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Biochemistry. 2015 Nov 24;54(46):6867-75. doi: 10.1021/acs.biochem.5b00977. Epub 2015 Nov 11.

Detection of RNA-Protein Interactions in Living Cells with SHAPE.

Author information

1
Department of Chemistry, University of North Carolina , Chapel Hill, North Carolina 27599-3290, United States.
2
Department of Pharmacology and Lineberger Comprehensive Cancer Center, University of North Carolina , Chapel Hill, North Carolina 27599, United States.

Abstract

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-protein 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-protein interactions. We then examine RNA-protein and RNA-substrate 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.

PMID:
26544910
PMCID:
PMC4900165
DOI:
10.1021/acs.biochem.5b00977
[Indexed for MEDLINE]
Free PMC Article

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