mRNA structure determines modification by pseudouridine synthase 1


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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Fig. 1: A high-throughput in vitro pseudouridylation assay.
Fig. 2: Identification of human PUS mRNA substrates in vitro.
Fig. 3: A structural motif associated with Pus1 mRNA targets in yeast.
Fig. 4: Kinetic analysis reveals sequence features important for mRNA pseudouridylation by Pus1.
Fig. 5: The rate of mRNA pseudouridylation depends on stem length and stability.
Fig. 6: The Pus1 structural motif is sufficient for pseudouridylation.

Data availability

Yeast strains and plasmids are available upon request. All sequencing data and oligonucleotide pool sequences have been deposited in GEO, accession GSE99487.

Code availability

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.).

Author information

T.M.C., B.Z., T.A.B. and W.V.G. conceived and designed the experiments. T.M.C., N.M.M., A.S., T.A.B. and W.V.G. performed the experiments. T.M.C., N.M.M. and C.S. performed the bioinformatic analyses. T.M.C. and W.V.G. interpreted the results and wrote the paper with input from all authors.

Correspondence to Wendy V. Gilbert.

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The authors declare no competing interests.

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Supplementary information

Supplementary Information

Supplementary Tables 1–2 and Supplementary Figures 1–7

Reporting Summary

Supplementary Dataset 1

Pseduo-seq signal for S. cerevisiae RNA pools treated with S100 extracts.

Supplementary Dataset 2

Pseudo-seq signal for H. sapiens RNA pools pseudouridylated with recombinant human PUS proteins.

Supplementary Dataset 3

Structural characteristics of S. cerevisiae Pus1 substrates.

Supplementary Dataset 4

Kinetic analysis of pseudouridylation of wild-type and mutant sequences by recombinant Pus1.

Supplementary Dataset 5

Kinetic analysis of pseudouridylation of wild-type and stem extension mutant sequences by recombinant Pus1.

Supplementary Dataset 6

Random forest classifier predicted Pus1 mRNA Ψs.

Supplementary Dataset 7

Summary of libraries contained in this study.

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