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.

A global survey of small RNA interactors identifies KhpA and KhpB as major RNA-binding proteins inFusobacterium nucleatum

“…We also recovered proteins involved in metabolic processes, consistent with prior RBP discovery studies that suggested that many metabolic enzymes moonlight as RBPs (45,60). For example, we find that the acyl carrier protein AcpP, which has a crucial role in bacterial fatty acid synthesis, was recovered by the majority of F. nucleatum sRNA baits.…”

“…For example, we find that the acyl carrier protein AcpP, which has a crucial role in bacterial fatty acid synthesis, was recovered by the majority of F. nucleatum sRNA baits. Curiously, the AcpP protein was also listed as a strong RBP candidate in E. coli based on analysis of the RNA bound proteome using global methods, such as TRAPP (total RNA-associated protein purification) (61) and RIC (RNA-interactome capture) (60). These observations suggest that AcpP may serve as an RBP in different bacteria, in addition to its core function in fatty acid biosynthesis.…”

RNA-binding proteins identified by R-DeeP/TripepSVM are involved in heterocyst differentiation

“…We used PNK assays 33 to test in vivo the RNA-binding capacity of proteins from two subgroups shown in Fig. 3d; proteins shifting with high SVM scores (putative new RBPs) and proteins shifting but with low SVM scores (RNA-associated proteins).…”

“…The PNK assays were carried out as previously described 33 with some modifications. 400 mL of cultures from reporter strains expressing 3xFLAG protein fusions were grown in BG11 supplemented with the appropriate antibiotic until they they reached a concentration of 2.5 µg chlorophyll/mL.…”

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).…”