Identification of streptococcal small RNAs that are putative targets of RNase III through bioinformatics analysis of RNA sequencing data

Abstract

BackgroundSmall noncoding regulatory RNAs (sRNAs) are post-transcriptional regulators, regulating mRNAs, proteins, and DNA in bacteria. One class of sRNAs, trans-acting sRNAs, are the most abundant sRNAs transcribed from the intergenic regions (IGRs) of the bacterial genome. In Streptococcus pyogenes, a common and potentially deadly pathogen, many sRNAs have been identified, but only a few have been studied. The goal of this study is to identify trans-acting sRNAs that can be substrates of RNase III. The endoribonuclease RNase III cleaves double stranded RNAs, which can be formed during the interaction between an sRNA and target mRNAs.ResultsFor this study, we created an RNase III null mutant of Streptococcus pyogenes and its RNA sequencing (RNA-Seq) data were analyzed and compared to that of the wild-type. First, we developed a custom script that can detect intergenic regions of the S. pyogenes genome. A differential expression analysis with Cufflinks and Stringtie was then performed to identify the intergenic regions whose expression was influenced by the RNase III gene deletion.ConclusionThis analysis yielded 12 differentially expressed regions with >|2| fold change and p ≤ 0.05. Using Artemis and Bamview genome viewers, these regions were visually verified leaving 6 putative sRNAs. This study not only expanded our knowledge on novel sRNAs but would also give us new insight into sRNA degradation.

RNase III-mediated processing of atrans-acting bacterial sRNA and itscis-encoded antagonist

“…While the RNase III domain-containing Dicer and Drosha play a central role in sRNA biogenesis in eukaryotes (Carthew & Sontheimer, 2009), its role in sRNA biogenesis in bacteria is less clear. However, its expropriation by Type-II CRISPR/Cas systems for biogenesis of CRISPR RNAs (Deltcheva et al, 2011;Dugar et al, 2018), as well as genome-wide studies of the RNase III targetome that report sRNAs as potential targets in Gram-negative as well as Gram-positive species (Altuvia et al, 2018;Lioliou et al, 2012Lioliou et al, , 2013Gordon et al, 2017;Le Rhun et al, 2017;Rath et al, 2017;Lybecker et al, 2014), indicate the potential for a broad role in sRNA processing in bacteria.…”

“…In S. aureus, RNase III generates RsaC sRNA from the 3′ UTR of the mntABC mRNA (Faubladier et al, 1990;Lalaouna et al, 2019). Moreover, its expropriation for biogenesis of CRISPR RNAs (Deltcheva et al, 2011;Dugar et al, 2018), as well as genome-wide studies of the RNase III targetome that report sRNAs as potential targets in Gram-negative as well as Gram-positive species (Altuvia et al, 2018;Gordon et al, 2017;Le Rhun et al, 2017;Lioliou et al, 2013Lioliou et al, , 2012Lybecker et al, 2014;Rath et al, 2017), indicate that RNase III might process sRNAs in diverse bacteria. However, most of these remain to be validated or studied.…”

RNase III participates in control of quorum sensing, pigmentation and oxidative stress resistance in Rhodobacter sphaeroides

“…In particular, the development of low‐cost high throughput RNA sequencing (RNA‐seq) methods in the beginning of the early 2000s and its applications revealed new functions of RNase III. RNase III was previously known to primarily affect maturation of ribosomal and transfer RNAs, but the spectrum of other RNAs recognised by RNase III has been largely expanded in the last decades (Altuvia et al, 2018; Gatewood et al, 2012; Ifill et al, 2021; Rath et al, 2017).…”

Comparison of Stranded and Non-stranded RNA-Seq in Predicting Small RNAs in a Non-model Bacterium

“…They can act as transcription terminators, potential inhibitors of translation initiation, or modulators of mRNA degradation. A trans-encoded sRNA interacts with its target mRNA by imperfect base pairing because such sRNA is coded by an intergenic region (ITR) and its coding sequence does not overlap with a sequence of the target gene [11]. This also means that trans-encoded sRNAs can be coded by the same strand as target genes and they have a wider range of regulatory mechanisms.…”