mRNA structure determines modification by pseudouridine synthase 1

Nat Chem Biol. 2019 Oct;15(10):966-974. doi: 10.1038/s41589-019-0353-z.

Thomas M Carlile ¹ ², Nicole M Martinez ³, Cassandra Schaening ¹, Amanda Su ¹ ⁴, Tristan A Bell ¹, Boris Zinshteyn ¹ ⁵, Wendy V Gilbert ⁶

Affiliations

1 Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA.
2 Biogen, Cambridge, MA, USA.
3 Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA.
4 University of California, Berkeley, Berkeley, CA, USA.
5 John Hopkins School of Medicine, Baltimore, MD, USA.
6 Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA. wendy.gilbert@yale.edu.

PMID: 31477916 DOI: 10.1038/s41589-019-0353-z

Abstract

Pseudouridine (Ψ) is a post-transcriptional RNA modification that alters RNA-RNA and RNA-protein interactions that affect gene expression. Messenger RNA pseudouridylation was recently discovered as a widespread and conserved phenomenon, but the mechanisms responsible for selective, regulated pseudouridylation of specific sequences within mRNAs were unknown. Here, we have revealed mRNA targets for five pseudouridine synthases and probed the determinants of mRNA target recognition by the predominant mRNA pseudouridylating Enzyme, Pus1, by developing high-throughput kinetic analysis of pseudouridylation in vitro. Combining computational prediction and rational mutational analysis revealed an RNA structural motif that is both necessary and sufficient for mRNA pseudouridylation. Applying this structural context information predicted hundreds of additional mRNA targets that were pseudouridylated in vivo. These results demonstrate a structure-dependent mode of mRNA target recognition by a conserved pseudouridine synthase and implicate modulation of RNA structure as the probable mechanism to regulate mRNA pseudouridylation.

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