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Cell. 2014 Sep 25;159(1):148-162. doi: 10.1016/j.cell.2014.08.028. Epub 2014 Sep 11.

Transcriptome-wide mapping reveals widespread dynamic-regulated pseudouridylation of ncRNA and mRNA.

Author information

1
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
2
Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA.
3
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Systems Biology, Harvard Medical School, Boston, MA 02114, USA.
4
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Biology, University of Puerto Rico, Rio Piedras Campus, San Juan 00931, Puerto Rico.
5
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Division of Health Sciences and Technology, MIT, Cambridge, MA 02139, USA.
6
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
7
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Systems Biology, Harvard Medical School, Boston, MA 02114, USA; Department of Biology, MIT, Cambridge, MA 02139, USA.
8
Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA; Department of Biology, MIT, Cambridge, MA 02139, USA. Electronic address: gfink@wi.mit.edu.
9
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Biology, MIT, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, 4000 Jones Bridge Road, Chevy Chase, MD 20815, USA. Electronic address: aregev@broad.mit.edu.

Abstract

Pseudouridine is the most abundant RNA modification, yet except for a few well-studied cases, little is known about the modified positions and their function(s). Here, we develop Ψ-seq for transcriptome-wide quantitative mapping of pseudouridine. We validate Ψ-seq with spike-ins and de novo identification of previously reported positions and discover hundreds of unique sites in human and yeast mRNAs and snoRNAs. Perturbing pseudouridine synthases (PUS) uncovers which pseudouridine synthase modifies each site and their target sequence features. mRNA pseudouridinylation depends on both site-specific and snoRNA-guided pseudouridine synthases. Upon heat shock in yeast, Pus7p-mediated pseudouridylation is induced at >200 sites, and PUS7 deletion decreases the levels of otherwise pseudouridylated mRNA, suggesting a role in enhancing transcript stability. rRNA pseudouridine stoichiometries are conserved but reduced in cells from dyskeratosis congenita patients, where the PUS DKC1 is mutated. Our work identifies an enhanced, transcriptome-wide scope for pseudouridine and methods to dissect its underlying mechanisms and function.

PMID:
25219674
PMCID:
PMC4180118
DOI:
10.1016/j.cell.2014.08.028
[Indexed for MEDLINE]
Free PMC Article

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