The YTHDF proteins ECT2 and ECT3 bind largely overlapping target sets and influence target mRNA abundance, not alternative polyadenylation

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Gene regulation via N6-methyladenosine (m6A) in mRNA involves RNA-binding proteins that recognize m6A via a YT521-B homology (YTH) domain. The plant YTH domain proteins ECT2 and ECT3 act genetically redundantly in stimulating cell proliferation during organogenesis, but several fundamental questions regarding their mode of action remain unclear. Here, we use HyperTRIBE (targets of RNA-binding proteins identified by editing) to show that most ECT2 and ECT3 targets overlap, with only few examples of preferential targeting by either of the two proteins. HyperTRIBE in different mutant backgrounds also provides direct views of redundant and specific target interactions of the two proteins. We also show that contrary to conclusions of previous reports, ECT2 does not accumulate in the nucleus. Accordingly, inactivation of ECT2, ECT3 and their surrogate ECT4 does not change patterns of polyadenylation site choice in ECT2/3 target mRNAs, but does lead to lower steady state accumulation of target mRNAs. In addition, mRNA and microRNA expression profiles show indications of stress response activation in ect2/ect3/ect4 mutants, likely via indirect effects. Thus, previous suggestions of control of alternative polyadenylation by ECT2 are not supported by evidence, and ECT2 and ECT3 act largely redundantly to regulate target mRNA, including its abundance, in the cytoplasm.

OriginalsprogEngelsk
Artikelnummere72377
TidsskrifteLife
Vol/bind10
Antal sider27
ISSN2050-084X
DOI
StatusUdgivet - 2021

Bibliografisk note

Funding Information:
We thank Lena Bjørn Johansson and Phillip Andersen for technical assistance in the construction of transgenic lines, Emilie Oksbjerg for preparing and sequencing small RNA libraries, Theo Bølsterli, René Hvidberg Petersen and their teams for plant care, and Anna Fossum and Rajesh Somasundaram for their assistance with FACS. Kim Rewitz and Kenneth Halberg are thanked for assistance with Airyscan microscopy. We acknowledge Mathias Tankmar, Alexander J.H. Andersen and Freja Asmussen for experimental support, and Tom Denyer and Marja Timmermans for their input and support in the analyses of ECT2/3 expression and target enrichment in their scRNAseq data. We are grateful to Norman R. Groves and Iris Meier for the kind donation of GFP-WIP1 seeds. This work was supported by a Consolidator Grant from the European Research Council (PATHORISC, ERC-2016-COG 726417) and a Research Grant from the Independent Research Fund Denmark (9040-00409B) to P.B.; a Starting Grant from the European Research Council (638173) and a Sapere Aude Starting Grant from the Independent Research Fund Denmark (6108-00038B) to R.A.; and a European Research Council under the European Union’s Horizon 2020 Research and Innovation Program grant 63788 to M.N.

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