1. Microbiology and Infectious Disease
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A plant-like mechanism coupling m6A reading to polyadenylation safeguards transcriptome integrity and developmental gene partitioning in Toxoplasma

  1. Dayana C Farhat
  2. Matthew W Bowler
  3. Guillaume Communie
  4. Dominique Pontier
  5. Lucid Belmudes
  6. Caroline Mas
  7. Charlotte Corrao
  8. Yohann Couté
  9. Alexandre Bougdour
  10. Thierry Lagrange
  11. Mohamed-ali Hakimi  Is a corresponding author
  12. Christopher Swale  Is a corresponding author
  1. Université Grenoble Alpes, France
  2. European Molecular Biology Laboratory, France
  3. Institut Laue-Langevin, France
  4. CNRS, France
Research Article
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Cite this article as: eLife 2021;10:e68312 doi: 10.7554/eLife.68312

Abstract

Correct 3'end processing of mRNAs is one of the regulatory cornerstones of gene expression. In a parasite that must adapt to the regulatory requirements of its multi-host life style, there is a need to adopt additional means to partition the distinct transcriptional signatures of the closely and tandemly-arranged stage specific genes. In this study, we report our findings in T. gondii of an m6A-dependent 3'end polyadenylation serving as a transcriptional barrier at these loci. We identify the core polyadenylation complex within T. gondii and establish CPSF4 as a reader for m6A-modified mRNAs, via a YTH domain within its C-terminus, a feature which is shared with plants. We bring evidence of the specificity of this interaction both biochemically, and by determining the crystal structure at high resolution of the T. gondii CPSF4-YTH in complex with an m6A modified RNA. We show that the loss of m6A, both at the level of its deposition or its recognition was associated with an increase in aberrantly elongated chimeric mRNAs emanating from impaired transcriptional termination, a phenotype previously noticed in the plant model Arabidopsis thaliana. Nanopore direct RNA sequencing shows the occurrence of transcriptional read-through breaching into downstream repressed stage-specific genes, in the absence of either CPSF4 or the m6A RNA methylase components in both T. gondii and A. thaliana. Taken together, our results shed light on an essential regulatory mechanism coupling the pathways of m6A metabolism directly to the cleavage and polyadenylation processes, one that interestingly seem to serve, in both T. gondii and A. thaliana, as a guardian against aberrant transcriptional read-throughs.

Data availability

The Nanopore RNAseq data have been deposited in NCBI's SRA data PRJNA705300. The MS proteomics data have been deposited to the ProteomeXchange Consortium through the PRIDE partner repository with the dataset identifier PXD024326. Sequencing data have been deposited in GEO under accession code GSE168155:

The following data sets were generated

Article and author information

Author details

  1. Dayana C Farhat

    Team Host-pathogen interactions and Immunity to Infection, Université Grenoble Alpes, Grenoble, France
    Competing interests
    The authors declare that no competing interests exist.
  2. Matthew W Bowler

    European Molecular Biology Laboratory, Grenoble, France
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0465-3351
  3. Guillaume Communie

    Institut Laue-Langevin, Institut Laue-Langevin, Grenoble, France
    Competing interests
    The authors declare that no competing interests exist.
  4. Dominique Pontier

    UMR5096, CNRS, Perpignan, France
    Competing interests
    The authors declare that no competing interests exist.
  5. Lucid Belmudes

    BIG-BGE, Université Grenoble Alpes, Grenoble, France
    Competing interests
    The authors declare that no competing interests exist.
  6. Caroline Mas

    Integrated Structural Biology Grenoble (ISBG), Université Grenoble Alpes, Grenoble, France
    Competing interests
    The authors declare that no competing interests exist.
  7. Charlotte Corrao

    Team Host-pathogen interactions and Immunity to Infection, Université Grenoble Alpes, Grenoble, France
    Competing interests
    The authors declare that no competing interests exist.
  8. Yohann Couté

    Institut de Biosciences et Biotechnologies de Grenoble, Université Grenoble Alpes, Grenoble, France
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3896-6196
  9. Alexandre Bougdour

    Institut de Biosciences et Biotechnologies de Grenoble, Université Grenoble Alpes, Grenoble, France
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5895-0020
  10. Thierry Lagrange

    UMR5096, CNRS, Grenoble, France
    Competing interests
    The authors declare that no competing interests exist.
  11. Mohamed-ali Hakimi

    Team Host-pathogen interactions and Immunity to Infection, Université Grenoble Alpes, Grenoble, France
    For correspondence
    mohamed-ali.hakimi@inserm.fr
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2547-8233
  12. Christopher Swale

    Team Host-pathogen interactions and Immunity to Infection, Université Grenoble Alpes, Grenoble, France
    For correspondence
    christopher.swale@univ-grenoble-alpes.fr
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9739-7774

Funding

Agence Nationale de la Recherche (Laboratoire d'Excellence (LabEx) ParaFrap [ANR-11-LABX-0024])

  • Dayana C Farhat
  • Mohamed-ali Hakimi
  • Christopher Swale

Agence Nationale de la Recherche (Project HostQuest,ANR-18-CE15-0023)

  • Charlotte Corrao
  • Alexandre Bougdour
  • Mohamed-ali Hakimi
  • Christopher Swale

European Research Council (ERC Consolidator Grant N{degree sign}614880 Hosting TOXO)

  • Mohamed-ali Hakimi

Fondation pour la Recherche Médicale (FRM FDT201904008364)

  • Dayana C Farhat
  • Mohamed-ali Hakimi

Agence Nationale de la Recherche (Proteomics French Infrastructure,Infrastructure Nationale en Biologie et Santé,ANR-10-INBS-08)

  • Lucid Belmudes
  • Yohann Couté

Agence Nationale de la Recherche (Laboratoires d'Excellences (LABEX) TULIP (ANR-10-LABX-41)")

  • Dominique Pontier
  • Thierry Lagrange

Agence Nationale de la Recherche (École Universitaire de Recherche (EUR)" TULIP-GS (ANR-18-EURE-0019)")

  • Dominique Pontier
  • Thierry Lagrange

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

Reviewing Editor

  1. Deborah Bourc'his, Institut Curie, France

Publication history

  1. Preprint posted: February 23, 2021 (view preprint)
  2. Received: March 11, 2021
  3. Accepted: July 13, 2021
  4. Accepted Manuscript published: July 15, 2021 (version 1)
  5. Version of Record published: July 26, 2021 (version 2)

Copyright

© 2021, Farhat 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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Further reading

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    Alexander O Pasternak et al.
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    Background:

    It remains unclear whether combination antiretroviral therapy (ART) regimens differ in their ability to fully suppress human immunodeficiency virus (HIV) replication. Here, we report the results of two cross-sectional studies that compared levels of cell-associated (CA) HIV markers between individuals receiving suppressive ART containing either a non-nucleoside reverse transcriptase inhibitor (NNRTI) or a protease inhibitor (PI).

    Methods:

    CA HIV unspliced RNA and total HIV DNA were quantified in two cohorts (n = 100, n = 124) of individuals treated with triple ART regimens consisting of two nucleoside reverse transcriptase inhibitors (NRTIs) plus either an NNRTI or a PI. To compare CA HIV RNA and DNA levels between the regimens, we built multivariable models adjusting for age, gender, current and nadir CD4+ count, plasma viral load zenith, duration of virological suppression, NRTI backbone composition, low-level plasma HIV RNA detectability, and electronically measured adherence to ART.

    Results:

    In both cohorts, levels of CA HIV RNA and DNA strongly correlated (rho = 0.70 and rho = 0.54) and both markers were lower in NNRTI-treated than in PI-treated individuals. In the multivariable analysis, CA RNA in both cohorts remained significantly reduced in NNRTI-treated individuals (padj = 0.02 in both cohorts), with a similar but weaker association between the ART regimen and total HIV DNA (padj = 0.048 and padj = 0.10). No differences in CA HIV RNA or DNA levels were observed between individual NNRTIs or individual PIs, but CA HIV RNA was lower in individuals treated with either nevirapine or efavirenz, compared to PI-treated individuals.

    Conclusions:

    All current classes of antiretroviral drugs only prevent infection of new cells but do not inhibit HIV RNA transcription in long-lived reservoir cells. Therefore, these differences in CA HIV RNA and DNA levels by treatment regimen suggest that NNRTIs are more potent in suppressing HIV residual replication than PIs, which may result in a smaller viral reservoir size.

    Funding:

    This work was supported by ZonMw (09120011910035) and FP7 Health (305522).

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    The replication of Plasmodium falciparum parasites within red blood cells (RBCs) causes severe disease in humans, especially in Africa. Deleterious alleles like hemoglobin S are well-known to confer strong resistance to malaria, but the effects of common RBC variation are largely undetermined. Here we collected fresh blood samples from 121 healthy donors, most with African ancestry, and performed exome sequencing, detailed RBC phenotyping, and parasite fitness assays. Over one third of healthy donors unknowingly carried alleles for G6PD deficiency or hemoglobinopathies, which were associated with characteristic RBC phenotypes. Among non-carriers alone, variation in RBC hydration, membrane deformability, and volume was strongly associated with P. falciparum growth rate. Common genetic variants in PIEZO1, SPTA1/SPTB, and several P. falciparum invasion receptors were also associated with parasite growth rate. Interestingly, we observed little or negative evidence for divergent selection on non-pathogenic RBC variation between Africans and Europeans. These findings suggest a model in which globally widespread variation in a moderate number of genes and phenotypes modulates P. falciparum fitness in RBCs.