Abstract

Specific recognition of N6-methyladenosine (m6A) in mRNA by RNA-binding proteins containing a YT521-B homology (YTH) domain is important in eukaryotic gene regulation. The Arabidopsis YTH-domain protein ECT2 is thought to bind to mRNA at URU(m6A)Y sites, yet RR(m6A)CH is the canonical m6A consensus site in all eukaryotes and ECT2 functions require m6A binding activity. Here, we apply iCLIP (individual-nucleotide resolution cross-linking and immunoprecipitation) and HyperTRIBE (targets of RNA-binding proteins identified by editing) to define high-quality target sets of ECT2, and analyze the patterns of enriched sequence motifs around ECT2 crosslink sites. Our analyses show that ECT2 does in fact bind to RR(m6A)CH. Pyrimidine-rich motifs are enriched around, but not at m6A-sites, reflecting a preference for N6-adenosine methylation of RRACH/GGAU islands in pyrimidine-rich regions. Such motifs, particularly oligo-U and UNUNU upstream of m6A sites, are also implicated in ECT2 binding via its intrinsically disordered region (IDR). Finally, URUAY-type motifs are enriched at ECT2 crosslink sites, but their distinct properties suggest function as sites of competition between binding of ECT2 and as yet unidentified RNA-binding proteins. Our study provides coherence between genetic and molecular studies of m6A-YTH function in plants, and reveals new insight into the mode of RNA recognition by YTH-domain-containing proteins.

Data availability

All sequencing data (iCLIP-seq, HyperTRIBE, mRNA-seq, small RNA-seq) have been deposited in the European Nucleotide Archive under accession code PRJEB44359.All code is available at GitHubhttps://github.com/sarah-ku/targets_arabidopsis

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. Tino Köster

    University of Bielefeld, Bielefeld, Germany
    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. Martin Lewinski

    University of Bielefeld, Bielefeld, Germany
    Competing interests
    The authors declare that no competing interests exist.
  6. Prof. Dr. Dorothee Staiger

    University of Bielefeld, Bielefeld, Germany
    For correspondence
    dorothee.staiger@uni-bielefeld.de
    Competing interests
    The authors declare that no competing interests exist.
  7. Robin Andersson

    University of Copenhagen, Copenhagen N, Denmark
    For correspondence
    robin@bio.ku.dk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1516-879X
  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 (ERC-2016-COG 726417)

  • Peter Brodersen

Independent Research Fund Denmark (9040-00409B)

  • Peter Brodersen

European Molecular Biology Organization (STF 7614)

  • Laura Arribas-Hernández

Deutsche Forschungsgemeinschaft (STA653/14-1)

  • Prof. Dr. Dorothee Staiger

H2020 European Research Council (638173)

  • Robin Andersson

Independent Research Fund Denmark (6108-00038B)

  • Robin Andersson

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. Preprint posted: April 18, 2021 (view preprint)
  2. Received: July 21, 2021
  3. Accepted: September 25, 2021
  4. Accepted Manuscript published: September 30, 2021 (version 1)
  5. 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. Tino Köster
  4. Carlotta Porcelli
  5. Martin Lewinski
  6. Prof. Dr. Dorothee Staiger
  7. Robin Andersson
  8. Peter Brodersen
(2021)
Principles of mRNA targeting via the Arabidopsis m6A-binding protein ECT2
eLife 10:e72375.
https://doi.org/10.7554/eLife.72375

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https://doi.org/10.7554/eLife.72375

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