SRA

Small RNAs have been found to control a broad range of bacterial phenotypes including tolerance to antibiotics. Vancomycin tolerance in multidrug resistance Staphylococcus aureus is correlated with dysregulation of small RNAs although their contribution to antibiotic tolerance in poorly understood. RNA-RNA interactome profiling techniques are expanding our understanding of sRNA-mRNA interactions in bacteria; however, determining the function of these interactions for hundreds of sRNA-mRNA pairs is a major challenge. At steady-state, protein and mRNA abundances are often highly correlated and lower than expected protein abundance may indicate translational repression of an mRNA. To identify sRNA-mRNA interactions that regulate mRNA translation, we examined the correlation between gene transcript abundance, ribosome occupancy, and protein levels. We used the machine learning technique self-organising maps (SOMS) to cluster genes with similar transcription and translation patterns and identified a cluster of mRNAs that appeared to be post-transcriptionally repressed. By integrating our clustering with sRNA-mRNA interactome data generated in vancomycin tolerant S. aureus by RNase III-CLASH, we identified sRNAs that may be mediating translational repression. We have confirmed sRNA-dependant post-transcriptional repression of several mRNAs in this cluster. Two of these interactions are mediated by RsaOI, a sRNA that is highly upregulated by vancomycin. We demonstrate regulation of HPr and the cell-wall autolysin Atl. These findings suggest RsaOI coordinates carbon metabolism and cell wall turnover during vancomycin treatment. Overall design: This study uses a metaomics approach to predict sRNA function on mRNA transcription and translation, and identify genes that are post-transcriptionally regulated. The technique CLASH was used to identify sRNA-mRNA interactions in the cell and is presented in this entry. RNaseIII-CLASH was performed on biological duplicate cultures grown in Muller Hinton media to an OD of 0.8 with or without treatment with 2ug/ml vancomycin for 10mins.