Christol, N. | Search bioRxiv

bioRxiv2026

RNase J2 is a specificity factor that governs global mRNA stability in Bacillus subtilis

Ribonucleases (RNases) play a key role in modulating gene expression at the post-transcriptional level, enabling bacteria to rapidly adapt to their environment. The B. subtilis genome encodes more than 20 RNases and understanding the role of each of these enzymes is crucial for a comprehensive knowledge of bacterial post-transcriptional adaptation mechanisms. Among these, RNase J1 and J2, belonging to the {beta}-lactamase enzyme family, form a heterodimer. While the primary function of RNase J1 as a 5'-exoribonuclease in B. subtilis has been known for some time, the role of RNase J2, encoded by the rnjB gene, has remained unclear. Indeed, it has been considered a minor degradation factor due its very weak 5'-exoribonuclease in vitro, the lack of a growth phenotype and the limited number of mRNAs with altered equilibrium levels in rnjB mutants. In this study, we demonstrate that the absence of RNase J2 influences pellicle and macro-colony biofilm formation, as well as swarming in B. subtilis. We show that RNase J2 plays a far more global role in governing mRNA half-lives than originally thought, by stimulating RNase J1-mediated degradation of a subset of RNase J1's mRNA substrates. An inter-subunit contact between RNase J1 and J2 is crucial for RNase J2 action. Altogether, our results suggest that RNase J2 acts primarily as a specificity co-factor for RNase J1 rather than as a ribonuclease per se.

microbiology↗

bioRxiv2023

New RoxS sRNA targets identified in B. subtilis by pulsed SILAC

Non-coding RNAs (sRNA) play a key role in controlling gene expression in bacteria, typically by base-pairing with ribosome binding sites to block translation. The modification of ribosome traffic along the mRNA generally affects its stability. However, a few cases have been described in bacteria where sRNAs can affect translation without a major impact on mRNA stability. To identify new sRNA targets in B. subtilis potentially belonging to this class of mRNAs, we used pulsed-SILAC (stable isotope labeling by amino acids in cell culture) to label newly synthesized proteins after short expression of the RoxS sRNA, the best characterized sRNA in this bacterium. RoxS sRNA was previously shown to interfere with the expression of genes involved in central metabolism, permitting control of the NAD+/NADH ratio in B. subtilis. In this study, we confirmed most of the known targets of RoxS, showing the efficiency of the method. We further expanded the number of mRNA targets encoding enzymes of the TCA cycle and identified new targets primarily regulated at the translational level. One of these is YcsA, a tartrate dehydrogenase that uses NAD+ as co-factor, in excellent agreement with the proposed role of RoxS in Firmicutes. ImportanceNon-coding RNA (sRNA) play an important role in bacterial adaptation and virulence. The identification of the most complete set of targets for these regulatory RNAs is key to fully identify the perimeter of its function(s). Most sRNAs modify both the translation (directly) and mRNA stability (indirectly) of their targets. However, sRNAs can also influence the translation efficiency of the target primarily, with little or no impact on mRNA stability. The characterization of these targets is challenging. We describe here the application of the pulsed SILAC method to identify these targets and obtain the most complete list of targets for a defined sRNA.

microbiology↗