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

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 a few examples of preferential targeting by either of the two proteins. HyperTRIBE in different mutant backgrounds also provides direct views of redundant, ectopic, 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.

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For small RNA-seq, 2 biological replicates were used. 3 replicates would have been preferred, but only two of each group (wild type and ect2/ect3/ect4 triple mutant) could be included in this experiment for quite trivial practical reasons related to the number of slots available for library preparation and in the sequencing lane.

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Group allocation does not apply to this study, other than in the most obvious sense: - plants of a specific genotype are treated as a group eLife Sciences Publications, Ltd is a limited liability non-profit non-stock corporation incorporated in the State of Delaware, USA, with company number 5030732, and is registered in the UK with company number FC030576 and branch number BR015634 at the address 1st Floor, 24 Hills Road, Cambridge CB2 1JP | August 2014 4 Code Relevant code is available at https://github.com/sarah-ku/targets_arabidopsis and https://github.com/Gregor-Mendel-Institute/nanoPARE Source data Figures  1-4: HyperTRIBE sequencing data have been deposited in the European Nucleotide Archive (accession number PRJEB44359) Figure  5, panel C: Source image files of the western blot and stained membrane have been uploaded to the eLife webpage.