Identification of RNAs bound by Hfq reveals widespread RNA partners and a sporulation regulator in the human pathogen <i>Clostridioides difficile</i>

Noncoding RNAs (ncRNA) have emerged as important components of regulatory networks governing bacterial physiology and virulence. Previous deep-sequencing analysis identified a large diversity of ncRNAs in the human enteropathogen <i>Clostridioides (Clostridium) difficile</i>. Some of them are <i>trans</i>-encoded RNAs that could require the RNA chaperone protein Hfq for their action. Recent analysis suggested a pleiotropic role of Hfq in <i>C. difficile</i> with the most pronounced effect on sporulation, a key process during the infectious cycle of this pathogen. However, a global view of RNAs interacting with <i>C. difficile</i> Hfq is missing. In the present study, we performed RNA immunoprecipitation high-throughput sequencing (RIP-Seq) to identify Hfq-associated RNAs in <i>C. difficile</i>. Our work revealed a large set of Hfq-interacting mRNAs and ncRNAs, including mRNA leaders and coding regions, known and potential new ncRNAs. In addition to <i>trans</i>-encoded RNAs, new categories of Hfq ligands were found including <i>cis</i>-antisense RNAs, riboswitches and CRISPR RNAs. ncRNA-mRNA and ncRNA-ncRNA pairings were postulated through computational predictions. Investigation of one of the Hfq-associated ncRNAs, RCd1, suggests that this RNA contributes to the control of late stages of sporulation in <i>C. difficile</i>. Altogether, these data provide essential molecular basis for further studies of post-transcriptional regulatory network in this enteropathogen.