Tissue-specific dynamic codon redefinition in .

Translational stop codon readthrough occurs in organisms ranging from viruses to mammals and is especially prevalent in decoding and viral mRNAs. Recoding of UGA, UAG, or UAA to specify an amino acid allows a proportion of the protein encoded by a single gene to be C-terminally extended. The extended product from mRNA is 160 kDa, whereas unextended Kelch protein, a subunit of a Cullin3-RING ubiquitin ligase, is 76 kDa.

Polysomes Bypass a 50-Nucleotide Coding Gap Less Efficiently Than Monosomes Due to Attenuation of a 5' mRNA Stem-Loop and Enhanced Drop-off.

Efficient translational bypassing of a 50-nt non-coding gap in a phage T4 topoisomerase subunit gene (gp60) requires several recoding signals. Here we investigate the function of the mRNA stem-loop 5' of the take-off codon, as well as the importance of ribosome loading density on the mRNA for efficient bypassing. We show that polysomes are less efficient at mediating bypassing than monosomes, both in vitro and in vivo, due to their preventing formation of a stem-loop 5' of the take-off codon and allowing greater peptidyl-tRNA drop off.

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.

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.

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.

Stimulation of stop codon readthrough: frequent presence of an extended 3' RNA structural element.

In Sindbis, Venezuelan equine encephalitis and related alphaviruses, the polymerase is translated as a fusion with other non-structural proteins via readthrough of a UGA stop codon. Surprisingly, earlier work reported that the signal for efficient readthrough comprises a single cytidine residue 3'-adjacent to the UGA. However, analysis of variability at synonymous sites revealed strikingly enhanced conservation within the ∼ 150 nt 3'-adjacent to the UGA, and RNA folding algorithms revealed the potential for a phylogenetically conserved stem-loop structure in the same region.

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.

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).

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.

NS1' of flaviviruses in the Japanese encephalitis virus serogroup is a product of ribosomal frameshifting and plays a role in viral neuroinvasiveness.

Flavivirus NS1 is a nonstructural protein involved in virus replication and regulation of the innate immune response. Interestingly, a larger NS1-related protein, NS1', is often detected during infection with the members of the Japanese encephalitis virus serogroup of flaviviruses. However, how NS1' is made and what role it performs in the viral life cycle have not been determined.

Translational bypassing without peptidyl-tRNA anticodon scanning of coding gap mRNA.

Half the ribosomes translating the mRNA for phage T4 gene 60 topoisomerase subunit bypass a 50 nucleotide coding gap between codons 46 and 47. The pairing of codon 46 with its cognate peptidyl-tRNA anticodon dissociates, and following mRNA slippage, peptidyl-tRNA re-pairs to mRNA at a matched triplet 5' adjacent to codon 47, where translation resumes. Here, in studies with gene 60 cassettes, it is shown that the peptidyl-tRNA anticodon does not scan the intervening sequence for potential complementarity.

A case for "StopGo": reprogramming translation to augment codon meaning of GGN by promoting unconventional termination (Stop) after addition of glycine and then allowing continued translation (Go).

When a eukaryotic mRNA sequence specifying an amino acid motif known as 2A is directly followed by a proline codon, two nonoverlapping proteins are synthesized. From earlier work, the second protein is known to start with this proline codon and is not created by proteolysis. Here we identify the C-terminal amino acid of an upstream 2A-encoded product from Perina nuda picorna-like virus that is glycine specified by the last codon of the 2A-encoding sequence.

The potential role of ribosomal frameshifting in generating aberrant proteins implicated in neurodegenerative diseases.

Aberrant forms of proteins ubiquitin B and beta-amyloid precusor protein, UBB+1 and APP+1, are implicated in human neurodegenerative diseases. They have their carboxyl-terminal regions derived from an alternative reading frame. Transcription slippage has been invoked to explain the production of these proteins from abnormal mRNA.

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.

P-site pairing subtleties revealed by the effects of different tRNAs on programmed translational bypassing where anticodon re-pairing to mRNA is separated from dissociation.

Programmed ribosomal bypassing occurs in decoding phage T4 gene 60 mRNA. Half the ribosomes bypass a 50 nucleotide gap between codons 46 and 47. Peptidyl-tRNA dissociates from the "take-off" GGA, codon 46, and re-pairs to mRNA at a matched GGA "landing site" codon directly 5' of codon 47 where translation resumes.

-1 frameshifting at a CGA AAG hexanucleotide site is required for transposition of insertion sequence IS1222.

The discovery of programmed -1 frameshifting at the hexanucleotide shift site CGA_AAG, in addition to the classical X_XXY_YYZ heptanucleotide shift sequences, prompted a search for instances among eubacterial insertion sequence elements. IS1222 has a CGA_AAG shift site. A genetic analysis revealed that frameshifting at this site is required for transposition.

Factors that influence selection of coding resumption sites in translational bypassing: minimal conventional peptidyl-tRNA:mRNA pairing can suffice.

This study investigates bypassing initiated from codons immediately 5' of a stop codon. The mRNA slips and is scanned by the peptidyl-tRNA for a suitable landing site, and standard decoding resumes at the next 3' codon. This work shows that landing sites with potentially strong base pairing between the peptidyl-tRNA anticodon and mRNA are preferred, but sites with little or no potential for Watson-Crick or wobble base pairing can also be utilized.

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.