Use of Aptamer Tagging to Identify In Vivo Protein Binding Partners of Small Regulatory RNAs

Institute for Molecular Infection Biology, University of Würzburg, Würzburg, Germany.
Methods in molecular biology (Clifton, N.J.) (Impact Factor: 1.29). 06/2012; 905:177-200. DOI: 10.1007/978-1-61779-949-5_11
Source: PubMed


Small regulatory RNAs (sRNAs) are short, generally noncoding RNAs that act posttranscriptionally to control target gene expression. Over the past 10 years there has been a rapid expansion in the discovery and characterization of sRNAs in a diverse range of bacteria. Paradigm shifts in our understanding of the breadth of posttranscriptional control by sRNAs were achieved in a number of pioneering studies that involved immunoprecipitation of a known RNA chaperone, the near-ubiquitous Hfq, followed by sequencing to identify novel putative regulators and targets. To perform the converse experiment, we previously developed a method which uses an aptamer-tagged sRNA to allow purification of in vivo assembled RNA-protein complexes and subsequent identification of bound proteins. We successfully implemented this protocol using the Hfq-associated sRNA, InvR, tagged with a tandem repeat of the commonly used MS2-aptamer. Incorporation of the aptamer had no effect on sRNA stability or activity. InvR-MS2 could be effectively purified along with associated proteins, such as Hfq, using maltose binding protein fused to the MS2 coat protein (MBP-MS2) immobilized on an amylose column. Mass-spectroscopy was also used to identify previously uncharacterized protein partners. These results have been described previously (Said et al., Nucleic Acids Res 37:e133, 2009) and thus the figures presented here are intended solely as an illustrative guide to complement this detailed step-by-step protocol.

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    • "We fused the 5'-end of ITS metZ-metW and ITS metW-metV with bacteriophage MS2 RNA stemloops, which are bound by the MS2 coat protein with high specificity. This highly specific interaction was previously shown to allow affinity co-purification of sRNA-bound proteins from bacterial extracts (Corcoran et al., 2012). As controls, we used untagged ITS metZ-metW and ITS metW-metV expressed under the same conditions. "
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