Zhang Xu, Zhu Qing, Tian Tian, Zhao Changlong, Zang Jianye, Xue Ting, Sun Baolin
School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, 230027, China.
School of Life Sciences, Anhui Agricultural University, Hefei, Anhui, 230036, China.
BMC Microbiol. 2015 May 15;15:102. doi: 10.1186/s12866-015-0435-3.
It 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.
Here 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.
This 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.
人们已经广泛认识到小RNA（sRNA）在细菌的生理和毒力控制中发挥着重要作用。在金黄色葡萄球菌中，已经鉴定出许多sRNA，并且其中一些已经进行了功能研究。由于难以鉴定RNA结合蛋白（RBP），因此对金黄色葡萄球菌中的RBP了解甚少，尤其是那些与sRNA相关的RBP。
在这里，我们采用基于tRNA支架链霉亲和素适体的下拉试验来鉴定金黄色葡萄球菌中的RBP。链霉亲和素磁珠成功捕获了拴系的RNA，并通过质谱分析了与RNAIII结合的蛋白质。我们鉴定出了81种蛋白质，并在大肠杆菌中异源表达了其中9种。通过电泳迁移率变动分析进一步分析了重组蛋白与RNAIII的结合能力，结果表明蛋白质CshA、RNase J2、Era、Hu、WalR、Pyk和FtsZ可以与RNAIII结合。
本研究表明，金黄色葡萄球菌中的一些蛋白质可以与RNA III结合，并可能参与RNA III的功能。基于tRSA的下拉试验是在细菌中寻找RBP的有效方法，这将有助于鉴定和研究不同细菌物种中的RBP及其功能。
