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

ABSTRACT: The genus Torovirus (order Nidovirales) includes a number of species that infect livestock. These viruses have a linear positive-sense ssRNA genome of approximately 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'.

SUBMITTER: Firth AE

PROVIDER: S-EPMC2749830 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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

Firth Andrew E AE Atkins John F JF

Virology journal 20090908

The genus Torovirus (order Nidovirales) includes a number of species that infect livestock. These viruses have a linear positive-sense ssRNA genome of approximately 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, ...[more]

PMID: 19737402

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Project description:BackgroundIn eukaryotic cells, each molecule of H/ACA small nucleolar RNA (snoRNA) assembles with four evolutionarily conserved core proteins to compose a specific ribonucleoprotein particle. One of the four core components has pseudouridine synthase activity and catalyzes the conversion of a selected uridine to pseudouridine. Members of the pseudouridine synthase family are highly conserved. In addition to catalyzing pseudouridylation of target RNAs, they carry out a variety of essential functions related to ribosome biogenesis and, in mammals, to telomere maintenance. To investigate further the molecular mechanisms underlying the expression of pseudouridine synthase genes, we analyzed the transcriptional activity of the Drosophila member of this family in great detail.ResultsThe Drosophila gene for pseudouridine synthase, minifly/Nop60b (mfl), encodes two novel mRNAs ending at a downstream poly(A) site. One species is characterized only by an extended 3'-untranslated region (3'UTR), while a minor mRNA encodes a variant protein that represents the first example of an alternative subform described for any member of the family to date. The rare spliced variant is detected mainly in females and is predicted to have distinct functional properties. We also report that a cluster comprising four isoforms of a C/D box snoRNA and two highly related copies of a small ncRNA gene of unknown function is intron-encoded at the gene-variable 3'UTRs. Because this arrangement, the alternative 3' ends allow mfl not only to produce two distinct protein subforms, but also to release different ncRNAs. Intriguingly, accumulation of all these intron-encoded RNAs was found to be sex-biased and quantitatively modulated throughout development and, within the ovaries, the ncRNAs of unknown function were found not ubiquitously expressed.ConclusionOur results expand the repertoire of coding/non-coding transcripts derived from the gene encoding Drosophila pseudouridine synthase. This gene exhibits a complex and interlaced organization, and its genetic information may be expressed as different protein subforms and/or ncRNAs that may potentially contribute to its biological functions.

Project description:Retroviruses transcribe messenger RNA for the overlapping Gag and Gag-Pol polyproteins, by using a programmed -1 ribosomal frameshift which requires a slippery sequence and an immediate downstream stem-loop secondary structure, together called frameshift stimulating signal (FSS). It follows that the molecular evolution of this genomic region of HIV-1 is highly constrained, since the retroviral genome must contain a slippery sequence (sequence constraint), code appropriate peptides in reading frames 0 and 1 (coding requirements), and form a thermodynamically stable stem-loop secondary structure (structure requirement).We describe a unique computational tool, RNAsampleCDS, designed to compute the number of RNA sequences that code two (or more) peptides p,q in overlapping reading frames, that are identical (or have BLOSUM/PAM similarity that exceeds a user-specified value) to the input peptides p,q. RNAsampleCDS then samples a user-specified number of messenger RNAs that code such peptides; alternatively, RNAsampleCDS can exactly compute the position-specific scoring matrix and codon usage bias for all such RNA sequences. Our software allows the user to stipulate overlapping coding requirements for all 6 possible reading frames simultaneously, even allowing IUPAC constraints on RNA sequences and fixing GC-content. We generalize the notion of codon preference index (CPI) to overlapping reading frames, and use RNAsampleCDS to generate control sequences required in the computation of CPI. Moreover, by applying RNAsampleCDS, we are able to quantify the extent to which the overlapping coding requirement in HIV-1 [resp. HCV] contribute to the formation of the stem-loop [resp. double stem-loop] secondary structure known as the frameshift stimulating signal. Using our software, we confirm that certain experimentally determined deleterious HCV mutations occur in positions for which our software RNAsampleCDS and RNAiFold both indicate a single possible nucleotide. We generalize the notion of codon preference index (CPI) to overlapping coding regions, and use RNAsampleCDS to generate control sequences required in the computation of CPI for the Gag-Pol overlapping coding region of HIV-1. These applications show that RNAsampleCDS constitutes a unique tool in the software arsenal now available to evolutionary biologists.Source code for the programs and additional data are available at http://bioinformatics.bc.edu/clotelab/RNAsampleCDS/ .

Dataset Information

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

Publications

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

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