An in vitro technique to identify the RNA binding-site sequences for RNA-binding proteins. (July 2017)

• Record Type: Journal Article
• Authors: Choi, SunKyung; Park, Chungoo; Kim, Kyoon Eon; Kim, Kee K.
• Is Part Of: Biotechniques. Volume 63:Number 1(2017)
• Page Start: 28
• Page End: 33
• Publication Date: 2017-07
• Subjects: RNA-binding protein (RBP) -- RBFOX2 -- FUS -- SAM68 -- SFPQ; Biology, Experimental -- Periodicals; Molecular biology -- Periodicals; Medical technology -- Periodicals; Biology, Experimental; Medical technology; Molecular biology; Clinical Laboratory Techniques -- Periodicals; Research -- Periodicals; Medical Laboratory Science -- Periodicals; Periodicals; Electronic journals; 570
• DOI: 10.2144/000114567
• Languages: English
• ISSNs: 0736-6205

Abstract

RNA–protein interactions play a major role in gene regulation. Although many techniques to analyze RNA–protein interactions have been developed, noteworthy challenges such as determining the RNA sequences that bind RNA-binding proteins (RBPs) remain unsolved. Here, we describe a novel technique using a 4-thio-uridine-incorporated RNA pool to identify the RBP-binding consensus sequences for RBPs produced by in vitro transcription and translation. To confirm the fidelity of this approach, we determined the consensus RBP-binding sequence for RBFOX2, UGC(A/U)(A/U)NU, which is very similar to the known RBFOX2-binding sequence, UGCAUG. Using our method, consensus RBP-binding sequences were determined for three RBPs, namely FUS (fused in sarcoma), SFPQ (splicing factor proline and glutamine rich), and SAM68 (Src-Associated substrate in Mitosis 68 kDa). The consensus RBP-binding sequences for these RBPs were confirmed by RNA–protein complex immunoprecipitation–PCR analysis.