title: Detection of RNA-Protein Interactions in Living Cells with SHAPE.
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.
authors.affiliation: Department of Chemistry, University of North Carolina , Chapel Hill, North Carolina 27599-3290, United States.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Smola
authors.affiliation: Department of Pharmacology and Lineberger Comprehensive Cancer Center, University of North Carolina , Chapel Hill, North Carolina 27599, United States.
authors.forename: J Mauro
authors.initials: JM
authors.lastname: Calabrese
authors.affiliation: Department of Chemistry, University of North Carolina , Chapel Hill, North Carolina 27599-3290, United States.
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Weeks
mesh_terms: D000215:Acylation; D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D012313:RNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D011506:Proteins; D012261:Ribonucleoproteins; D012313:RNA