RNA interactome capture in Escherichia coli globally identifies RNA-binding proteins

• Published in: Nucleic acids research 2023-05, Vol.51 (9), p.4572-4587
• Main Authors: Stenum, Thomas Søndergaard, Kumar, Ankith D, Sandbaumhüter, Friederike A, Kjellin, Jonas, Jerlström-Hultqvist, Jon, Andrén, Per E, Koskiniemi, Sanna, Jansson, Erik T, Holmqvist, Erik
• Format: Article
• Language: English
• Subjects: Chromatin Immunoprecipitation Sequencing; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - analysis; Escherichia coli Proteins - metabolism; Eukaryota; Polyadenylation; Polynucleotide Adenylyltransferase - metabolism; Protein Binding; RNA and RNA-protein complexes; RNA, Bacterial - genetics; RNA, Bacterial - metabolism; RNA-Binding Proteins - analysis; RNA-Binding Proteins - metabolism; Transcriptome
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/gkad216

Abstract RNA-binding proteins (RPBs) are deeply involved in fundamental cellular processes in bacteria and are vital for their survival. Despite this, few studies have so far been dedicated to direct and global identification of bacterial RBPs. We have adapted the RNA interactome capture (RIC) technique, originally developed for eukaryotic systems, to globally identify RBPs in bacteria. RIC takes advantage of the base pairing potential of poly(A) tails to pull-down RNA–protein complexes. Overexpressing poly(A) polymerase I in Escherichia coli drastically increased transcriptome-wide RNA polyadenylation, enabling pull-down of crosslinked RNA–protein complexes using immobilized oligo(dT) as bait. With this approach, we identified 169 putative RBPs, roughly half of which are already annotated as RNA-binding. We experimentally verified the RNA-binding ability of a number of uncharacterized RBPs, including YhgF, which is exceptionally well conserved not only in bacteria, but also in archaea and eukaryotes. We identified YhgF RNA targets in vivo using CLIP-seq, verified specific binding in vitro, and reveal a putative role for YhgF in regulation of gene expression. Our findings present a simple and robust strategy for RBP identification in bacteria, provide a resource of new bacterial RBPs, and lay the foundation for further studies of the highly conserved RBP YhgF.