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

Ethan C. Rath, Stephanie Pitman, Kyu Hong Cho, Yongsheng Bai

Small 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. For 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. This 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.