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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Yeast strains and plasmids are available upon request. All sequencing data and oligonucleotide pool sequences have been deposited in GEO, accession GSE99487.
Custom Bash and Python codes used for analysis are available on request.
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We thank Y. Motorin (Université de Lorraine) for the yeast Pus1 expression plasmid and R. Stroud (University of California, San Francisco) for human Pus1 expression plasmids. We also thank G. Mawla and C. Mason for technical assistance and members of the Gilbert lab for critical reading of the manuscript. This work was supported by the National Institutes of Health (grant no. GM101316) (W.V.G.), American Cancer Society (grant no. RSG-13-396-01-RMC) (W.V.G.), American Cancer Society postdoctoral fellowship (T.M.C.), Jane Coffin Childs postdoctoral fellowship (N.M.M.), NSF graduate research fellowship (C.S.) and National Institutes of Health predoctoral training grant (no. T32GM007287) (T.A.B.).
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Tables 1–2 and Supplementary Figures 1–7
Pseduo-seq signal for S. cerevisiae RNA pools treated with S100 extracts.
Pseudo-seq signal for H. sapiens RNA pools pseudouridylated with recombinant human PUS proteins.
Structural characteristics of S. cerevisiae Pus1 substrates.
Kinetic analysis of pseudouridylation of wild-type and mutant sequences by recombinant Pus1.
Kinetic analysis of pseudouridylation of wild-type and stem extension mutant sequences by recombinant Pus1.
Random forest classifier predicted Pus1 mRNA Ψs.
Summary of libraries contained in this study.