Mycobacterium tuberculosis 6C sRNA binds multiple mRNA targets via C-rich loops independent of RNA chaperones

Abstract

Bacterial small regulatory RNAs (sRNAs) are the most abundant class of post-transcriptional regulators and have been well studied in Gram-negative bacteria. Little is known about the functions and mechanisms of sRNAs in high GC Gram-positive bacteria including Mycobacterium and Streptomyces . Here, we performed an in-depth study of 6C sRNA of Mycobacterium tuberculosis , which is conserved among high GC Gram-positive bacteria. Forty-seven genes were identified as possible direct targets of 6C sRNA and 15 of them were validated using an in vivo translational lacZ fusion system. We found that 6C sRNA plays a pleotropic role and regulates genes involved in various cellular processes, including DNA replication and protein secretion. Mapping the interactions of 6C sRNA with mRNA targets panD and dnaB revealed that the C-rich loops of 6C sRNA act as direct binding sites to mRNA targets. Unlike in Gram-negative bacteria where RNA binding proteins Hfq and ProQ are required, the interactions of 6C sRNA with mRNAs appear to be independent of RNA chaperones. Our findings suggest that the multiple G–C pairings between single stranded regions are sufficient to establish stable interactions between 6C sRNA and mRNA targets, providing a mechanism for sRNAs in high GC Gram-positive bacteria.

Transposon mutagenesis in Mycobacterium kansasii links a small RNA gene to colony morphology and biofilm formation and identifies 9,885 intragenic insertions that do not compromise colony outgrowth

“…Our cross‐species genome sequence similarity analysis revealed that the B11 gene locus is highly conserved in the Mycobacterium genus (Appendix 2, Figure A4a) and in many representative members of other genera of Actinobacteria (not shown). These findings are consistent with reported analyses (DiChiara et al, ; Mai et al, ; Weinberg et al, ). The conservation of this gene suggests that its sRNA product has a critical function in the biology of Actinobacteria .…”

Small RNAs as Fundamental Players in the Transference of Information During Bacterial Infectious Diseases

“…The role of sRNAs has been long established, and there is a growing body of knowledge on the topic. However, as reported by Mai et al (2019), there are considerable gaps of knowledge regarding small regulatory RNAs. The field has discovered until now a wide set of these molecules, but bigger efforts to elucidate their molecular mechanisms of action are needed.…”

“…The field has discovered until now a wide set of these molecules, but bigger efforts to elucidate their molecular mechanisms of action are needed. The sRNA 6C (six cytosine residues) was hypothesized, by the authors of this study, to play an important role in regulation of cell division of Mycobacterium tuberculosis (M. tb) (Mai et al, 2019). In their experiments, the authors used a vector to overexpress 6C sRNA of M. tb, and transformed Mycobacterium smegmatis.…”

Small RNA Mcr11 requires the transcription factor AbmR for stable expression and regulates genes involved in the central metabolism of Mycobacterium tuberculosis

“…It also has been observed that overexpression of some sRNAs leads to slow or delayed growth in mycobacteria (Arnvig & Young, 2009;Ignatov et al, 2015). Roles for the sRNAs Mcr7, MsrI and 6C/B11 in gene regulation in Mtb or Mycobacterium smegmatis (Msm) have been reported (Gerrick et al, 2018;Mai et al, 2019;Solans et al, 2014). However, the importance of these differentially expressed sRNAs to the adaptation and survival of Mtb during periods of stress has not been fully addressed.…”