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

Abstract

Bacterial small RNAs (sRNAs) are widespread post‐transcriptional regulators that control bacterial stress responses and virulence. Nevertheless, little is known about how they arise and evolve. Homologs can be difficult to identify beyond the strain level using sequence‐based approaches, and similar functionalities can arise by convergent evolution. Here, we found that the virulence‐associated CJnc190 sRNA of the foodborne pathogen Campylobacter jejuni resembles the RepG sRNA from the gastric pathogen Helicobacter pylori. However, while both sRNAs bind G‐rich sites in their target mRNAs using a C/U‐rich loop, they largely differ in their biogenesis. RepG is transcribed from a stand‐alone gene and does not require processing, whereas CJnc190 is transcribed from two promoters as precursors that are processed by RNase III and also has a cis‐encoded antagonist, CJnc180. By comparing CJnc190 homologs in diverse Campylobacter species, we show that RNase III‐dependent processing of CJnc190 appears to be a conserved feature even outside of C. jejuni. We also demonstrate the CJnc180 antisense partner is expressed in C. coli, yet here might be derived from the 3’UTR (untranslated region) of an upstream flagella‐related gene. Our analysis of G‐tract targeting sRNAs in Epsilonproteobacteria demonstrates that similar sRNAs can have markedly different biogenesis pathways.

Complementary Ribo-seq approaches map the translatome and provide a small protein census in the foodborne pathogenCampylobacter jejuni

“…In general, our “standard” C. jejuni Ribo-seq dataset successfully differentiated between known coding and non-coding regions. For example, cDNA reads for the sRNA CJnc180 (Dugar et al, 2013; Svensson and Sharma, 2022, 2021) were mainly restricted to the RNA-seq (transcriptome) library prepared in parallel, while the adjacent ORF Cj1650 had both RNA-seq and Ribo-seq coverage ( Fig. 1B ).…”

RNase III-mediated processing of a trans-acting bacterial sRNA and its cis-encoded antagonist

“…While we found that CJnc180 was not required for CJnc190 processing, several C. jejuni and C. coli strains express this antisense RNA ( Dugar et al, 2013 ; Riedel et al, 2020 ; Svensson and Sharma, 2021 ), suggesting it has a conserved function. Because of its extensive complementarity to CJnc190, as well as its co-processing, we hypothesized that it might serve as a CJnc190 antagonist and indirectly affect ptmG regulation.…”

“…Thus, CJnc190 binding likely interferes with translation initiation, resembling the canonical mode of sRNA-mediated target repression ( Storz et al, 2011 ). The structure of mature CJnc190 is reminiscent of RepG sRNA from Helicobacter pylori ( Svensson and Sharma, 2021 ), which uses a C/U-rich loop to target a phase-variable G-repeat in the 5’ UTR of a chemotaxis receptor mRNA ( Pernitzsch et al, 2014 ). Although CJnc190 and RepG share C/U-rich loop regions, their biogenesis is strikingly different: while RepG is transcribed as a separate standing gene corresponding to the mature sRNA ( Pernitzsch et al, 2014 ), CJnc190 is transcribed opposite to another sRNA (CJnc180) and is processed into the mature form from diverse precursors in C. jejuni and related C. coli ( Svensson and Sharma, 2021 ).…”

“…The structure of mature CJnc190 is reminiscent of RepG sRNA from Helicobacter pylori ( Svensson and Sharma, 2021 ), which uses a C/U-rich loop to target a phase-variable G-repeat in the 5’ UTR of a chemotaxis receptor mRNA ( Pernitzsch et al, 2014 ). Although CJnc190 and RepG share C/U-rich loop regions, their biogenesis is strikingly different: while RepG is transcribed as a separate standing gene corresponding to the mature sRNA ( Pernitzsch et al, 2014 ), CJnc190 is transcribed opposite to another sRNA (CJnc180) and is processed into the mature form from diverse precursors in C. jejuni and related C. coli ( Svensson and Sharma, 2021 ). It is interesting to imagine how CJnc190 (and CJnc180) might have arisen - de novo ( Jose et al, 2019 , Updegrove et al, 2015 ), or from a degenerate type I toxin-antitoxin system as might be the case for the H. pylori sRNA NikS ( Eisenbart et al, 2020 ).…”

Transcriptome fine-mapping in Fusobacterium nucleatum reveals FoxJ, a new σ E -dependent small RNA with unusual mRNA activation activity

“…Poly-cytosine stretches, such as in FoxJ, are often found in bacterial sRNAs and might represent a hallmark of regulatory RNAs in bacteria with low GC content genomes such as F. nucleatum ( 38 , 39 ). In fact, FoxJ’s companion sRNA FoxI uses a C-stretch to repress the translation of its targets ( 8 , 25 ).…”