iDRiP for the systematic discovery of proteins bound directly to noncoding RNA

Autor: Jeannie T. Lee, Yunfei Chen, Robert Morris, Myriam Boukhali, Anand Minajigi, Chia-Yu Guh, Wilhelm Haas, Hsueh-Ping Chu, Yu-Hung Hsieh
Rok vydání: 2021
Předmět:
Zdroj: Nature Protocols. 16:3672-3694
ISSN: 1750-2799
1754-2189
Popis: More than 90% of the human genome is transcribed into noncoding RNAs, but their functional characterization has lagged behind. A major bottleneck in the understanding of their functions and mechanisms has been a dearth of systematic methods for identifying interacting protein partners. There now exist several methods, including identification of direct RNA interacting proteins (iDRiP), chromatin isolation by RNA purification (ChIRP), and RNA antisense purification, each previously applied towards identifying a proteome for the prototype noncoding RNA, Xist. iDRiP has recently been modified to successfully identify proteomes for two additional noncoding RNAs of interest, TERRA and U1 RNA. Here we describe the modified protocol in detail, highlighting technical differences that facilitate capture of various noncoding RNAs. The protocol can be applied to short and long RNAs in both cultured cells and tissues, and requires ~1 week from start to finish. Here we also perform a comparative analysis between iDRiP and ChIRP. We obtain partially overlapping profiles, but find that iDRiP yields a greater number of specific proteins and fewer mitochondrial contaminants. With an increasing number of essential long noncoding RNAs being described, robust RNA-centric protein capture methods are critical for the probing of noncoding RNA function and mechanism. This protocol describes how to identify direct protein targets of noncoding RNAs—Xist, TERRA and U1—and outlines modifications specific to each noncoding RNA and its partners.
Databáze: OpenAIRE
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