RNA binding proteins (RBPs) play critical roles in controlling transcription and translation in the cell. Recent profiling of the RNA-binding proteome in both prokaryotes and eukaryotes has revealed a surprisingly large number of RBPs suggesting that we are far from a complete understanding of protein-RNA interactions that modulate gene expression. To understand how the human pathogen, Enterohaemorrhagic E. coli (EHEC), utilises RNA-binding proteins to regulate virulence gene expression we have applied an organic extraction and silica bead enrichment technique termed Total RNA Associated Protein Purification (TRAPP) to capture the RNA-binding proteome. TRAPP enriched for 443 RNA-binding proteins >2-fold (FDR<0.05) including 35 proteins encoded within EHEC-specific pathogenicity islands. One of these RBPs, RhsFI is a type 6 secretion system (T6SS) immunity protein. Promoter mapping using dRNA-seq indicates that the immunity protein is transcribed independently of the core T6SS. Using CLIP-seq, RNA interaction sites for RhsFI were identified and validated using a filter binding assay. These analyses indicated a similar binding activity to the small RNA chaperone Hfq. Deletion of rhsF-rhsFI results in dysregulation of 300 mRNAs and we find Shiga toxin 1 expression is strongly suppressed through stabilisation of the Shiga toxin small RNA, StxS.
Collectively, our data indicates that EHEC contains hundreds of novel RNA binding proteins including many found within horizontally acquired pathogenicity islands. The T6SS immunity protein RhsFI affects the accumulation of a broad range of sRNAs with concomittent effects on virulence gene expression.