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

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.

Advantages and limitations of UV cross‐linking analysis of protein–RNA interactomes in microbes

“…Since this technique specifically detects proteins bound to eukaryotic messenger RNAs, it is unsuitable to examine RBPs recognising transcripts lacking (long) poly-A tails (e.g., eukaryotic ribosomal, transfer and small nucleolar RNAs as well as prokaryotic transcripts in general). However, this issue can be overcome by transiently overexpressing a poly-A polymerase (Stenum et al, 2023).…”

“…Since this technique specifically detects proteins bound to eukaryotic messenger RNAs, it is unsuitable to examine RBPs recognising transcripts lacking (long) poly‐A tails (e.g., eukaryotic ribosomal, transfer and small nucleolar RNAs as well as prokaryotic transcripts in general). However, this issue can be overcome by transiently overexpressing a poly‐A polymerase (Stenum et al, 2023). Click chemistry‐based methods (click chemistry‐assisted RNA‐interactome capture, CARIC; RNA interactome using click chemistry, RICK) have sought to circumvent this drawback by treating cells with synthetic uridine analogues (Bao et al, 2018; Huang et al, 2018).…”