Global discovery of bacterial RNA-binding proteins by RNase-sensitive gradient profiles reports a new FinO domain protein

Abstract

RNA-binding proteins (RBPs) play important roles in bacterial gene expression and physiology but their true number and functional scope remain little understood even in model microbes. To advance global RBP discovery in bacteria, we here establish glycerol gradient sedimentation with RNase treatment and mass spectrometry (GradR). Applied to Salmonella enterica, GradR confirms many known RBPs such as CsrA, Hfq, and ProQ by their RNase-sensitive sedimentation profiles, and discovers the FopA protein as a new member of the emerging family of FinO/ProQ-like RBPs. FopA, encoded on resistance plasmid pCol1B9, primarily targets a small RNA associated with plasmid replication. The target suite of FopA dramatically differs from the related global RBP ProQ, revealing context-dependent selective RNA recognition by FinOdomain RBPs. Numerous other unexpected RNase-induced changes in gradient profiles suggest that cellular RNA helps to organize macromolecular complexes in bacteria. By enabling poly(A)-independent generic RBP discovery, GradR provides an important element in the quest to build a comprehensive catalog of microbial RBPs.

A Grad-seq View of RNA and Protein Complexes in Pseudomonas aeruginosa under Standard and Bacteriophage Predation Conditions

“…Normalization and dose-response curves. Relative protein abundance in gradient fractions were calculated by iBAQ, as described previously (75). Relative protein abundance was estimated by correcting for alterations in digestion and C 18 purification in fractions by normalization to human albumin (Table S1, norm.…”

“…Oligonucleotide probe labeling and Northern blotting. Oligonucleotides were radioactively labeled at 59-OH with [g-32 P]ATP by the T4-polynucleotide kinase, as reported previously (75). Northern probing was performed as previously reported (98).…”

The World of Stable Ribonucleoproteins and Its Mapping With Grad-Seq and Related Approaches

“…The ProQ research is now booming: from an obscure protein (unfairly) commanding only passing interest it has grown into a major topic of bacterial RNA biology ( Olejniczak and Storz, 2017 ; Holmqvist et al, 2020 ). ProQ homologues have been found in a number of proteobacteria ( Attaiech et al, 2016 ; Smirnov et al, 2016 ) and implicated in key physiological processes, including competence ( Sexton and Vogel, 2004 ; Attaiech et al, 2016 ; Durieux et al, 2019 ), virulence, chemotaxis, motility and biofilm formation ( Sheidy and Zielke, 2013 ; Westermann et al, 2019 ), metabolism ( Kunte et al, 1999 ; Smirnov et al, 2016 ), stress resistance, plasmid and core genome maintenance ( Smirnov et al, 2016 ; Bauriedl et al, 2020 ; Gerovac et al, 2020 ). Many known base-pairing sRNAs repressing their targets in- cis or in- trans turned out to be avid ProQ binders, but only in a handful of cases the role of this protein in regulation has been solved mechanistically ( Smirnov et al, 2017b ; Silva et al, 2019 ; Westermann et al, 2019 ), and an even larger number of ProQ-dependent sRNAs remain totally uncharacterized.…”

“…Their role as new RNA matchmakers, mechanistically distinct from Hfq, is being intensively studied, but the molecular details of the interplay between ProQ and its natural RNA ligands only begin to emerge ( Melamed et al, 2020 ). Structural and functional studies highlighted the importance of the conserved N-terminal FinO-like domain, which harbors most of the RNA-binding activity and places ProQ into a larger family of FinO-like RNA chaperones ( Chaulk et al, 2010 , 2011 ; Glover et al, 2015 ; Gonzalez et al, 2017b ; Eidelpes et al, 2020 ; Gerovac et al, 2020 , 2021 ; Immer et al, 2020 ; Pandey et al, 2020 ; Stein et al, 2020 ). However, the role of the C-terminal “Tudor-like” domain ( Figure 6 ), that contributes to the RNA chaperone activities of ProQ ( Chaulk et al, 2011 ; Gonzalez et al, 2017b ), does not cease to intrigue researchers.…”

An RNA-centric view on gut Bacteroidetes

“…As of now, we do not know whether the function of Bacteroidetes sRNAs depends on assisting chaperones or if regulatory RNAs work in a protein-independent manner in this phylum. Recent technological breakthroughs (Gerovac et al 2020;Queiroz et al 2019;Shchepachev et al 2019;Smirnov et al 2017;Urdaneta et al 2019) led to the identification of novel RBPs even in species that have served us as RNA research models for decades (Attaiech et al 2016;Pagliuso et al 2019;Smirnov et al 2016) and should likewise foster RBP discovery in Bacteroidetes (Figure 4c).…”