Journal article
Multiplexed profiling facilitates robust m6A quantification at site, gene and sample resolution
Nature methods, Vol.18(9), pp.1060-1067
03/Sep/2021
PMID: 34480159
Abstract
N6-methyladenosine (m6A) is the most prevalent modification of messenger RNA in mammals. To interrogate its functions and dynamics, there is a critical need to quantify m6A at three levels: site, gene and sample. Current approaches address these needs in a limited manner. Here we develop m6A-seq2, relying on multiplexed m6A-immunoprecipitation of barcoded and pooled samples. m6A-seq2 allows a big increase in throughput while reducing technical variability, requirements of input material and cost. m6A-seq2 is furthermore uniquely capable of providing sample-level relative quantitations of m6A, serving as an orthogonal alternative to mass spectrometry-based approaches. Finally, we develop a computational approach for gene-level quantitation of m6A. We demonstrate that using this metric, roughly 30% of the variability in RNA half life in mouse embryonic stem cells can be explained, establishing m6A as a main driver of RNA stability. m6A-seq2 thus provides an experimental and analytic framework for dissecting m6A-mediated regulation at three different levels.
Details
- Title
- Multiplexed profiling facilitates robust m6A quantification at site, gene and sample resolution
- Creators
- David Dierks - 972WIS_INST___112Miguel Angel Garcia-Campos - 972WIS_INST___111Anna Uzonyi - 972WIS_INST___111Modi Safra - 972WIS_INST___111Sarit Edelheit - 972WIS_INST___111Alice Rossi - The Francis Crick InstituteTheodora Sideri - The Francis Crick InstituteRadhika A Varier - The Francis Crick InstituteAlexander Brandis - 972WIS_INST___113Yonatan Stelzer - 972WIS_INST___122Folkert van Werven - The Francis Crick InstituteRuth Scherz-Shouval - 972WIS_INST___112Schraga Schwartz (Corresponding Author) - 972WIS_INST___111
- Resource Type
- Journal article
- Publication Details
- Nature methods, Vol.18(9), pp.1060-1067; 03/Sep/2021
- Number of pages
- 19
- Publisher
- NATURE PORTFOLIO
- Language
- English
- DOI
- https://doi.org/10.1038/s41592-021-01242-z
- PMID
- 34480159
- Grants
- Grant note
- D.D., M.A.G.C., A.U., M.S., S.E. and S.S. have received funding from the Israel Science Foundation (grant no. 543165), the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant no. 714023 and ERC-POC 899122), the Estate of Emile Mimran. S.S. is the incumbent of the Robert Edward and Roselyn Rich Manson Career Development Chair in Perpetuity. R.S.S. is supported by ISF grant nos. 401/17 and 1384/1, ERC grant no. 754320, and the Laura Gurwin Flug Family Fund. R.S.S. is the incumbent of the Ernst and Kaethe Ascher Career Development Chair in Life Sciences. F.v.W., T.S., R.A.V. and A.R. are supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (grant no. FC001203), the UK Medical Research Council (grant no. FC001203) and the Wellcome Trust (grant no. FC001203). Contributions: D.D. and S.S. conceived the project. M.S., S.E., A.U. and D.D. conducted all genomic experiments. A.B. performed the conducted mass-spectroscopy analysis. F.v.W., T.S., R.A.V. and A.R. generated and conducted the experiment of yeast inducible perturbations. A.U. and Y.S. conducted the experiments of the partial KO experiments of m6A writers in mESC. M.A.G.C. provided and created the in-house generated scripts that were used to perform the analysis. D.D. and S.S. performed the data analysis. R.S.S. and S.S. acquired funding. D.D., R.S.S. and S.S. wrote the paper, with input from all authors.
- Record Identifier
- 993336115603596
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