Ribosomal frameshifting in decoding antizyme mRNAs from yeast and protists to humans: close to 300 cases reveal remarkable diversity despite underlying conservation

The protein antizyme is a negative regulator of intracellular polyamine levels. Ribosomes synthesizing antizyme start in one ORF and at the codon 5′ adjacent to its stop codon, shift +1 to a second and partially overlapping ORF which encodes most of the protein. The ribosomal frameshifting is a sensor and effector of an autoregulatory circuit which is conserved in animals, fungi and protists. Stimulatory signals encoded 5′ and 3′ of the shift site act to program the frameshifting. Despite overall conservation, many individual branches have evolved specific features surrounding the frameshift site. Among these are RNA pseudoknots, RNA stem-loops, conserved primary RNA sequences, nascent peptide sequences and branch-specific ‘shifty’ codons

A case for a CUG-initiated coding sequence overlapping torovirus ORF1a and encoding a novel 30 kDa product

The genus Torovirus (order Nidovirales) includes a number of species that infect livestock. These viruses have a linear positive-sense ssRNA genome of ~25-30 kb, encoding a large polyprotein that is expressed from the genomic RNA, and several additional proteins expressed from a nested set of 3'-coterminal subgenomic RNAs. In this brief report, we describe the bioinformatic discovery of a new, apparently coding, ORF that overlaps the 5' end of the polyprotein coding sequence, ORF1a, in the +2 reading frame. The new ORF has a strong coding signature and, in fact, is more conserved at the amino acid level than the overlapping region of ORF1a. We propose that the new ORF utilizes a non-AUG initiation codon - namely a conserved CUG codon in a strong Kozak context - upstream of the ORF1a AUG initiation codon, resulting in a novel 258 amino acid protein, dubbed '30K'

A conserved predicted pseudoknot in the NS2A-encoding sequence of West Nile and Japanese encephalitis flaviviruses suggests NS1' may derive from ribosomal frameshifting

Japanese encephalitis, West Nile, Usutu and Murray Valley encephalitis viruses form a tight subgroup within the larger Flavivirus genus. These viruses utilize a single-polyprotein expression strategy, resulting in ~10 mature proteins. Plotting the conservation at synonymous sites along the polyprotein coding sequence reveals strong conservation peaks at the very 5' end of the coding sequence, and also at the 5' end of the sequence encoding the NS2A protein. Such peaks are generally indicative of functionally important non-coding sequence elements. The second peak corresponds to a predicted stable pseudoknot structure whose biological importance is supported by compensatory mutations that preserve the structure. The pseudoknot is preceded by a conserved slippery heptanucleotide (Y CCU UUU), thus forming a classical stimulatory motif for -1 ribosomal frameshifting. We hypothesize, therefore, that the functional importance of the pseudoknot is to stimulate a portion of ribosomes to shift -1 nt into a short (45 codon), conserved, overlapping open reading frame, termed foo. Since cleavage at the NS1-NS2A boundary is known to require synthesis of NS2A in cis, the resulting transframe fusion protein is predicted to be NS1-NS2AN-term-FOO. We hypothesize that this may explain the origin of the previously identified NS1 'extension' protein in JEV-group flaviviruses, known as NS1'

Bioinformatic analysis suggests that the Cypovirus 1 major core protein cistron harbours an overlapping gene

Members of the genus Cypovirus (family Reoviridae) are common pathogens of insects. These viruses have linear dsRNA genomes divided into 10–11 segments, which have generally been assumed to be monocistronic. Here, bioinformatic evidence is presented for a short overlapping coding sequence (CDS) in the cypovirus genome segment encoding the major core capsid protein VP1, overlapping the 5'-terminal region of the VP1 ORF in the +1 reading frame. In Cypovirus type 1 (CPV-1), a 62-codon AUG-initiated open reading frame (hereafter ORFX) is present in all four available segment 1 sequences. The pattern of base variations across the sequence alignment indicates that ORFX is subject to functional constraints at the amino acid level (even when the constraints due to coding in the overlapping VP1 reading frame are taken into account; MLOGD software). In fact the translated ORFX shows greater amino acid conservation than the overlapping region of VP1. The genomic location of ORFX is consistent with translation via leaky scanning. A 62–64 codon AUG-initiated ORF is present in a corresponding location and reading frame in other available cypovirus sequences (2 CPV-14, 1 CPV-15) and an 87-codon ORFX homologue may also be present in Aedes pseudoscutellaris reovirus. The ORFX amino acid sequences are hydrophilic and basic, with between 12 and 16 Arg/Lys residues in each though, at 7.5–10.2 kDa, the putative ORFX product is too small to appear on typical published protein gels

Transcriptional frameshifting rescues Citrobacter rodentium Type VI secretion by the production of two length variants from the prematurely interrupted tssM gene

The Type VI secretion system (T6SS) mediates toxin delivery into both eukaryotic and prokaryotic cells. It is composed of a cytoplasmic structure resembling the tail of contractile bacteriophages anchored to the cell envelope through a membrane complex composed of the TssL and TssM inner membrane proteins and of the TssJ outer membrane lipoprotein. The C-terminal domain of TssM is required for its interaction with TssJ, and for the function of the T6SS. In Citrobacter rodentium, the tssM1 gene does not encode the C-terminal domain. However, the stop codon is preceded by a run of 11 consecutive adenosines. In this study, we demonstrate that this poly-A tract is a transcriptional slippery site that induces the incorporation of additional adenosines, leading to frameshifting, and hence the production of two TssM1 variants, including a full-length canonical protein. We show that both forms of TssM1, and the ratio between these two forms, are required for the function of the T6SS in C. rodentium. Finally, we demonstrate that the tssM gene associated with the Yersinia pseudotuberculosis T6SS-3 gene cluster is also subjected to transcriptional frameshifting

Translational autoregulation of BZW1 and BZW2 expression by modulating the stringency of start codon selection.

The efficiency of start codon selection during ribosomal scanning in eukaryotic translation initiation is influenced by the context or flanking nucleotides surrounding the AUG codon. The levels of eukaryotic translation initiation factors 1 (eIF1) and 5 (eIF5) play critical roles in controlling the stringency of translation start site selection. The basic leucine zipper and W2 domain-containing proteins 1 and 2 (BZW1 and BZW2), also known as eIF5-mimic proteins, are paralogous human proteins containing C-terminal HEAT domains that resemble the HEAT domain of eIF5. We show that translation of mRNAs encoding BZW1 and BZW2 homologs in fungi, plants and metazoans is initiated by AUG codons in conserved unfavorable initiation contexts. This conservation is reminiscent of the conserved unfavorable initiation context that enables autoregulation of EIF1. We show that overexpression of BZW1 and BZW2 proteins enhances the stringency of start site selection, and that their poor initiation codons confer autoregulation on BZW1 and BZW2 mRNA translation. We also show that overexpression of these two proteins significantly diminishes the effect of overexpressing eIF5 on stringency of start codon selection, suggesting they antagonize this function of eIF5. These results reveal a surprising role for BZW1 and BZW2 in maintaining homeostatic stringency of start codon selection, and taking into account recent biochemical, genetic and structural insights into eukaryotic initiation, suggest a model for BZW1 and BZW2 function

Pyrrolysine and Selenocysteine Use Dissimilar Decoding Strategies

Selenocysteine (Sec) and pyrrolysine (Pyl) are known as the 21st and 22nd amino acids in protein. Both are encoded by codons that normally function as stop signals. Sec specification by UGA codons requires the presence of a cis-acting selenocysteine insertion sequence (SECIS) element. Similarly, it is thought that Pyl is inserted by UAG codons with the help of a putative pyrrolysine insertion sequence (PYLIS) element. Herein, we analyzed the occurrence of Pyl-utilizing organisms, Pyl-associated genes, and Pyl-containing proteins. The Pyl trait is restricted to several microbes, and only one organism has both Pyl and Sec. We found that methanogenic archaea that utilize Pyl have few genes that contain in-frame UAG codons, and many of these are followed with nearby UAA or UGA codons. In addition, unambiguous UAG stop signals could not be identified. This bias was not observed in Sec-utilizing organisms and non-Pyl-utilizing archaea, as well as with other stop codons. These observations as well as analyses of the coding potential of UAG codons, overlapping genes, and release factor sequences suggest that UAG is not a typical stop signal in Pyl-utilizing archaea. On the other hand, searches for conserved Pyl-containing proteins revealed only four protein families, including methylamine methyltransferases and transposases. Only methylamine methyltransferases matched the Pyl trait and had conserved Pyl, suggesting that this amino acid is used primarily by these enzymes. These findings are best explained by a model wherein UAG codons may have ambiguous meaning and Pyl insertion can effectively compete with translation termination for UAG codons obviating the need for a specific PYLIS structure. Thus, Sec and Pyl follow dissimilar decoding and evolutionary strategies

Transcriptional slippage in bacteria: distribution in sequenced genomes and utilization in IS element gene expression

BACKGROUND: Transcription slippage occurs on certain patterns of repeat mononucleotides, resulting in synthesis of a heterogeneous population of mRNAs. Individual mRNA molecules within this population differ in the number of nucleotides they contain that are not specified by the template. When transcriptional slippage occurs in a coding sequence, translation of the resulting mRNAs yields more than one protein product. Except where the products of the resulting mRNAs have distinct functions, transcription slippage occurring in a coding region is expected to be disadvantageous. This probably leads to selection against most slippage-prone sequences in coding regions. RESULTS: To find a length at which such selection is evident, we analyzed the distribution of repetitive runs of A and T of different lengths in 108 bacterial genomes. This length varies significantly among different bacteria, but in a large proportion of available genomes corresponds to nine nucleotides. Comparative sequence analysis of these genomes was used to identify occurrences of 9A and 9T transcriptional slippage-prone sequences used for gene expression. CONCLUSIONS: IS element genes are the largest group found to exploit this phenomenon. A number of genes with disrupted open reading frames (ORFs) have slippage-prone sequences at which transcriptional slippage would result in uninterrupted ORF restoration at the mRNA level. The ability of such genes to encode functional full-length protein products brings into question their annotation as pseudogenes and in these cases is pertinent to the significance of the term 'authentic frameshift' frequently assigned to such genes

Transcriptional slippage in the positive-sense RNA virus family Potyviridae.

The family Potyviridae encompasses ~30% of plant viruses and is responsible for significant economic losses worldwide. Recently, a small overlapping coding sequence, termed pipo, was found to be conserved in the genomes of all potyvirids. PIPO is expressed as part of a frameshift protein, P3N-PIPO, which is essential for virus cell-to-cell movement. However, the frameshift expression mechanism has hitherto remained unknown. Here, we demonstrate that transcriptional slippage, specific to the viral RNA polymerase, results in a population of transcripts with an additional "A" inserted within a highly conserved GAAAAAA sequence, thus enabling expression of P3N-PIPO. The slippage efficiency is ~2% in Turnip mosaic virus and slippage is inhibited by mutations in the GAAAAAA sequence. While utilization of transcriptional slippage is well known in negative-sense RNA viruses such as Ebola, mumps and measles, to our knowledge this is the first report of its widespread utilization for gene expression in positive-sense RNA viruses.Work in the AEF laboratory was funded by grants from the WellcomeTrust [088789], [106207] and Biotechnology and Biological ResearchCouncil (BBSRC) [BB/J007072/1], [BB/J015652/1]. Work in the JPC laboratorywas funded by BBSRC grants [BB/J015652/1], [BB/J011762/1]. BYWC wassupported by a Sir Henry Wellcome Postdoctoral Fellowship [096082]and an EMBL long-term postdoctoral fellowship

Identification of the nature of reading frame transitions observed in prokaryotic genomes

Our goal was to identify evolutionary conserved frame transitions in protein coding regions and to uncover an underlying functional role of these structural aberrations. We used the ab initio frameshift prediction program, GeneTack, to detect reading frame transitions in 206 991 genes (fs-genes) from 1106 complete prokaryotic genomes. We grouped 102 731 fs-genes into 19 430 clusters based on sequence similarity between protein products (fs-proteins) as well as conservation of predicted position of the frameshift and its direction. We identified 4010 pseudogene clusters and 146 clusters of fs-genes apparently using recoding (local deviation from using standard genetic code) due to possessing specific sequence motifs near frameshift positions. Particularly interesting was finding of a novel type of organization of the dnaX gene, where recoding is required for synthesis of the longer subunit, tau. We selected 20 clusters of predicted recoding candidates and designed a series of genetic constructs with a reporter gene or affinity tag whose expression would require a frameshift event. Expression of the constructs in Escherichia coli demonstrated enrichment of the set of candidates with sequences that trigger genuine programmed ribosomal frameshifting; we have experimentally confirmed four new families of programmed frameshifts