Identification of RNAIII-binding proteins in Staphylococcus aureus using tethered RNAs and streptavidin aptamers based pull-down assay.

Xu Zhang,Qing Zhu,Baolin Sun,Changlong Zhao,Ting Xue,Jianye Zang,Tian Tian

doi:10.1186/s12866-015-0435-3

Abstract

BackgroundIt has been widely recognized that small RNAs (sRNAs) play important roles in physiology and virulence control in bacteria. In Staphylococcus aureus, many sRNAs have been identified and some of them have been functionally studied. Since it is difficult to identify RNA-binding proteins (RBPs), very little has been known about the RBPs in S. aureus, especially those associated with sRNAs.ResultsHere we adopted a tRNA scaffold streptavidin aptamer based pull-down assay to identify RBPs in S. aureus. The tethered RNA was successfully captured by the streptavidin magnetic beads, and proteins binding to RNAIII were isolated and analyzed by mass spectrometry. We have identified 81 proteins, and expressed heterologously 9 of them in Escherichia coli. The binding ability of the recombinant proteins with RNAIII was further analyzed by electrophoresis mobility shift assay, and the result indicates that proteins CshA, RNase J2, Era, Hu, WalR, Pyk, and FtsZ can bind to RNAIII.ConclusionsThis study suggests that some proteins can bind to RNA III in S. aureus, and may be involved in RNA III function. And tRSA based pull-down assay is an effective method to search for RBPs in bacteria, which should facilitate the identification and functional study of RBPs in diverse bacterial species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0435-3) contains supplementary material, which is available to authorized users.

Highlights

It has been widely recognized that small RNAs play important roles in physiology and virulence control in bacteria
The tethered RNAs were successfully captured by Streptavidin MagneSphere Paramagnetic Particles (SA-PMPs), and those proteins binding to RNAIII were isolated and analyzed by mass spectrometry (MS)
Streptavidin aptamer fusion in the anti-code site of tRNA was captured by streptavidin PMPs, while RNAIII attached to the 3′ site of tRNA can act as a bait for RNA-binding proteins (RBPs) (Fig. 1A)

Summary

Introduction

It has been widely recognized that small RNAs (sRNAs) play important roles in physiology and virulence control in bacteria. The high infection ability of the bacterium depends on the production of many virulence factors, which are under control of multiple regulatory pathways. These regulators include transcriptional regulator proteins, two-component systems, and small RNAs (sRNAs). Hundreds of sRNAs have been identified in S. aureus, but very little has been known about RNA-binding proteins (RBPs) involved in sRNA regulation in this bacterium. RNase III is the only ribonuclease proved to be important for sRNA regulation in S. aureus, and it degrades double-stranded RNA formed by base-pairing of RNAIII with its mRNA targets such as spa, coa, and rot [6].

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