Grad‐seq in a Gram‐positive bacterium reveals exonucleolytic sRNA activation in competence control

RNA–protein interactions are the crucial basis for many steps of bacterial gene expression, including post‐transcriptional control by small regulatory RNAs (sRNAs). In stark contrast to recent progress in the analysis of Gram‐negative bacteria, knowledge about RNA–protein complexes in Gram‐positive species remains scarce. Here, we used the Grad‐seq approach to draft a comprehensive landscape of such complexes in Streptococcus pneumoniae , in total determining the sedimentation profiles of ~ 88% of the transcripts and ~ 62% of the proteins of this important human pathogen. Analysis of in‐gradient distributions and subsequent tag‐based protein capture identified interactions of the exoribonuclease Cbf1/YhaM with sRNAs that control bacterial competence for DNA uptake. Unexpectedly, the nucleolytic activity of Cbf1 stabilizes these sRNAs, thereby promoting their function as repressors of competence. Overall, these results provide the first RNA/protein complexome resource of a Gram‐positive species and illustrate how this can be utilized to identify new molecular factors with functions in RNA‐based regulation of virulence‐relevant pathways. Synopsis Application of Grad‐seq analysis to Streptococcus pneumoniae provides the first cellular RNA/protein complexome resource for a Gram‐positive bacterium, and uncovers a specific exonuclease as a new player in the competence regulon for DNA uptake and pneumococcal virulence. Grad‐seq analyses predict complexes for thousands of Streptococcus pneumoniae RNAs and proteins. Grad‐seq‐predicted complexes aid functional characterization also for other Gram‐positive bacteria. Cbf1 is identified as an exonuclease that trims and stabilizes small RNAs in the competence regulon. Cbf1 acts as a negative regulator of competence in S. pneumoniae . Graphical Abstract Comprehensive assessment of protein‐RNA complexes in Streptococcus pneumoniae via Grad‐seq uncovers an unexpected role for the exoribonuclease Cbf1 in stabilizing sRNAs that control bacterial competence for DNA uptake.