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

Area of Science:

• Bacteriology
• Molecular Biology
• Genomics

Background:

• RNA-binding proteins (RBPs) are essential for bacterial survival and cellular processes.
• Global identification of bacterial RBPs remains challenging, with limited studies available.

Purpose of the Study:

• To adapt and apply the RNA interactome capture (RIC) technique for global RBP identification in bacteria.
• To discover novel bacterial RBPs and characterize the conserved protein YhgF.

Main Methods:

• Adapted eukaryotic RNA interactome capture (RIC) technique for bacterial systems.
• Utilized overexpressed poly(A) polymerase I in Escherichia coli to enhance RNA polyadenylation.
• Employed immobilized oligo(dT) for pull-down of crosslinked RNA-protein complexes.
• Used CLIP-seq for in vivo identification of YhgF RNA targets.

Main Results:

• Identified 169 putative bacterial RBPs, with approximately half being previously uncharacterized.
• Experimentally validated RNA-binding capabilities of several novel RBPs, including the highly conserved YhgF.
• Confirmed YhgF's RNA targets in vivo and in vitro, suggesting a role in gene expression regulation.

Conclusions:

• Presented a robust strategy for global bacterial RBP identification.
• Established a valuable resource of newly identified bacterial RBPs.
• Laid the groundwork for further investigation into the conserved RBP YhgF and its regulatory functions.