One Gene and Two Proteins: a Leaderless mRNA Supports the Translation of a Shorter Form of the
            <i>Shigella</i>
            VirF Regulator

Martino, Maria Letizia Di; Romilly, Cédric; Wagner, E. Gerhart H.; Colonna, Bianca; Prosseda, Gianni

doi:10.1128/mbio.01860-16

Cited by 33 publications

(43 citation statements)

References 46 publications

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“…Nevertheless, the actual production of proteins derived from intraRNAs has been little explored. Coding potential of intraRNAs have been explicitly suggested in internal open reading frames (intraORFs) and detection of peptides attributed to them has been observed in Caulobacter crescentus [15], Bradyrhizobium japonicum [16] and Shigella flexneri [17]. …”

Section: Introductionmentioning

confidence: 99%

Internal RNAs overlapping coding sequences can drive the production of alternative proteins in archaea

et al. 2018

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Prokaryotic genomes show a high level of information compaction often with different molecules transcribed from the same locus. Although antisense RNAs have been relatively well studied, RNAs in the same strand, internal RNAs (intraRNAs), are still poorly understood. The question of how common is the translation of overlapping reading frames remains open. We address this question in the model archaeon Halobacterium salinarum. In the present work we used differential RNA-seq (dRNA-seq) in H. salinarum NRC-1 to locate intraRNA signals in subsets of internal transcription start sites (iTSS) and establish the open reading frames associated to them (intraORFs). Using C-terminally flagged proteins, we experimentally observed isoforms accurately predicted by intraRNA translation for kef1, acs3 and orc4 genes. We also recovered from the literature and mass spectrometry databases several instances of protein isoforms consistent with intraRNA translation such as the gas vesicle protein gene gvpC1. We found evidence for intraRNAs in horizontally transferred genes such as the chaperone dnaK and the aerobic respiration related cydA in both H. salinarum and Escherichia coli. Also, intraRNA translation evidence in H. salinarum, E. coli and yeast of a universal elongation factor (aEF-2, fusA and eEF-2) suggests that this is an ancient phenomenon present in all domains of life.

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Section: Introductionmentioning

confidence: 99%

Internal RNAs overlapping coding sequences can drive the production of alternative proteins in archaea

et al. 2018

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“…Nevertheless, native protein expression could not be investigated immunologically and natural occurrence of the protein Pop remains unclear. Pop might be stable, degraded or exist in different isoforms, a phenomenon reported for some bacterial proteins recently (Waters et al, 2011;Di Martino et al, 2016;Nakahigashi et al, 2016;Vanderhaeghen et al, 2018;Meydan et al, 2019).…”

Section: Discussionmentioning

confidence: 87%

A Novel pH-Regulated, Unusual 603 bp Overlapping Protein Coding Gene pop Is Encoded Antisense to ompA in Escherichia coli O157:H7 (EHEC)

et al. 2020

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Antisense transcription is well known in bacteria. However, translation of antisense RNAs is typically not considered, as the implied overlapping coding at a DNA locus is assumed to be highly improbable. Therefore, such overlapping genes are systematically excluded in prokaryotic genome annotation. Here we report an exceptional 603 bp long open reading frame completely embedded in antisense to the gene of the outer membrane protein ompA. An active σ 70 promoter, transcription start site (TSS), Shine-Dalgarno motif and rho-independent terminator were experimentally validated, providing evidence that this open reading frame has all the structural features of a functional gene. Furthermore, ribosomal profiling revealed translation of the mRNA, the protein was detected in Western blots and a pH-dependent phenotype conferred by the protein was shown in competitive overexpression growth experiments of a translationally arrested mutant versus wild type. We designate this novel gene pop (pH-regulated overlapping protein-coding gene), thus adding another example to the growing list of overlapping, protein coding genes in bacteria.

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“…Leaderless genes are translated by a different mechanism than genes with a leader sequence, as is the case for virF in Shigella spp. [104].…”

Section: Discussionmentioning

confidence: 99%

Complete genome sequence and annotation of the laboratory reference strain Shigella flexneri serotype 5a M90T and genome-wide transcriptional start site determination

2020

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Background: Shigella is a Gram-negative facultative intracellular bacterium that causes bacillary dysentery in humans. Shigella invades cells of the colonic mucosa owing to its virulence plasmid-encoded Type 3 Secretion System (T3SS), and multiplies in the target cell cytosol. Although the laboratory reference strain S. flexneri serotype 5a M90T has been extensively used to understand the molecular mechanisms of pathogenesis, its complete genome sequence is not available, thereby greatly limiting studies employing high-throughput sequencing and systems biology approaches. Results: We have sequenced, assembled, annotated and manually curated the full genome of S. flexneri 5a M90T. This yielded two complete circular contigs, the chromosome and the virulence plasmid (pWR100). To obtain the genome sequence, we have employed long-read PacBio DNA sequencing followed by polishing with Illumina RNA-seq data. This provides a new hybrid strategy to prepare gapless, highly accurate genome sequences, which also cover AT-rich tracks or repetitive sequences that are transcribed. Furthermore, we have performed genome-wide analysis of transcriptional start sites (TSS) and determined the length of 5′ untranslated regions (5′-UTRs) at typical culture conditions for the inoculum of in vitro infection experiments. We identified 6723 primary TSS (pTSS) and 7328 secondary TSS (sTSS). The S. flexneri 5a M90T annotated genome sequence and the transcriptional start sites are integrated into RegulonDB (http://regulondb.ccg.unam. mx) and RSAT (http://embnet.ccg.unam.mx/rsat/) databases to use their analysis tools in the S. flexneri 5a M90T genome. Conclusions: We provide the first complete genome for S. flexneri serotype 5a, specifically the laboratory reference strain M90T. Our work opens the possibility of employing S. flexneri M90T in high-quality systems biology studies such as transcriptomic and differential expression analyses or in genome evolution studies. Moreover, the catalogue of TSS that we report here can be used in molecular pathogenesis studies as a resource to know which genes are transcribed before infection of host cells. The genome sequence, together with the analysis of transcriptional start sites, is also a valuable tool for precise genetic manipulation of S. flexneri 5a M90T. Further, we present a new hybrid strategy to prepare gapless, highly accurate genome sequences. Unlike currently used hybrid strategies combining long-and short-read DNA sequencing technologies to maximize accuracy, our workflow using long-read DNA sequencing and short-read RNA sequencing provides the added value of using non-redundant technologies, which yield distinct, exploitable datasets.

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One Gene and Two Proteins: a Leaderless mRNA Supports the Translation of a Shorter Form of the Shigella VirF Regulator

Cited by 33 publications

References 46 publications

Internal RNAs overlapping coding sequences can drive the production of alternative proteins in archaea

Internal RNAs overlapping coding sequences can drive the production of alternative proteins in archaea

A Novel pH-Regulated, Unusual 603 bp Overlapping Protein Coding Gene pop Is Encoded Antisense to ompA in Escherichia coli O157:H7 (EHEC)

Complete genome sequence and annotation of the laboratory reference strain Shigella flexneri serotype 5a M90T and genome-wide transcriptional start site determination

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