Choi SunKyung, Park Chungoo, Kim Kyoon Eon, Kim Kee K
Department of Biochemistry, College of Natural Sciences, Chungnam National University, Daejeon, Republic of Korea.
School of Biological Sciences and Technology, Chonnam National University, Gwangju, Republic of Korea.
Biotechniques. 2017 Jul 1;63(1):28-33. doi: 10.2144/000114567.
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.
RNA-蛋白质相互作用在基因调控中起着重要作用。尽管已经开发了许多分析RNA-蛋白质相互作用的技术，但诸如确定与RNA结合蛋白（RBP）结合的RNA序列等值得注意的挑战仍然未得到解决。在这里，我们描述了一种使用掺入4-硫代尿苷的RNA文库的新技术，以鉴定通过体外转录和翻译产生的RBP的RBP结合共有序列。为了确认这种方法的准确性，我们确定了RBFOX2的共有RBP结合序列UGC(A/U)(A/U)NU，它与已知的RBFOX2结合序列UGCAUG非常相似。使用我们的方法，确定了三种RBP的共有RBP结合序列，即FUS（肉瘤融合蛋白）、SFPQ（富含脯氨酸和谷氨酰胺的剪接因子）和SAM68（有丝分裂68 kDa的Src相关底物）。这些RBP的共有RBP结合序列通过RNA-蛋白质复合物免疫沉淀-PCR分析得到了证实。
