Widespread premature transcription termination of Arabidopsis thaliana NLR genes by the spen protein FPA

  1. Matthew T Parker
  2. Katarzyna Knop
  3. Vasiliki Zacharaki
  4. Anna V Sherwood
  5. Daniel Tome
  6. Xuhong Yu
  7. Pascal GP Martin
  8. Jim Beynon
  9. Scott Michaels
  10. Geoffrey John Barton
  11. Gordon Grant Simpson  Is a corresponding author
  1. University of Dundee, United Kingdom
  2. University of Warwick, United Kingdom
  3. Indiana University, United States

Abstract

Genes involved in disease resistance are some of the fastest evolving and most diverse components of genomes. Large numbers of nucleotide-binding, leucine-rich repeat (NLR) genes are found in plant genomes and are required for disease resistance. However, NLRs can trigger autoimmunity, disrupt beneficial microbiota or reduce fitness. It is therefore crucial to understand how NLRs are controlled. Here we show that the RNA-binding protein FPA mediates widespread premature cleavage and polyadenylation of NLR transcripts, thereby controlling their functional expression and impacting immunity. Using long-read Nanopore direct RNA sequencing, we resolved the complexity of NLR transcript processing and gene annotation. Our results uncover a co-transcriptional layer of NLR control with implications for understanding the regulatory and evolutionary dynamics of NLRs in the immune responses of plants.

Data availability

IVI-MS data is available from the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD022684. FPA and Pol II ChIP-Seq data is available from ENA accession PRJNA449914. Col-0 nanopore DRS data is available from ENA accession PRJEB32782. fpa-8 and 35S::FPA:YFP nanopore DRS data is available from ENA accession PRJEB41451. hen2-2 nanopore DRS data is available from ENA accession PRJEB41381. Col-0, fpa-8 and 35S::FPA:YFP Helicos DRS data is available from Zenodo DOI 10.5281/zenodo.4309752 ahead of submission to ENA. Col-0, fpa-8 and 35S::FPA:YFP Illumina RNA-Seq data is available from ENA accession PRJEB41455.

The following data sets were generated
The following previously published data sets were used

Article and author information

Author details

  1. Matthew T Parker

    School of Life Sciences, University of Dundee, Dundee, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0891-8495
  2. Katarzyna Knop

    School of Life Sciences, University of Dundee, Dundee, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2636-9450
  3. Vasiliki Zacharaki

    School of Life Sciences, University of Dundee, Dundee, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5543-2332
  4. Anna V Sherwood

    School of Life Sciences, University of Dundee, Dundee, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  5. Daniel Tome

    School of Life Sciences, University of Warwick, Coventry, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  6. Xuhong Yu

    Department of Biology, Indiana University, Bloomington, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Pascal GP Martin

    Department of Biology, Indiana University, Bloomington, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4271-658X
  8. Jim Beynon

    School of Life Sciences, University of Warwick, Coventry, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  9. Scott Michaels

    Department of Biology, Indiana University, Bloomington, United States
    Competing interests
    The authors declare that no competing interests exist.
  10. Geoffrey John Barton

    School of Life Sciences, University of Dundee, Dundee, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9014-5355
  11. Gordon Grant Simpson

    School of Life Sciences, University of Dundee, Dundee, United Kingdom
    For correspondence
    g.g.simpson@dundee.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6744-5889

Funding

Biotechnology and Biological Sciences Research Council (BB/M010066/1)

  • Geoffrey John Barton
  • Gordon Grant Simpson

Biotechnology and Biological Sciences Research Council (BB/J00247X/1)

  • Geoffrey John Barton
  • Gordon Grant Simpson

Biotechnology and Biological Sciences Research Council (BB/M004155/1)

  • Geoffrey John Barton
  • Gordon Grant Simpson

H2020 Marie Skłodowska-Curie Actions (799300)

  • Katarzyna Knop

Wellcome (097945/B/11/Z)

  • Geoffrey John Barton
  • Gordon Grant Simpson

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

Reviewing Editor

  1. Hao Yu, National University of Singapore & Temasek Life Sciences Laboratory, Singapore

Version history

  1. Received: December 7, 2020
  2. Accepted: April 26, 2021
  3. Accepted Manuscript published: April 27, 2021 (version 1)
  4. Version of Record published: May 12, 2021 (version 2)
  5. Version of Record updated: June 4, 2021 (version 3)

Copyright

© 2021, Parker 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.

Metrics

  • 3,302
    views
  • 494
    downloads
  • 35
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Matthew T Parker
  2. Katarzyna Knop
  3. Vasiliki Zacharaki
  4. Anna V Sherwood
  5. Daniel Tome
  6. Xuhong Yu
  7. Pascal GP Martin
  8. Jim Beynon
  9. Scott Michaels
  10. Geoffrey John Barton
  11. Gordon Grant Simpson
(2021)
Widespread premature transcription termination of Arabidopsis thaliana NLR genes by the spen protein FPA
eLife 10:e65537.
https://doi.org/10.7554/eLife.65537

Share this article

https://doi.org/10.7554/eLife.65537

Further reading

    1. Chromosomes and Gene Expression
    Rupam Choudhury, Anuroop Venkateswaran Venkatasubramani ... Axel Imhof
    Research Article

    Eukaryotic chromatin is organized into functional domains, that are characterized by distinct proteomic compositions and specific nuclear positions. In contrast to cellular organelles surrounded by lipid membranes, the composition of distinct chromatin domains is rather ill described and highly dynamic. To gain molecular insight into these domains and explore their composition, we developed an antibody-based proximity-biotinylation method targeting the RNA and proteins constituents. The method that we termed Antibody-Mediated-Proximity-Labelling-coupled to Mass Spectrometry (AMPL-MS) does not require the expression of fusion proteins and therefore constitutes a versatile and very sensitive method to characterize the composition of chromatin domains based on specific signature proteins or histone modifications. To demonstrate the utility of our approach we used AMPL-MS to characterize the molecular features of the chromocenter as well as the chromosome territory containing the hyperactive X-chromosome in Drosophila. This analysis identified a number of known RNA binding proteins in proximity of the hyperactive X and the centromere, supporting the accuracy of our method. In addition, it enabled us to characterize the role of RNA in the formation of these nuclear bodies. Furthermore, our method identified a new set of RNA molecules associated with the Drosophila centromere. Characterization of these novel molecules suggested the formation of R-loops in centromeres, which we validated using a novel probe for R-loops in Drosophila. Taken together, AMPL-MS improves the selectivity and specificity of proximity ligation allowing for novel discoveries of weak protein-RNA interactions in biologically diverse domains.

    1. Cancer Biology
    2. Chromosomes and Gene Expression
    Gregory Caleb Howard, Jing Wang ... William P Tansey
    Research Article

    The chromatin-associated protein WD Repeat Domain 5 (WDR5) is a promising target for cancer drug discovery, with most efforts blocking an arginine-binding cavity on the protein called the ‘WIN’ site that tethers WDR5 to chromatin. WIN site inhibitors (WINi) are active against multiple cancer cell types in vitro, the most notable of which are those derived from MLL-rearranged (MLLr) leukemias. Peptidomimetic WINi were originally proposed to inhibit MLLr cells via dysregulation of genes connected to hematopoietic stem cell expansion. Our discovery and interrogation of small-molecule WINi, however, revealed that they act in MLLr cell lines to suppress ribosome protein gene (RPG) transcription, induce nucleolar stress, and activate p53. Because there is no precedent for an anticancer strategy that specifically targets RPG expression, we took an integrated multi-omics approach to further interrogate the mechanism of action of WINi in human MLLr cancer cells. We show that WINi induce depletion of the stock of ribosomes, accompanied by a broad yet modest translational choke and changes in alternative mRNA splicing that inactivate the p53 antagonist MDM4. We also show that WINi are synergistic with agents including venetoclax and BET-bromodomain inhibitors. Together, these studies reinforce the concept that WINi are a novel type of ribosome-directed anticancer therapy and provide a resource to support their clinical implementation in MLLr leukemias and other malignancies.