Martins, DiogoSalgueiro, BrunoSobral, DanielGragera, MarcosHensel, ZachHenriques, Adriano O.Romão, Célia V.Serrano, Mónica2025-12-032025-12-032025-07-08Nucleic Acids Res. 2025 Jul 8;53(13):gkaf644. doi: 10.1093/nar/gkaf6440305-1048http://hdl.handle.net/10400.18/10635Clostridioides difficile CD25890 is a YicC-like endoribonuclease involved in regulating sporulation initiation, a process critical for the host-host transmission of this anaerobic pathogen. Using comparative transcriptomics we identified a small RNA, SQ528, that accumulates at higher levels in a CD25890 deletion mutant and we show that purified CD25890 cleaves SQ528 in a metal-dependent manner. Moreover, the overexpression of SQ528 increases sporulation under certain nutritional conditions phenocopying a CD25890 deletion mutant. CD25890 is an hexamer in solution and in vivo. An N-terminal domain, which self-interacts as assessed by size exclusion chromatography and a two hybrid assay, is essential for oligomerization of CD25890. A C-terminal domain harbours residues H230, E254, and E258, conserved among orthologues, important for catalysis. AlphaFold2 modelling and cryo-EM suggest an elongated barrel-like structure with an internal cavity lined with basic residues that may aid in RNA binding. We show that CD25890 forms a complex with polynucleotide phosphorylase which combines the endoribonuclease activity of the first with the exonucleolytic activity of the latter and leads to the complete degradation of SQ528. This study identifies a native substrate for the YicC-family of ribonucleases and advances our understanding of the role of CD25890 in sporulation initiation in C. difficile.engClostridioides DifficileEndoribonucleasesBacterial ProteinsGene Expression RegulationBacterialRNAGeneticsGenómica Funcional e Estruturaljournal article10.1093/nar/gkaf6441362-496240650975