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.

High-efficiency translational bypassing of non-coding nucleotides specified by mRNA structure and nascent peptide.

The gene product 60 (gp60) of bacteriophage T4 is synthesized as a single polypeptide chain from a discontinuous reading frame as a result of bypassing of a non-coding mRNA region of 50 nucleotides by the ribosome. To identify the minimum set of signals required for bypassing, we recapitulated efficient translational bypassing in an in vitro reconstituted translation system from Escherichia coli. We find that the signals, which promote efficient and accurate bypassing, are specified by the gene 60 mRNA sequence. Systematic analysis of the mRNA suggests unexpected contributions of sequences upstream and downstream of the non-coding gap region as well as of the nascent peptide. During bypassing, ribosomes glide forward on the mRNA track in a processive way. Gliding may have a role not only for gp60 synthesis, but also during regular mRNA translation for reading frame selection during initiation or tRNA translocation during elongation.

Programmed ribosomal frameshifting in the expression of the regulator of intestinal stem cell proliferation, adenomatous polyposis coli (APC).

A programmed ribosomal frameshift (PRF) in the decoding of APC (adenomatous polyposis coli) mRNA has been identified and characterized in Caenorhabditis worms, Drosophila and mosquitoes. The frameshift product lacks the C-terminal approximately one-third of the product of standard decoding and instead has a short sequence encoded by the -1 frame which is just 13 residues in C. elegans, but is 125 in D. melanogaster. The frameshift site is A_AA.A_AA.C in Caenorhabditids, fruit flies and the mosquitoes studied while a variant A_AA.A_AA.A is found in some other nematodes. The predicted secondary RNA structure of the downstream stimulators varies considerably in the species studied. In the twelve sequenced Drosophila genomes, it is a long stem with a four-way junction in its loop. In the five sequenced Caenorhabditis species, it is a short RNA pseudoknot with an additional stem in loop 1. The efficiency of frameshifting varies significantly, depending on the particular stimulator within the frameshift cassette, when tested with reporter constructs in rabbit reticulocyte lysates. Phylogenetic analysis of the distribution of APC programmed ribosomal frameshifting cassettes suggests it has an ancient origin and raises questions about a possibility of synthesis of alternative protein products during expression of APC in other organisms such as humans. The origin of APC as a PRF candidate emerged from a prior study of evolutionary signatures derived from comparative analysis of the 12 fly genomes. Three other proposed PRF candidates (Xbp1, CG32736, CG14047) with switches in conservation of reading frames are likely explained by mechanisms other than PRF.

Discovery of a small arterivirus gene that overlaps the GP5 coding sequence and is important for virus production.

The arterivirus family (order Nidovirales) of single-stranded, positive-sense RNA viruses includes porcine respiratory and reproductive syndrome virus and equine arteritis virus (EAV). Their replicative enzymes are translated from their genomic RNA, while their seven structural proteins are encoded by a set of small, partially overlapping genes in the genomic 3'-proximal region. The latter are expressed via synthesis of a set of subgenomic mRNAs that, in general, are functionally monocistronic (except for a bicistronic mRNA encoding the E and GP2 proteins). ORF5, which encodes the major glycoprotein GP5, has been used extensively for phylogenetic analyses. However, an in-depth computational analysis now reveals the arterivirus-wide conservation of an additional AUG-initiated ORF, here termed ORF5a, that overlaps the 5' end of ORF5. The pattern of substitutions across sequence alignments indicated that ORF5a is subject to functional constraints at the amino acid level, while an analysis of substitutions at synonymous sites in ORF5 revealed a greatly reduced frequency of substitution in the portion of ORF5 that is overlapped by ORF5a. The 43-64 aa ORF5a protein and GP5 are probably expressed from the same subgenomic mRNA, via a translation initiation mechanism involving leaky ribosomal scanning. Inactivation of ORF5a expression by reverse genetics yielded a severely crippled EAV mutant, which displayed lower titres and a tiny plaque phenotype. These defects, which could be partially complemented in ORF5a-expressing cells, indicate that the novel protein, which may be the eighth structural protein of arteriviruses, is expressed and important for arterivirus infection.

Evidence for ribosomal frameshifting and a novel overlapping gene in the genomes of insect-specific flaviviruses.

Flaviviruses have a positive-sense, single-stranded RNA genome of approximately 11 kb, encoding a large polyprotein that is cleaved to produce approximately 10 mature proteins. Cell fusing agent virus, Kamiti River virus, Culex flavivirus and several recently discovered flaviviruses have no known vertebrate host and apparently infect only insects. We present compelling bioinformatic evidence for a 253-295 codon overlapping gene (designated fifo) conserved throughout these insect-specific flaviviruses and immunofluorescent detection of its product. Fifo overlaps the NS2A/NS2B coding sequence in the -1/+2 reading frame and is most likely expressed as a trans-frame fusion protein via ribosomal frameshifting at a conserved GGAUUUY slippery heptanucleotide with 3'-adjacent RNA secondary structure (which stimulates efficient frameshifting in vitro). The discovery bears striking parallels to the recently discovered ribosomal frameshifting site in the NS2A coding sequence of the Japanese encephalitis serogroup of flaviviruses and suggests that programmed ribosomal frameshifting may be more widespread in flaviviruses than currently realized.

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.

Comparative studies of frameshifting and nonframeshifting RNA pseudoknots: a mutational and NMR investigation of pseudoknots derived from the bacteriophage T2 gene 32 mRNA and the retroviral gag-pro frameshift site.

Mutational and NMR methods were used to investigate features of sequence, structure, and dynamics that are associated with the ability of a pseudoknot to stimulate a -1 frameshift. In vitro frameshift assays were performed on retroviral gag-pro frameshift-stimulating pseudoknots and their derivatives, a pseudoknot from the gene 32 mRNA of bacteriophage T2 that is not naturally associated with frameshifting, and hybrids of these pseudoknots. Results show that the gag-pro pseudoknot from human endogenous retrovirus-K10 (HERV) stimulates a -1 frameshift with an efficiency similar to that of the closely related retrovirus MMTV. The bacteriophage T2 mRNA pseudoknot was found to be a poor stimulator of frameshifting, supporting a hypothesis that the retroviral pseudoknots have distinctive properties that make them efficient frameshift stimulators. A hybrid, designed by combining features of the bacteriophage and retroviral pseudoknots, was found to stimulate frameshifting while retaining significant structural similarity to the nonframeshifting bacteriophage pseudoknot. Mutational analyses of the retroviral and hybrid pseudoknots were used to evaluate the effects of an unpaired (wedged) adenosine at the junction of the pseudoknot stems, changing the base pairs near the junction of the two stems, and changing the identity of the loop 2 nucleotide nearest the junction of the stems. Pseudoknots both with and without the wedged adenosine can stimulate frameshifting, though the identities of the nucleotides near the stem1/stem2 junction do influence efficiency. NMR data showed that the bacteriophage and hybrid pseudoknots are similar in their local structure at the junction of the stems, indicating that pseudoknots that are similar in this structural feature can differ radically in their ability to stimulate frameshifting. NMR methods were used to compare the internal motions of the bacteriophage T2 pseudoknot and representative frameshifting pseudoknots. The stems of the investigated pseudoknots are similarly well ordered on the time scales to which nitrogen-15 relaxation data are sensitive; however, solvent exchange rates for protons at the junction of the two stems of the nonframeshifting bacteriophage pseudoknot are significantly slower than the analogous protons in the representative frameshifting pseudoknots.