Understanding the roles of non-coding RNAs in Enterohaemorhaggic E. coli pathogenesis
Expression of virulence genes in pathogenic bacteria is tightly regulated in response to environmental cues at both the transcriptional and post-transcriptional level. RNAs that do not encode proteins (non-coding RNAs) are now appreciated to play important roles in post-transcriptional gene regulation by interacting with mRNAs and modulating translation and stability. High throughput sequencing studies are now uncovering hundreds of non-coding RNAs in pathogenic bacteria and the challenge now is to understand the function of these RNA species.
A major subclass of bacterial non-coding RNA, termed small RNAs (sRNAs), requires the RNA chaperone Hfq to anneal to mRNA targets and effect regulation. Using UV-crosslinking and NextGen sequencing techniques (CRAC or CLIP-Seq) we have generated high resolution maps of Hfq-RNA interactions in the human pathogen Enterohaemorhaggic E. coli (EHEC). Within this dataset of Hfq binding sites we have identified 55 new sRNAs (Tree et al Molecular Cell) and we are now looking to identify the mRNA targets of these sRNAs and understand their role in pathogenesis.
Recently it has been demonstrated that RNA-RNA interactions can be extracted from CLIP-Seq data allowing ncRNAs to be sequencing in complex with their mRNA targets (a technique termed CLASH). This analysis gives insights into the function of ncRNAs in vivo. Small RNAs have been shown to recruit the RNA endonuclease, RNase E, when duplexed with an mRNA target and we have recently demonstrated that sRNA-mRNA interactions can be sequenced from RNaseE CLIP-Seq data. We have confirmed a subset of these interactions using translational GFP fusions. Using this dataset we have identified mRNA targets for our newly identified EHEC sRNAs and have begun assigning functions to some of these novel RNA species. We have found that the EHEC specific sRNA, Esr41, represses translation of select iron uptake receptors indicating a role in modulating iron availability.