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

Abstract

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.

Data availability

Accession numbersThe raw and processed data for ECT3-HyperTRIBE, Smart-seq2 from root protoplasts and RNA-seq from root tips have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under the accession number PRJEB44359.Code availabilityThe code for running the hyperTRIBER pipeline is available at https://github.com/sarah-ku/targets_arabidopsis,and the nanoPARE pipeline for PAS analysis can be found at https://github.com/Gregor-Mendel-Institute/nanoPARE.

The following data sets were generated

Article and author information

Author details

  1. Laura Arribas-Hernández

    University of Copenhagen, Copenhagen N, Denmark
    For correspondence
    laura.arribas@bio.ku.dk
    Competing interests
    The authors declare that no competing interests exist.
  2. Sarah Rennie

    University of Copenhagen, Copenhagen N, Denmark
    Competing interests
    The authors declare that no competing interests exist.
  3. Michael Schon

    Gregor Mendel Institute, Vienna, Austria
    Competing interests
    The authors declare that no competing interests exist.
  4. Carlotta Porcelli

    Biology, University of Copenhagen, Copenhagen, Denmark
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4675-4898
  5. Balaji Enugutti

    Gregor Mendel Institute, Vienna, Austria
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0816-024X
  6. Robin Andersson

    University of Copenhagen, Copenhagen N, Denmark
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1516-879X
  7. Michael D Nodine

    Gregor Mendel Institute, Vienna, Austria
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6204-8857
  8. Peter Brodersen

    University of Copenhagen, Copenhagen N, Denmark
    For correspondence
    PBrodersen@bio.ku.dk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1083-1150

Funding

H2020 European Research Council (PATHORISC,ERC-2016-COG 726417)

  • Peter Brodersen

Independent Research Fund Denmark (9040-00409B)

  • Peter Brodersen

H2020 European Research Council (638173)

  • Robin Andersson

Independent Research Fund Denmark (6108-00038B)

  • Robin Andersson

H2020 European Research Council (63788)

  • Michael D Nodine

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Pablo A Manavella, Universidad Nacional del Litoral-CONICET, Argentina

Version history

  1. Received: July 21, 2021
  2. Accepted: September 25, 2021
  3. Accepted Manuscript published: September 30, 2021 (version 1)
  4. Version of Record published: January 25, 2022 (version 2)

Copyright

© 2021, Arribas-Hernández et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Laura Arribas-Hernández
  2. Sarah Rennie
  3. Michael Schon
  4. Carlotta Porcelli
  5. Balaji Enugutti
  6. Robin Andersson
  7. Michael D Nodine
  8. Peter Brodersen
(2021)
The YTHDF proteins ECT2 and ECT3 bind largely overlapping target sets and influence target mRNA abundance, not alternative polyadenylation
eLife 10:e72377.
https://doi.org/10.7554/eLife.72377

Share this article

https://doi.org/10.7554/eLife.72377

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