Detection of RNA–Protein Interactions in Living Cells with SHAPE
| Autor: | J. Mauro Calabrese, Matthew J. Smola, Kevin M. Weeks |
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| Jazyk: | angličtina |
| Rok vydání: | 2015 |
| Předmět: |
Models
Molecular Protein Conformation Acylation Computational biology Biology medicine.disease_cause Biochemistry Article Mice Protein structure medicine Animals Binding site Nucleic acid structure Cells Cultured Embryonic Stem Cells Ribonucleoprotein Genetics Mutation Binding Sites Proteins RNA Embryonic stem cell Ribonucleoproteins Cytoplasm Nucleic Acid Conformation |
| DOI: | 10.17615/9wsd-qw92 |
| Popis: | 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 enable 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-minute 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 non-coding 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 pre-existing knowledge of the proteins or RNAs involved. |
| Databáze: | OpenAIRE |
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