Abstract
sRNAs are a taxonomically-restricted but transcriptomically-abundant class of post-transcriptional regulators. While of major importance for adaption to the environment, we currently lack global-scale methodology enabling target identification, especially in species without known RNA hub proteins (e.g. Hfq). Using psoralen RNA cross-linking and Illumina-sequencing we identify RNA–RNA interacting pairs in vivo in Bacillus subtilis, resolving previously well-described interactants. Although sRNA–sRNA pairings are rare (compared with sRNA–mRNA), we identify a robust example involving the conserved sRNA RoxS and an unstudied sRNA RosA (Regulator of sRNA A). We show RosA to be the first confirmed RNA sponge described in a Gram-positive bacterium. RosA interacts with at least two sRNAs, RoxS and FsrA. The RosA/RoxS interaction not only affects the levels of RoxS but also its processing and regulatory activity. We also found that the transcription of RosA is repressed by CcpA, the key regulator of carbon-metabolism in B. subtilis. Since RoxS is already known to be transcriptionally controlled by malate via the transcriptional repressor Rex, its post-transcriptional regulation by CcpA via RosA places RoxS in a key position to control central metabolism in response to varying carbon sources.
Highlights
To adapt to changing environments and survive exposure to harsh conditions, organisms have evolved complicated metabolic and genetic regulatory networks to ensure that a homeostatic balance is maintained [1,2]
WT FsrA still bound to RosAGRR2/4U but failed to form a complex with RosAGRR1/2U. These results show that the CRR2 sequence of FsrA and the GRR1 region of RosA, the site of the most stable interaction predicted by the IntaRNA program [42], are most likely to be involved in the pairing between the two RNAs
In this study we report the use of in vivo RNA cross-linking using the psoralen AMT to globally identify RNA–RNA interactions occurring in the Gram-positive model organism B. subtilis
Summary
To adapt to changing environments and survive exposure to harsh conditions, organisms have evolved complicated metabolic and genetic regulatory networks to ensure that a homeostatic balance is maintained [1,2]. At the RNA synthesis level, gene expression can be modulated through combinations of transcription factors controlling genes required for growth and survival under specific conditions [3,4,5]. At the post-transcriptional level, small regulatory RNAs (sRNAs) act to temper gene expression by short imperfect base pairing with their mRNA targets, altering the level of protein production by increasing or decreasing access to the ribosome-binding site, or by facilitating or blocking the access to the mRNA by ribonucleases (RNases) [6,7]. Most regulatory RNAs are independently expressed under the control of specific transcription factors. Regulation by RNA is an important mechanism for finetuning gene expression in the Gram-positive model bacterium Bacillus subtilis, recently reviewed in [10].
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