A census of RNA/protein complexes in a model Gram-positive bacterium reveals exonuclease-mediated sRNA activation in competence regulation

RNA-protein interactions crucially underlie many steps of bacterial gene expression including post-transcriptional control by small regulatory RNAs (sRNAs). In stark contrast with recent progress in Gram-negative bacteria, knowledge about RNA and protein complexes in Gram-positive species remains scarce. Here, we used Grad-seq to draft a landscape of such complexes in Streptococcus pneumoniae, 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 (a.k.a. YhaM) with sRNAs that control bacterial competence. Contrary to expectation, the nucleolytic activity of Cbf1 stabilized these sRNAs, thereby promoting their function as repressors of competence. These results illustrate how this first RNA/protein complexome resource for a Gram-positive species can be utilized to identify new molecular factors in RNA-based regulation of pathways with relevance to bacterial virulence. 22 total RNA samples from S. pneumoniae wild type resolved on a glycerol gradient resulting in 20 fractions and a pellet fraction. A lysate sample is given as input control. Fraction numbers are sequential: 1 is the fraction with the low molecular weight complexes and with increasing sample number the molecular weight of the complexes increases.