Detection of RNA鈥揚rotein Interactions in Living Cells with SHAPE

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• 作者：Matthew J. Smola ; J. Mauro Calabrese ; Kevin M. Weeks
• 刊名：Biochemistry
• 出版年：2015
• 出版时间：November 24, 2015
• 年：2015
• 卷：54
• 期：46
• 页码：6867-6875
• 全文大小：610K
• ISSN：1520-4995

文摘

SHAPE-MaP is unique among RNA structure probing strategies in that it both measures flexibility at single-nucleotide resolution and quantifies the uncertainties in these measurements. We report a straightforward analytical framework that incorporates these uncertainties to allow detection of RNA structural differences between any two states, and we use it here to detect RNA鈥損rotein interactions in healthy mouse trophoblast stem cells. We validate this approach by analysis of three model cytoplasmic and nuclear ribonucleoprotein complexes, in 2 min in-cell probing experiments. In contrast, data produced by alternative in-cell SHAPE probing methods correlate poorly (r = 0.2) with those generated by SHAPE-MaP and do not yield accurate signals for RNA鈥損rotein interactions. We then examine RNA鈥損rotein and RNA鈥搒ubstrate interactions in the RNase MRP complex and, by comparing in-cell interaction sites with disease-associated mutations, characterize these noncoding mutations in terms of molecular phenotype. Together, these results reveal that SHAPE-MaP can define true interaction sites and infer RNA functions under native cellular conditions with limited preexisting knowledge of the proteins or RNAs involved.