Pull-down of Biotinylated RNA and Associated Proteins.
| Autor: | Uppala JK; Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, 3209 N Maryland Ave, WI-53211, USA., Ghosh C; Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, 3209 N Maryland Ave, WI-53211, USA., Sabat G; Biotechnology Center, University of Wisconsin-Madison, Madison, Wisconsin, 53706, USA., Dey M; Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, 3209 N Maryland Ave, WI-53211, USA. |
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| Jazyk: | angličtina |
| Zdroj: | Bio-protocol [Bio Protoc] 2022 Feb 20; Vol. 12 (4), pp. e4331. Date of Electronic Publication: 2022 Feb 20 (Print Publication: 2022). |
| DOI: | 10.21769/BioProtoc.4331 |
| Abstrakt: | Mapping networks of RNA-protein interactions in cells is essential for understanding the inner workings of many biological processes, including RNA processing, trafficking, and translation. Current in vivo methods for studying protein-RNA interactions rely mostly on purification of poly(A) transcripts, which represent only ~2-3% of total RNAs ( Figure 1 ). Alternate robust methods for tagging RNA molecules with an RNA aptamer ( e.g. , MS2-, U1A- and biotin-RNA aptamer) and capturing the RNA-protein complex by the respective aptamer-specific partner are not extensively studied. Here, we describe a protocol ( Figure 2 ) in which a biotin-RNA aptamer, referred to as the RNA mimic of biotin (RMB), was conjugated separately to two small RNA secondary structures that contribute to trafficking and translating HAC1 mRNA in the budding yeast Saccharomyces cerevisiae . The RMB-tagged RNA was expressed in yeast cells from a constitutive promoter. The biotinylated RNA bound to proteins was pulled down from the cell lysate by streptavidin agarose beads. RNA was detected by RT-PCR ( Figure 3 ) and associated proteins by mass spectrometry ( Figure 4 ). Our findings show that an RNA aptamer tag to RNA molecule is an effective method to explore the functional roles of RNA-protein networks in vivo . Competing Interests: Competing interestsAuthors declare that they have no conflict of interests. (Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC.) |
| Databáze: | MEDLINE |
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