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

Abstract

Small RNAs (sRNAs) are emerging as important and diverse post-transcriptional gene expression regulators in bacterial stress responses and virulence. While originally identified mainly in intergenic regions, genome-wide approaches have revealed sRNAs encoded in diverse contexts, such as processed from parental transcripts by RNase E. Despite its well-known roles in rRNA processing, RNA decay, cleavage of sRNA-mRNA duplexes, the role of RNase III in sRNA biogenesis is less well understood. Here, we show that a pair of cis-encoded sRNAs (CJnc190 and CJnc180) are processed by RNase III in the foodborne pathogen Campylobacter jejuni. While CJnc180 processing requires CJnc190, RNase III cleaves an intramolecular duplex in CJnc190, independent of CJnc180. Moreover, we demonstrate that CJnc190 directly represses translation of the colonization factor PtmG by binding its G-rich ribosome binding site, and show that CJnc180 is a cis-acting antagonist of CJnc190, thereby indirectly affecting ptmG regulation. Our results expand the diversity of known genomic locations of bacterial sRNA sponges and highlight a role for bacterial RNase III that parallels miRNA processing by related eukaryotic Dicer and Drosha.

Small RNAs that target G‐rich sequences are generated by diverse biogenesis pathways in Epsilonproteobacteria

“…In our recent work, we showed that processed CJnc190 encoded in the CJnc190/180 sRNA locus of C. jejuni strain NCTC11168 represses ptmG translation by base‐pairing with its G‐rich translation initiation region using a single‐stranded C/U‐rich sequence within its second stem‐loop (SL2; Figure 1a; Svensson & Sharma, 2021). We noticed that the mature CJnc190 sRNA resembles the H. pylori sRNA RepG, which also uses a C/U‐rich loop to bind to a G‐rich sequence in the 5’UTR of its target mRNA tlpB ‐HP0102 (Pernitzsch et al., 2014, 2021), in structure and function.…”

“…RepG is encoded as a stand‐alone sRNA that is transcribed from a single promoter and does not require processing for its expression or stability (Pernitzsch et al., 2014). In contrast, CJnc190 is transcribed as precursors from two promoters (P1 & P2), which are then processed by RNase III into the mature, stable, and/or active form of the sRNA (Svensson & Sharma, 2021; Figure 1a,b). In addition, an antisense RNA, CJnc180, is transcribed from the opposite strand of CJnc190 and antagonizes its regulation of ptmG .…”

“… C. jejuni CJnc190 and H. pylori RepG share a C/U‐rich loop that targets G‐rich sequences. (a) CJnc190 from C. jejuni strain NCTC11168 uses a C/U‐rich loop to repress PtmG expression via base‐pairing with the G‐rich RBS of ptmG mRNA (Svensson & Sharma, 2021). (b,c) Secondary structure, genomic context, and biogenesis of C. jejuni CJnc190 and H. pylori RepG (Pernitzsch et al., 2014; Svensson & Sharma, 2021).…”

“…(a) CJnc190 from C. jejuni strain NCTC11168 uses a C/U‐rich loop to repress PtmG expression via base‐pairing with the G‐rich RBS of ptmG mRNA (Svensson & Sharma, 2021). (b,c) Secondary structure, genomic context, and biogenesis of C. jejuni CJnc190 and H. pylori RepG (Pernitzsch et al., 2014; Svensson & Sharma, 2021). (Left) CJnc190 is transcribed as precursors from two promoters (P1/P2) and is independently processed by RNase III.…”

“…Moreover, we recently reported that the sRNA pair CJnc180/190 affects C. jejuni adherence and internalization in a tissue‐engineered model of the human intestine (Alzheimer et al., 2020). CJnc190 represses expression of the colonization factor PtmG by binding to a G‐rich sequence in the ptmG mRNA using a C/U‐rich loop (Svensson & Sharma, 2021). CJnc190 is generated by a unique biogenesis pathway involving processing by RNase III and is also antagonized by its cis ‐encoded sRNA partner, CJnc180.…”