Programmed +1 frameshifting stimulated by complementarity between a downstream mRNA sequence and an error-correcting region of rRNA

Like most retroviruses and retrotransposons, the retrotransposon Ty3 expresses its pol gene analog (POL3) as a translational fusion to the upstream gag analog (GAG3). The Gag3-Pol3 fusion occurs by frameshifting during translation of the mRNA that encodes the two separate but overlapping ORFs. We showed previously that the shift occurs by out-of-frame binding of a normal aminoacyl-tRNA in the ribosomal A site caused by an aberrant codonoanticodon interaction in the P site. This event is unlike all previously described programmed translational frameshifts because it does not require tRNA slippage between cognate or near-cognate codons in the mRNA. A sequence of 15 nt distal to the frameshift site stimulates frameshifting 7.5-fold. Here we show that the Ty3 stimulator acts as an unstructured region to stimulate frameshifting. Its function depends on strict spacing from the site of frameshifting. Finally, the stimulator increases frameshifting dependent on sense codon-induced pausing, but has no effect on frameshifting dependent on pauses induced by nonsense codons. Complementarity between the stimulator and a portion of the accuracy center of the ribosome, Helix 18, implies that the stimulator may directly disrupt error correction by the ribosome.     

Identification of a new antizyme mRNA +1 frameshifting stimulatory pseudoknot in a subset of diverse invertebrates and its apparent absence in intermediate species

The expression of eukaryotic antizyme genes requires +1 translational frameshifting. The frameshift in decoding most vertebrate antizyme mRNAs is stimulated by an RNA pseudoknot 3' of the frameshift site. Although the frameshifting event itself is conserved in a wide variety of organisms from yeast to mammals, until recently no corresponding 3' RNA pseudoknot was known in invertebrate antizyme mRNAs. A pseudoknot, different in structure and origin from its vertebrate counterparts, is now shown to be encoded by the antizyme genes of distantly related invertebrates. Identification of the 3' frameshifting stimulator in intermediate species or other invertebrates remains unresolved.     

Evolutionary specialization of recoding: frameshifting in the expression of S. cerevisiae antizyme mRNA is via an atypical antizyme shift site but is still +1

An autoregulatory translational shift to the +1 frame is required for the expression of ornithine decarboxylase antizyme from fungi to mammals. In most eukaryotes, including all vertebrates and a majority of the studied fungi/yeast, the site on antizyme mRNA where the shift occurs is UCC-UGA. The mechanism of the frameshift on this sequence likely involves nearly universal aspects of the eukaryotic translational machinery. Nevertheless, a mammalian antizyme frameshift cassette yields predominantly -2 frameshift in Saccharomyces cerevisiae, instead of the +1 in mammals. The recently identified endogenous S. cerevisiae antizyme mRNA has an atypical shift site: UGC-GCG-UGA. It is shown here that endogenous S. cerevisiae antizyme frameshifting is +1 rather than -2. We discuss how antizyme frameshifting in budding yeasts exploits peculiarities of their tRNA balance, and relate this to prior studies on Ty frameshifting.     

The interplay of mRNA stimulatory signals required for AUU-mediated initiation and programmed -1 ribosomal frameshifting in decoding of transposable element IS911

The IS911 bacterial transposable element uses -1 programmed translational frameshifting to generate the protein required for its mobility: translation initiated in one gene (orfA) shifts to the -1 frame and continues in a second overlapping gene (orfB), thus generating the OrfAB transposase. The A-AAA-AAG frameshift site of IS911 is flanked by two stimulatory elements, an upstream Shine-Dalgarno sequence and a downstream stem-loop. We show here that, while they can act independently, these stimulators have a synergistic effect when combined. Mutagenic analyses revealed features of the complex stem-loop that make it a low-efficiency stimulator. They also revealed the dual role of the upstream Shine-Dalgarno sequence as (i) a stimulator of frameshifting, by itself more potent than the stem-loop, and (ii) a mandatory determinant of initiation of OrfB protein synthesis on an AUU codon directly preceding the A6G motif. Both roles rely on transient base pairing of the Shine-Dalgarno sequence with the 3' end of 16S rRNA. Because of its effect on frameshifting, the Shine-Dalgarno sequence is an important determinant of the level of transposase in IS911-containing cells, and hence of the frequency of transposition.     

Pulling the ribosome out of frame by +1 at a programmed frameshift site by cognate binding of aminoacyl-tRNA.

Programmed translational frameshifts efficiently alter a translational reading frame by shifting the reading frame during translation. A +1 frameshift has two simultaneous requirements: a translational pause which occurs when either an inefficiently recognized sense or termination codon occupies the A site, and the presence of a special peptidyl-tRNA occupying the P site during the pause. The special nature of the peptidyl-tRNA reflects its ability to slip +1 on the mRNA or to facilitate binding of an incoming aminoacyl-tRNA out of frame in the A site. This second mechanism suggested that in some cases the first +1 frame tRNA could have an active role in frameshifting. We found that overproducing this tRNA can drive frameshifting, surprisingly regardless of whether frameshifting occurs by peptidyl-tRNA slippage or out-of-frame binding of aminoacyl-tRNA. This finding suggests that in both cases, the shift in reading frame occurs coincident with formation of a cognate codon-anticodon interaction in the shifted frame.     

Efficient stimulation of site-specific ribosome frameshifting by antisense oligonucleotides

Evidence is presented that morpholino, 2'-O-methyl, phosphorothioate, and RNA antisense oligonucleotides can direct site-specific -1 translational frameshifting when annealed to mRNA downstream from sequences where the P- and A-site tRNAs are both capable of repairing with -1 frame codons. The efficiency of ribosomes shifting into the new frame can be as high as 40%, determined by the sequence of the frameshift site, as well as the location, sequence composition, and modification of the antisense oligonucleotide. These results demonstrate that a perfect duplex formed by complementary oligonucleotides is sufficient to induce high level -1 frameshifting. The implications for the mechanism of action of natural programmed translational frameshift stimulators are discussed.     

Novel Gag-Pol Frameshift Site in Human Immunodeficiency Virus Type 1 Variants Resistant to Protease Inhibitors

Human immunodeficiency virus type 1 (HIV-1) variants resistant to protease inhibitors have been shown to contain a mutation in the p1/p6 Gag precursor cleavage site. At the messenger RNA level, this mutation generates a U UUU UUU sequence that is reminiscent of the U UUU UUA sequence required for ribosomal frameshifting and Gag-Pol synthesis. To test whether the p1/p6 cleavage site mutation was generating a novel frameshift site, HIV sequences were inserted in translation vectors containing a chloramphenicol acetyltransferase (CAT) reporter gene requiring −1 frameshifting for expression. All sequences containing the original HIV frameshift site supported the synthesis of CAT but expression was increased 3- to 11-fold in the presence of the mutant p1/p6 sequence. When the original frameshift site was abolished by mutation, expression remained unchanged when using constructs containing the mutant p1/p6 sequence, whereas it was decreased 2- to 4.5-fold when using wild-type p1/p6 constructs. Similarly, when introduced into HIV molecular clones, the p1/p6 mutant sequence supported Gag-Pol synthesis and protease activity in the absence of the original frameshift site, indicating that this sequence could also promote ribosomal frameshifting in virus-expressing cells.     

Special peptidyl-tRNA molecules can promote translational frameshifting without slippage.

Recently we described an unusual programmed +1 frameshift event in yeast retrotransposon Ty3. Frameshifting depends on the presence of peptidyl-tRNA(AlaCGC) on the GCG codon in the ribosomal P site and on a translational pause stimulated by the slowly decoded AGU codon. Frameshifting occurs on the sequence GCG-AGU-U by out-of-frame binding of a valyl-tRNA to GUU without slippage of peptidyl-tRNA(AlaCGC). This mechanism challenges the conventional understanding that frameshift efficiency must correlate with the ability of mRNA-bound tRNA to slip between cognate or near-cognate codons. Though frameshifting does not require slippery tRNAs, it does require special peptidyl-tRNAs. We show that overproducing a second isoacceptor whose anticodon had been changed to CGC eliminated frameshifting; peptidyl-tRNA(AlaCGC) must have a special capacity to induce +1 frameshifting in the adjacent ribosomal A site. In order to identify other special peptidyl-tRNAs, we tested the ability of each of the other 63 codons to replace GCG in the P site. We found no correlation between the ability to stimulate +1 frameshifting and the ability of the cognate tRNA to slip on the mRNA--several codons predicted to slip efficiently do not stimulate frameshifting, while several predicted not to slip do stimulate frameshifting. By inducing a severe translational pause, we identified eight tRNAs capable of inducing measurable +1 frameshifting, only four of which are predicted to slip on the mRNA. We conclude that in Saccharomyces cerevisiae, special peptidyl-tRNAs can induce frameshifting dependent on some characteristic(s) other than the ability to slip on the mRNA.     

Analyses of frameshifting at UUU-pyrimidine sites.

Others have recently shown that the UUU phenylalanine codon is highly frameshift-prone in the 3'(rightward) direction at pyrimidine 3'contexts. Here, several approaches are used to analyze frameshifting at such sites. The four permutations of the UUU/C (phenylalanine) and CGG/U (arginine) codon pairs were examined because they vary greatly in their expected frameshifting tendencies. Furthermore, these synonymous sites allow direct tests of the idea that codon usage can control frameshifting. Frameshifting was measured for these dicodons embedded within each of two broader contexts: the Escherichia coli prfB (RF2 gene) programmed frameshift site and a 'normal' message site. The principal difference between these contexts is that the programmed frameshift contains a purine-rich sequence upstream of the slippery site that can base pair with the 3'end of 16 S rRNA (the anti-Shine-Dalgarno) to enhance frameshifting. In both contexts frameshift frequencies are highest if the slippery tRNAPhe is capable of stable base pairing in the shifted reading frame. This requirement is less stringent in the RF2 context, as if the Shine-Dalgarno interaction can help stabilize a quasi-stable rephased tRNA:message complex. It was previously shown that frameshifting in RF2 occurs more frequently if the codon 3'to the slippery site is read by a rare tRNA. Consistent with that earlier work, in the RF2 context frameshifting occurs substantially more frequently if the arginine codon is CGG, which is read by a rare tRNA. In contrast, in the 'normal' context frameshifting is only slightly greater at CGG than at CGU. It is suggested that the Shine-Dalgarno-like interaction elevates frameshifting specifically during the pause prior to translation of the second codon, which makes frameshifting exquisitely sensitive to the rate of translation of that codon. In both contexts frameshifting increases in a mutant strain that fails to modify tRNA base A37, which is 3'of the anticodon. Thus, those base modifications may limit frameshifting at UUU codons. Finally, statistical analyses show that UUU Ynn dicodons are extremely rare in E.coli genes that have highly biased codon usage.     

Frameshift autoregulation in the gene for Escherichia coli release factor 2: partly functional mutants result in frameshift enhancement.

The regulation of release factor 2 (RF-2) synthesis in Escherichia coli occurs, at least in part, through autoregulatory feedback exerted at a unique frameshifting step required during RF-2 translation. We have constructed fusions between the genes for RF-2 and E. coli trpE which make direct measurement of frameshifting efficiency possible since both products of regulation, the termination product and the frameshift product, are stable. The addition of purified RF-2 to in vitro expressions of these fusion genes was found to result in decreased frameshifting and increased termination at the regulation site. The frame-shifted trpE-RF-2 products synthesized from these fusions are unique with respect to their functional release factor activities; when tested in assays of two intermediate steps of translational termination, they were found to be partially active for the function of ribosome binding, but inactive for peptidyl-tRNA hydrolysis (release). These are the first examples of release factor mutants selectively active for only one of these function. In vivo these chimeric proteins promote large increases in frameshifting at the RF-2 frameshift region, thereby reversing normal negative autoregulatory feedback and instead supporting fully efficient frameshifting in their own synthesis. This activity provides new evidence for the importance of ribosomal pausing in directing efficient frameshifting at the RF-2 frameshift region.     

Identification of the -1 translational frameshift sites using a liquid chromatography-tandem mass spectrometric approach

Translational frameshifting, a ubiquitous mechanism used to produce alternative proteins for different biological purposes, appears in a variety of genes in probably all organisms. In the past, the combinational use of sophisticated expression vectors, specific endopeptidases, and Edman degradation has been the main approach for identification of the translational frameshift sites. Although Edman degradation is highly reliable, it is also time-consuming and costly. In this article, we report a new liquid chromatography-tandem mass spectrometric (LC-MS/MS) approach for identifying the -1 translational frameshift sites. The approach consists of three steps: (i) LC-MS/MS analysis of the protein digests, (ii) primary data analysis using the known mRNA sequence, and (iii) advanced data analysis using a new database containing distinct mRNA sequences with single insertion at particular positions. We first validated our approach by analyzing the previously documented slippery sequence, A4G, from IS3. With this approach, we further determined whether the TTTTTTG (T6G) sequence of IS1372 from Streptomyces lividans had the -1 translational frameshifting potential. The identified amino acid sequence of the transframe peptide indicated that the -1 frameshifting occurred at the T6G motif, as predicted previously. The results on IS3 (A4G) and IS1372 (T6G) suggested that this approach is effective for the translational frameshifting studies.     

A Nascent Peptide Signal Responsive to Endogenous Levels of Polyamines Acts to Stimulate Regulatory Frameshifting on Antizyme mRNA

The protein antizyme is a negative regulator of cellular polyamine concentrations from yeast to mammals. Synthesis of functional antizyme requires programmed +1 ribosomal frameshifting at the 3′ end of the first of two partially overlapping ORFs. The frameshift is the sensor and effector in an autoregulatory circuit. Except for Saccharomyces cerevisiae antizyme mRNA, the frameshift site alone only supports low levels of frameshifting. The high levels usually observed depend on the presence of cis-acting stimulatory elements located 5′ and 3′ of the frameshift site. Antizyme genes from different evolutionary branches have evolved different stimulatory elements. Prior and new multiple alignments of fungal antizyme mRNA sequences from the Agaricomycetes class of Basidiomycota show a distinct pattern of conservation 5′ of the frameshift site consistent with a function at the amino acid level. As shown here when tested in Schizosaccharomyces pombe and mammalian HEK293T cells, the 5′ part of this conserved sequence acts at the nascent peptide level to stimulate the frameshifting, without involving stalling detectable by toe-printing. However, the peptide is only part of the signal. The 3′ part of the stimulator functions largely independently and acts at least mostly at the nucleotide level. When polyamine levels were varied, the stimulatory effect was seen to be especially responsive in the endogenous polyamine concentration range, and this effect may be more general. A conserved RNA secondary structure 3′ of the frameshift site has weaker stimulatory and polyamine sensitizing effects on frameshifting.     

Evolution of +1 Programmed Frameshifting Signals and Frameshift-Regulating tRNAs in the Order Saccharomycetales

Programmed translational frameshifting is a ubiquitous but rare mechanism of gene expression in which mRNA sequences cause the translational machinery to shift reading frames with extreme efficiency, up to at least 50%. The mRNA sequences responsible are deceptively simple; the sequence CUU-AGG-C causes about 40% frameshifting when inserted into an mRNA in the yeast Saccharomyces cerevisiae. The high efficiency of this site depends on a set of S. cerevisiae tRNA isoacceptors that perturb the mechanism of translation to cause the programmed translational error. The simplicity of the system might suggest that it could evolve frequently and perhaps be lost as easily. We have investigated the history of programmed +1 frameshifting in fungi. We find that frameshifting has persisted in two structural genes in budding yeasts, ABP140 and EST3 for about 150 million years. Further, the tRNAs that stimulate the event are equally old. Species that diverged from the lineage earlier both do not employ frameshifting and have a different complement of tRNAs predicted to be inimical to frameshifting. The stability of the coevolution of protein coding genes and tRNAs suggests that frameshifting has been selected for during the divergence of these species. [Reviewing Editor: Dr. Niles Lehman]     

The structure of the frameshift stimulatory signal in HIV-1 RNA: a potential target for the treatment of patients infected with HIV

Ribosomal frameshift is used by HIV-1 to synthesize the precursor of its enzymes. The frameshift stimulator is a peculiar structure in the viral messenger RNA coding for this precursor, which increases the probability that this frameshift occurs. It was proposed to be either a triplex structure or an irregular stem-loop. Recently, two NMR groups independently showed that the frameshift stimulatory signal of HIV-1 is an extended stem-loop, with an internal three-purine bulge separating two helical regions. However, it remains unclear how such a structure promotes frameshifting. It is proposed that frameshifting results from a specific interaction between the stimulatory signal and either a hypothetical protein factor or the ribosome. The characterization of the structure of the frameshift stimulatory signal paves the way to the rational design of novel antiviral drugs, which, by binding to this signal, could interfere with frameshifting and viral replication.     

A functional -1 ribosomal frameshift signal in the human paraneoplastic Ma3 gene

A bioinformatics approach to finding new cases of -1 frameshifting in the expression of human genes revealed a classical retrovirus-like heptanucleotide shift site followed by a potential structural stimulator in the paraneoplastic antigen Ma3 and Ma5 genes. Analysis of the sequence 3' of the shift site demonstrated that an RNA pseudoknot in Ma3 is important for promoting efficient -1 frame-shifting. Ma3 is a member of a family of six genes in humans whose protein products contain homology to retroviral Gag proteins. The -1 frameshift site and pseudoknot structure are conserved in other mammals, but there are some sequence differences. Although the functions of the Ma genes are unknown, the serious neurological effects of ectopic expression in tumor cells indicate their importance in the brain.     

Characterization of ribosomal frameshifting for expression of pol gene products of human T-cell leukemia virus type I.

For study of the pol gene expression of human T-cell leukemia virus type I (HTLV-I), RNA was transcribed in vitro from proviral DNA and translated in rabbit reticulocyte lysates. This cell-free translation resulted in two major translation products representing the Gag and Gag-Pro polyproteins. By contrast, the Gag-Pro-Pol polyprotein could be readily observed only when translation was performed with mutant mRNA in which the protease (pro) reading frame was aligned to gag to eliminate the frameshifting event in the gag-pro overlap. The results indicated that two independent ribosomal frameshifting events are required for expression of the HTLV-I pol gene product. Studies with mutant DNAs facilitated the characterization of the primary structure of the HTLV-I mRNA responsible for the ribosomal frameshift in the pro-pol overlap and demonstrated that the frameshift occurs at the signal sequence UUUAAAC. Direct amino acid sequencing of the transframe protein localized the site of the frameshift to the asparagine codon AAC.     

Prokaryotic ribosomes recode the HIV-1 gag-pol-1 frameshift sequence by an E/P site post-translocation simultaneous slippage mechanism.

The mechanism favoured for -1 frameshifting at typical retroviral sites is a pre-translocation simultaneous slippage model. An alternative post-translocation mechanism would also generate the same protein sequence across the frameshift site and therefore in this study the strategic placement of a stop codon has been used to distinguish between the two mechanisms. A 26 base pair frameshift sequence from the HIV-1 gag-pol overlap has been modified to include a stop codon immediately 3' to the heptanucleotide frameshift signal, where it often occurs naturally in retroviral recoding sites. Stop codons at the 3'-end of the heptanucleotide sequence decreased the frame-shifting efficiency on prokaryote ribosomes and the recording event was further depressed when the levels of the release factors in vivo were increased. In the presence of elevated levels of a defective release factor 2, frameshifting efficiency in vivo was increased in the constructs containing the stop codons recognized specifically by that release factor. These results are consistent with the last six nucleotides of the heptanucleotide slippery sequence occupying the ribosomal E and P sites, rather than the P and A sites, with the next codon occupying the A site and therefore with a post-translocation rather than a pre-translocation -1 slippage model.