Abstract
The epitranscriptome has emerged as a new fundamental layer of control of gene expression. Nevertheless, the determination of the transcriptome-wide occupancy and function of RNA modifications remains challenging. Here we have developed Rho-seq, an integrated pipeline detecting a range of modifications through differential modification-dependent rhodamine labeling. Using Rho-seq, we confirm that the reduction of uridine to dihydrouridine (D) by the Dus reductase enzymes targets tRNAs in E. coli and fission yeast. We find that the D modification is also present on fission yeast mRNAs, particularly those encoding cytoskeleton-related proteins, which is supported by large-scale proteome analyses and ribosome profiling. We show that the α-tubulin encoding mRNA nda2 undergoes Dus3-dependent dihydrouridylation, which affects its translation. The absence of the modification on nda2 mRNA strongly impacts meiotic chromosome segregation, resulting in low gamete viability. Applying Rho-seq to human cells revealed that tubulin mRNA dihydrouridylation is evolutionarily conserved.
Original language | English |
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Pages (from-to) | 404-419.e9 |
Journal | Molecular Cell |
Volume | 82 |
Issue number | 2 |
Early online date | 18 Nov 2021 |
DOIs | |
Publication status | Published - 20 Jan 2022 |
Funding
We are grateful to Eric Phizicky for reagents and Marc Graille for comments on the manuscript. Research in the lab of D.L.J.L. is supported by the Fonds de la Recherche Nationale (F.R.S./FNRS), the Universit\u00E9 Libre de Bruxelles (ULB), the European Joint Programme on Rare Diseases (EJP-RD; RiboEurope and DBAGeneCure), the R\u00E9gion Wallone (RiboCancer), and the International Brachet Stift\u00FCng (IBS). High-throughput sequencing was performed by the NGS platform of Institut Curie, supported by grants from the Agence Nationale de la Recherche (investissements d\u2019avenir; ANR-10-EQPX-03 and ANR10-INBS-09-08 ) and by the Cancerop\u00F4le Ile-de-France . This work was also supported by NIH grants ES031529 and AG063341 (to P.D.) and PDR T.0012.14 , CDR J.0066.16 , and PDR T.0112.21 (to D.H.). O. F. and C.Y.-S. were supported by a FRIA fellowship. D.L.J.L and D.H. are FNRS Directors of Research. We are grateful to Eric Phizicky for reagents and Marc Graille for comments on the manuscript. Research in the lab of D.L.J.L. is supported by the Fonds de la Recherche Nationale (F.R.S./FNRS), the Universit\u00E9 Libre de Bruxelles (ULB), the European Joint Programme on Rare Diseases (EJP-RD; RiboEurope and DBAGeneCure), the R\u00E9gion Wallone (RiboCancer), and the International Brachet Stift\u00FCng (IBS). High-throughput sequencing was performed by the NGS platform of Institut Curie, supported by grants from the Agence Nationale de la Recherche (investissements d'avenir; ANR-10-EQPX-03 and ANR10-INBS-09-08) and by the Cancerop\u00F4le Ile-de-France. This work was also supported by NIH grants ES031529 and AG063341 (to P.D.) and PDR T.0012.14, CDR J.0066.16, and PDR T.0112.21 (to D.H.). O. F. and C.Y.-S. were supported by a FRIA fellowship. D.L.J.L and D.H. are FNRS Directors of Research. O.F. C.Y.-S. L.K.K. M.L. A.N. M.R. J.S. and L.W. designed, performed, and analyzed the experiments. V.M. performed the in vitro translation assays and related experiments. F.G.M.E. and D.L.J.L. designed, performed, and analyzed the HPLC, ribosome profiling, and human CRISPR experiments. P.T. designed the life imaging. P.D. designed and analysed the MS data. M.W. and A.M. contributed to the design and funding of the Rho-seq datasets. D.H. designed the project, acquired funding, supervised the experiments, and wrote the paper with input from all authors. The authors declare no competing interests.
Funders | Funder number |
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Cancéropôle Ile de France | |
Fonds de la Recherche Nationale | |
Waalse Gewest | |
DBAGeneCure | |
European Joint Programme on Rare Diseases | |
Fonds De La Recherche Scientifique - FNRS | |
Fonds pour la Formation à la Recherche dans l’Industrie et dans l’Agriculture | |
F.R.S. | |
Université Libre de Bruxelles | |
National Institute of Environmental Health Sciences | R01ES031529, R01ES026856 |
Agence Nationale de la Recherche | ANR-10-EQPX-03, ANR10-INBS-09-08 |
National Institutes of Health | PDR T.0112.21, PDR T.0012.14, CDR J.0066.16 |
Fundação para a Ciência e a Tecnologia | PTDC/CCI-BIO/29266/2017 |
National Institute on Aging | R01AG063341 |
Keywords
- DUS
- dihydrouridine
- epitranscriptomics
- meiosis
- yeast