iDRiP for the systematic discovery of proteins bound directly to noncoding RNA
Journal
Nature Protocols
Journal Volume
16
Journal Issue
7
Pages
3672 - 3694
Date Issued
2021
Author(s)
Minajigi, Anand
Chen, Yunfei
Morris, Robert
Guh, Chia-Yu
Hsieh, Yu-Hung
Boukhali, Myriam
Haas, Wilhelm
Lee, Jeannie T.
Abstract
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. ? 2021, The Author(s), under exclusive licence to Springer Nature Limited.
SDGs
Type
review