Poster Presentation BACPATH 2022

The RNA interactome of methicillin-resistant Staphylococcus aureus reveals a role for regulatory mRNAs in coordinating gene expression (#161)

Daniel G Mediati 1 , Julia Wong 1 , Wei Gao 2 , Stuart MacKellar 3 , Winton Wu 1 , Ian R Monk 2 , Timothy P Stinear 2 , Sander Granneman 3 , Jai J Tree 1
  1. School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, Australia
  2. Department of Microbiology and Immunology, Peter Doherty Institute, University of Melbourne, Melbourne, VIC, Australia
  3. Centre for Systems and Synthetic Biology, University of Edinburgh, Edinburgh, United Kingdom

Staphylococcus aureus is a major cause of skin, soft tissue, respiratory and endovascular infections. The emergence of antibiotic resistant strains, including methicillin-resistant S. aureus (MRSA), has limited the treatment options to last-line antibiotics like glycopeptides. Transcriptional profiling of MRSA isolates following antibiotic treatment indicate that non-coding small RNA (sRNA) form key coordinated responses to antibiotic stresses. Hundreds of regulatory sRNAs litter the transcriptome but the precise function of the majority of these ubiquitous gene regulators are unknown.

Here we have performed UV crosslinking, ligation, and sequencing of hybrids (CLASH) to profile the in vivo RNA interactome associated with the endoribonuclease RNase III and identify functions for regulatory non-coding RNA in the MRSA isolate JKD6009. To accurately map these interactions to transcriptome features, we used dRNA-seq and term-seq techniques to map RNA 5’ and 3’ boundary ends. We uncovered hundreds of novel sRNA-mRNA interactions allowing functional characterisation of many sRNA for the first time, some of which were verified experimentally confirming the robustness of our approach. Surprisingly, we recovered 543 statistically significant mRNA-mRNA interactions. One of the most abundant mRNA-mRNA interactions uncovered was a novel interaction within the yabJ-spoVG bicistronic operon involved in methicillin and glycopeptide resistance. Our data indicate that the 5’UTR of the yabJ mRNA anneals to its own 3’UTR incorporating the intergenic region of spoVG, and we confirm this direct interaction in vitro. Annealing of the UTRs appears to create an RNase III cleavage site that releases spoVG mRNA from the bicistronic transcript and allows sub-operonic control of spoVG expression. Future work will aim to verify the mechanism of spoVG release and effect on antibiotic resistance in MRSA. Our results demonstrate the utility of CLASH for identifying new regulatory non-coding RNA and indicate for the first time the extensive use of regulatory mRNAs to coordinate gene expression in a prokaryote.