RNA binding proteins (RBPs) play critical roles in controlling both 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. Four RBPs were verified by using polynucleotide kinase to end label RNA crosslinked to the protein. Surprisingly, one of these validated RBPs, RhsFI, is a type 6 secretion system (T6SS) immunity protein. Using CLIP-seq, RNA interaction sites for RhsFI were identified and validated using a filter binding assay. These data indicate that RhsFI has a similar RNA-binding activity to the small RNA chaperone, Hfq. Promoter mapping using dRNA-seq indicates that the immunity protein is transcribed independently of the T6SS effector and suggests that RhsFI may have been co-opted for gene regulation in EHEC.
In ongoing work, the function of RhsFI will be explored using transcriptome profiling of rhsFI mutants. It is not clear whether RhsFI interactions with small RNAs will compete for Hfq binding and effect the stability of target RNAs. To address this, we will determine target RNA half-lives in our mutant and wild type strains and assess competition with Hfq using filter binding assays.
Collectively, our data indicate that EHEC contains hundreds of novel RNA binding proteins including many found within horizontally acquired pathogenicity islands. The T6SS immunity protein RhsFI appears to be expressed independently of the cognate T6 effector in EHEC and has been re-purposed for regulatory sRNA interactions.