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Proc Natl Acad Sci U S A. 2016 Sep 13;113(37):10322-7. doi: 10.1073/pnas.1600008113. Epub 2016 Aug 30.

SHAPE reveals transcript-wide interactions, complex structural domains, and protein interactions across the Xist lncRNA in living cells.

Author information

1
Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599;
2
Department of Pharmacology and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599;
3
Department of Molecular Genetics and Microbiology, Duke University, Durham, NC 27708.
4
Department of Pharmacology and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599; weeks@unc.edu jmcalabr@med.unc.edu.
5
Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599; weeks@unc.edu jmcalabr@med.unc.edu.

Abstract

The 18-kb Xist long noncoding RNA (lncRNA) is essential for X-chromosome inactivation during female eutherian mammalian development. Global structural architecture, cell-induced conformational changes, and protein-RNA interactions within Xist are poorly understood. We used selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) to examine these features of Xist at single-nucleotide resolution both in living cells and ex vivo. The Xist RNA forms complex well-defined secondary structure domains and the cellular environment strongly modulates the RNA structure, via motifs spanning one-half of all Xist nucleotides. The Xist RNA structure modulates protein interactions in cells via multiple mechanisms. For example, repeat-containing elements adopt accessible and dynamic structures that function as landing pads for protein cofactors. Structured RNA motifs create interaction domains for specific proteins and also sequester other motifs, such that only a subset of potential binding sites forms stable interactions. This work creates a broad quantitative framework for understanding structure-function interrelationships for Xist and other lncRNAs in cells.

KEYWORDS:

RNA structure; RNA–protein interaction; SHAPE-MaP; X-inactivation

PMID:
27578869
PMCID:
PMC5027438
DOI:
10.1073/pnas.1600008113
[Indexed for MEDLINE]
Free PMC Article

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