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Nat Methods. 2017 Jan;14(1):75-82. doi: 10.1038/nmeth.4057. Epub 2016 Nov 7.

DMS-MaPseq for genome-wide or targeted RNA structure probing in vivo.

Author information

1
Department of Cellular and Molecular Pharmacology, California Institute of Quantitative Biology, Center for RNA Systems Biology, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, California, USA.
2
Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.
3
Institute for Cellular and Molecular Biology, Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas, USA.

Abstract

Coupling of structure-specific in vivo chemical modification to next-generation sequencing is transforming RNA secondary structure studies in living cells. The dominant strategy for detecting in vivo chemical modifications uses reverse transcriptase truncation products, which introduce biases and necessitate population-average assessments of RNA structure. Here we present dimethyl sulfate (DMS) mutational profiling with sequencing (DMS-MaPseq), which encodes DMS modifications as mismatches using a thermostable group II intron reverse transcriptase. DMS-MaPseq yields a high signal-to-noise ratio, can report multiple structural features per molecule, and allows both genome-wide studies and focused in vivo investigations of even low-abundance RNAs. We apply DMS-MaPseq for the first analysis of RNA structure within an animal tissue and to identify a functional structure involved in noncanonical translation initiation. Additionally, we use DMS-MaPseq to compare the in vivo structure of pre-mRNAs with their mature isoforms. These applications illustrate DMS-MaPseq's capacity to dramatically expand in vivo analysis of RNA structure.

PMID:
27819661
PMCID:
PMC5508988
DOI:
10.1038/nmeth.4057
[Indexed for MEDLINE]
Free PMC Article
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