[SCAN] A novel endoribonuclease modulates sporulation initiation in Clostridioides difficile

Title: A novel endoribonuclease modulates sporulation initiation in
Clostridioides difficile

Speaker: Mónica Serrano

Abstract: The YicC-like family of proteins is widespread in eubacteria and includes Escherichia coli YicC and Bacillus subtilis YloC. This family includes poorly characterized proteins that play a role in survival during the stationary phase of growth. The E. coli and B. subtilis proteins were recently described as a novel type of endoribonucleases with a role in silencing small RNAs. We show that a Clostridioides difficile YicC/YloC homologue, CsiA, is involved in the control of sporulation initiation. Comparative transcriptomic analysis identifies two small RNAs that are upregulated in a csiA deletion mutant compared to the wild type, suggesting that they are CsiA substrates. While with a predicted MW of 35.4 kDa, we show that purified CsiA exists in solution predominantly as a 245 kDa oligomer, presumably an hexamer. These results were confirmed by CryoEM and further supported by bacterial two hybrid assays that detected strong self-interaction of CsiA. Moreover, CsiA also accumulates as an hexamer in vivo. Activity assays show that the oligomeric CsiA has endoribonuclease activity and importantly, it cleaves one of the small RNAs upregulated in the csiA mutant. In agreement with the CryoEM studies, limited trypsin proteolysis indicates that CsiA has an N terminal domain which we show binds RNA and a C-terminal domain that harbors the catalytic center. Ongoing work aims at understanding the molecular details of action of this new family of endoribonucleases.