiae. Here, we present evidence that pokeweed antiviral protein (PAP), a single-chain ribosomal inhibitory protein that depurinates an adenine residue in the α-sarcin loop of 25S rRNA and inhibits translocation, specifically inhibits Ty1-directed +1 ribosomal frameshifting in intact yeast cells and in an in vitro assay system. Using an in vivo assay for Ty1 retrotransposition, we show that PAP specifically inhibits Ty1 retrotransposition, suggesting that Ty1 viral particle morphogenesis is inhibited in infected cells. PAP does not affect programmed −1 ribosomal frameshift efficiencies, nor does it have a noticeable impact on the ability of cells to maintain the M₁-dependent killer virus phenotype, suggesting that −1 ribosomal frameshifting does not occur after the peptidyltransferase reaction. These results provide the first evidence that PAP has viral RNA-specific effects in vivo which may be responsible for the mechanism of its antiviral activity.