Tombusvirusp19 Captures RNase III-Cleaved Double-Stranded RNAs Formed by Overlapping Sense and Antisense Transcripts in Escherichia coli

Abstract

Antisense transcription is widespread in bacteria. By base pairing with overlapping sense RNAs, antisense RNAs (asRNA) can form double-stranded RNAs (dsRNA), which are cleaved by RNase III, a dsRNA endoribonuclease. The ectopic expression of plant Tombusvirus p19 in Escherichia coli stabilizes ∼21-nucleotide (nt) dsRNA RNase III decay intermediates, which enabled us to characterize otherwise highly unstable asRNA by deep sequencing of p19-captured dsRNA. RNase III-produced small dsRNA were formed at most bacterial genes in the bacterial genome and in a plasmid. We classified the types of asRNA in genomic clusters producing the most abundant p19-captured dsRNA and confirmed RNase III regulation of asRNA and sense RNA decay at three type I toxin-antitoxin loci and at a coding gene, rsd. Furthermore, we provide potential evidence for the RNase III-dependent regulation of CspD protein by asRNA. The analysis of p19-captured dsRNA revealed an RNase III sequence preference for AU-rich sequences 3 nucleotides on either side of the cleavage sites and for GC-rich sequences in the 2-nt overhangs. Unexpectedly, GC-rich sequences were enriched in the middle section of p19-captured dsRNA, suggesting some unexpected sequence bias in p19 protein binding. Nonetheless, the ectopic expression of p19 is a sensitive method for identifying antisense transcripts and RNase III cleavage sites in dsRNA formed by overlapping sense and antisense transcripts in bacteria.

RNase III Participates in the Adaptation to Temperature Shock and Oxidative Stress in Escherichia coli

“…As we observed a significant reduction in rpoH expression in the rnc mutant during a heat shock, we investigated the importance of RNase III on rpoH mRNA stability after a shift at 45 • C. We observed that RNase III inactivation led to a slight reduction in rpoH stability after a 15 min heat shock from 30 to 45 • C (from 4.01 to 3.39 min, Table 1 and Figure S4A), suggesting that RNase III has only a modest effect on the stability of the rpoH mRNA during a heat shock. However, it should be remembered that the inactivation of RNase III leads to the accumulation of the exoribonuclease PNPase, as previously shown at the mRNA [22] and protein level [23,31]. We observed that the stability of rpoH mRNA after a heat shock in a PNPase inactivated mutant (pnp) is reduced (from 4.01 to 3.14 min, Table 1 and Figure S4A).…”

“…The transcription of rpoH was shown to be dependent on the sigma factors RpoD, RpoE, RpoN and RpoS and regulated by transcriptional factors DnaA, ZraR, CpxR, IHF, CRP and CytR (Ecocyc database [41]). Remarkably, the details of the transcriptional induction of rpoH during a heat shock remains, to our knowledge, non-elucidated [31]. We compared the induction level of the rpoH mRNA after 15 min heat shock from 30 to 45 • C in mutants inactivated for CRP and/or CytR and/or RNase III.…”

“…The application of RNA-seq methodologies has demonstrated an even wider role of RNase III in the control of gene expression [29][30][31]. One study, combining stability experiments with RNA-seq (i.e., sequencing after rifampicin treatment), reported 2054 RNase III-dependent cleavage sites (11 were verified on purified RNA fragments by in vitro RNase III cleavage assays) [32].…”

Bacterial 3′UTRs: A Useful Resource in Post-transcriptional Regulation

“…Since bacterial genomes are compact and the distance between CDSs is often short, when transcriptional terminator signals between convergent genes are missing and/or transcriptional read-through occurs, long antisense overlapping 3′UTRs are produced (Hernández et al, 2006 ; Toledo-Arana et al, 2009 ; Arnvig et al, 2011 ; Lasa et al, 2011 ; Nicolas et al, 2012 ; Moody et al, 2013 ; Mäder et al, 2016 ; Stazic and Voss, 2016 ; Bidnenko et al, 2017 ; Huang et al, 2020 ; Toledo-Arana and Lasa, 2020 ) ( Figure 1A ). In addition, more complex transcriptional organizations like operons containing a gene(s) that is transcribed in the opposite direction to the rest of the operon are also known for generating long overlapping transcripts (Lasa et al, 2011 ; Sáenz-Lahoya et al, 2019 ).…”

“…In addition, more complex transcriptional organizations like operons containing a gene(s) that is transcribed in the opposite direction to the rest of the operon are also known for generating long overlapping transcripts (Lasa et al, 2011 ; Sáenz-Lahoya et al, 2019 ). The outcome is usually the development of large double-stranded RNA regions that are processed by RNase III in both Gram-positive and Gram-negative bacteria (Lasa et al, 2011 , 2012 ; Gatewood et al, 2012 ; Lioliou et al, 2012 ; Lybecker et al, 2014 ; Huang et al, 2020 ). Interestingly, overlapping 3′UTRs are widely distributed in bacteria and constitute an abundant source of antisense RNAs (Arnvig et al, 2011 ; Lasa et al, 2011 ; Ruiz de Los Mozos et al, 2013 ).…”