Identification of RNA-RNA interactions in Bacillus Subtilis by in vivo UV crosslinking

Small RNAs (sRNAs) are a taxonomically-restricted but transcriptomically-abundant class of post-transcriptional regulators. While potentially of importance, we know the function of few. This is in no small part because we lack global-scale methodology enabling target identification, this being especially acute in species without known RNA meeting point proteins (e.g. Hfq). We apply a combination of psoralen RNA cross-linking and Illumina-sequencing to 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 unusually conserved sRNA (RoxS/RsaE) and an unstudied sRNA that we term Regulator of small RNA A (RosA). This interaction is found in independent samples across multiple conditions. Given the possibility of a novel associated regulatory mechanism, and the rarity of well-characterised bacterial sRNA-sRNA interactions, we mechanistically dissect RosA and its interactants. RosA we show to be a sponge RNA, the first to be described in a Gram-positive bacterium. RosA interacts with at least two sRNAs, RoxS and FsrA. Unexpectedly, it acts differently on each. As expected of a sponge RNA, FsrA is sequestered by RosA. The RosA/RoxS interaction is more complex affecting not only the level of RoxS but also its processing and efficacy. Importantly, RosA provides the condition-dependent intermediary between CcpA, the key regulator of carbon metabolism, and RoxS. This not only provides evidence for a novel, and functionally important, regulatory mechanism, but in addition, provides the missing link between transcriptional and post-transcriptional regulation of central metabolism.

RNA sequencing, High-throughput sequencing
Microbial sciences
Omic sciences and technologies

Cite this dataset as:
Denham, E., Li, S., 2021. Identification of RNA-RNA interactions in Bacillus Subtilis by in vivo UV crosslinking. BioStudies.


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Emma Denham
University of Bath

Stephen Li
University of Warwick


University of Bath
Rights Holder


Data collection method:

Sample collection: Bacteria were grown to the required O.D before 10 O.D 600 nm units were harvested by centrifugation (4000 g, 5 minutes, 4C). Bacteria were resuspended in 2 ml PBS either containing no AMT (to identify background and levels of spurious interactions) or 0.7mM AMT. Bacteria were incubated for 10 minutes at 37C for 10 minutes before being transferred to a 6 well plate. The bacteria were exposed to UV 365 nm at 0.120 Jcm-2in a for 10 minutes before being added to 1 ml of ice cold killing buffer (20 mM Tris-HCl [pH 7.5], 5 mM MgCl2, 20 mM Na-azide). The bacteria were harvested by centrifugation at X g, the supernatant was discarded and the pellet flash frozen in liquid nitrogen. We determined the in vivo RNA interactome of B. subtilis grown in M9 minimal media supplemented with 0.3% glucose at three points in the growth curve (exponential phase O.D.600nm 0.5, stationary phase O.D.600nm 1.4 and just after lysis had started to occur, and in LB at mid-exponential phase (O.D.600nm of 1.0). Samples were prepared in duplicate. Nucleic acid extraction: The RNA was extracted by resuspending the cell pellet in 800 µl LETS buffer (10 mM Tris-HCl [pH 8.0], 50 mM LiCl, 10 mM EDTA, 1% sodium dodecyl sulfate [SDS]) and bead beating in a FastPrep using 0.1 µm glass beads for three rounds of 40 seconds. The tubes were transferred to ice in between cycles. The tubes were briefly spun to remove the bubbles created during bead beating. Two rounds of phenol chloroform isoamyl alcohol extraction and one round of choloroform isoamyl alcohol extraction were carried out. Before the addition of 10 % v/v NaAcetate and 1 ml Isopropanyl and precipitation of RNA overnight at -20C. The RNA was pelleted by centrifugation at maximum speed at 4C and the pellet was washed with 70% Ethanol before being air dried and resuspended in water. The RNA was quantified using the Qubit kit (Fisher Life Science). 10 µg of RNA was treated with Turbo DNase (Fisher Scientific) to remove contaminating DNA. Ribosomal RNA was removed using Ribozero (Illumina) according to the manufacturer’s instructions. To form the chimeric RNAs between RNAs crosslinked with AMT the protocol described by Sharma et al was followed. The only modification was the use of CircDNAligase (Epicentre) instead of CircRNAligase as this has been discontinued. Nucleic acid library construction: Following uncrosslinking at UV 254 nm, RNA was purified and resuspended in 10 µl H2O and processed through the TruSeq RNAseq kit (Illumina) according to the manufacturers instructions. Nucleic acid sequencing: The prepared libraries were sequenced on the MiSeq (Illumina). Growth: Growth experiments were performed in LB, M9 medium supplemented with glucose at a final concentration of 0.3%. Normalization data transformation: The output Sam files and Ban files were generated by aligning the fastq files to the Bacillus genome sequence using STAR aligner (with single end).


Biotechnology and Biological Sciences Research Council (BBSRC)

Understanding the role of small regulatory RNAs in the Gram-positive model organism Bacillus subtilis

University of Warwick (Warwick)

Noreen Murray Award

Medical Research Council (MRC)

PhD studentship

Centre National de la Recherche Scientifique (CNRS)

Université de Paris (University of Paris)


Agence Nationale de la Recherche (ANR)

Publication details

Publication date: 25 August 2021
by: BioStudies

Version: 1

Official landing page URL:

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Related papers and books

Durand, S., Callan-Sidat, A., McKeown, J., Li, S., Kostova, G., Hernandez-Fernaud, J. R., Alam, M. T., Millard, A., Allouche, D., Constantinidou, C., Condon, C., and Denham, E. L., 2021. Identification of an RNA sponge that controls the RoxS riboregulator of central metabolism in Bacillus subtilis. Nucleic Acids Research, 49(11), 6399-6419. Available from:

Contact information

Please contact the Research Data Service in the first instance for all matters concerning this item.

Contact person: Emma Denham


Life Sciences
Biology & Biochemistry