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Published September 25, 2014 | Supplemental Material + Accepted Version
Journal Article Open

Transcriptome-wide Mapping Reveals Widespread Dynamic-Regulated Pseudouridylation of ncRNA and mRNA

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.

Additional Information

© 2014 Elsevier Inc. Received: July 30, 2014; Revised: August 21, 2014; Accepted: August 22, 2014; Published: September 11, 2014. Work was supported by NHGRI P50HG006193, Pioneer Award, HHMI (A.R.), NHGRI U54 HG003067 (E.S.L.) Broad Institute Funds, NIH GM035010 (G.F.), HFSP fellowship (S.S.), NIH 1F32HD075541-01 (R.S.), and fellowships of the Swiss National Science Foundation and the Marie Curie IOF (M.J.). Author Contributions: S.S., D.B., M.R.M., E.S.L., G.F., and A.R. conceived and designed the study. S.S. led the development of experimental and computational methods. D.A.B. developed and executed all yeast experiments. M.R.M. developed and conducted all genomic assays and mammalian experiments. M.J. conducted proteomics experiments. R.H.B., R.S., and B.X.L.-R. analyzed data. J.M.E. and M.G. developed the RNA hybrid selection protocol. E.S.L., G.F., and A.R. guided work. S.S. and A.R. wrote the manuscript with input from all authors. Accession Numbers: Sequencing data have been deposited into the Gene Expression Omnibus (GEO) under the accession number GSE60047.

Attached Files

Accepted Version - nihms624611.pdf

Supplemental Material - mmc1.xlsx

Supplemental Material - mmc2.xlsx

Supplemental Material - mmc3.xlsx

Supplemental Material - mmc4.pdf

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Additional details

Created:
August 20, 2023
Modified:
October 17, 2023