Systemic impact of small regulatory RNAs

  • MicroRNAs ("miRNAs") are small post-transcriptional regulators. The function of these small RNAs in animals has been well characterized at a molecular level, but their role is less well known at the macroscopic scale: how could miRNAs have any biological function if they repress most of their targets less than 2-fold (while inter-individual gene expression fluctuation typically exceeds 2-fold, and is buffered by homeostatic mechanisms)?
    According to the current dogma, each miRNA regulates tens or hundreds of targets, yet several observations suggest miRNAs have a much weaker impact on animal biology. Our recent work also suggests that both experimental and computational miRNA target identification methods are heavily contaminated with false positives: these false positives may be truly repressed by miRNAs at the molecular scale, but such a small repressive effect fails to translate into a macroscopic phenotype for most genes.
    Our work thus suggests that the biological role of miRNAs has been largely over-estimated. We are currently exploring practical consequences of this new theoretical framework, measuring the contribution of individual miRNA/target interactions to global in vivo phenotypes.
    More generally, we are proposing a new vision of gene regulation: a regulatory target is not simply a gene that is affected by a regulatory pathway; it is a gene that is affected enough by the pathway – the extent of a measured regulation needs to be confronted to the robustness of biological systems to fluctuations.

    figure 3 fr

    To date, our research is funded by:

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    Members

    Isabelle Busseau
    Busseau Isabelle
    Hervé Seitz
    Seitz Hervé
    Sophie Mockly
    Mockly Sophie

    Publications

    Functional lability of RNA-dependent RNA polymerases in animals.

    Pinzón N, Bertrand S, Subirana L, Busseau I, Escrivá H, Seitz H

    2019 - PLoS Genet, 15(2):e1007915

    Request for full article30779744

    Amphioxus functional genomics and the origins of vertebrate gene regulation.

    Marlétaz F, Firbas PN, Maeso I, Tena JJ, Bogdanovic O, Perry M, Wyatt CDR, de la Calle-Mustienes E, Bertrand S, Burguera D, Acemel RD, van Heeringen SJ, Naranjo S, Herrera-Ubeda C, Skvortsova K, Jimenez-Gancedo S, Aldea D, Marquez Y, Buono L, Kozmikova I, Permanyer J, Louis A, Albuixech-Crespo B, Le Petillon Y, Leon A, Subirana L, Balwierz PJ, Duckett PE, Farahani E, Aury JM, Mangenot S, Wincker P, Albalat R, Benito-Gutiérrez È, Cañestro C, Castro F, D'Aniello S, Ferrier DEK, Huang S, Laudet V, Marais GAB, Pontarotti P, Schubert M, Seitz H, Somorjai I, Takahashi T, Mirabeau O, Xu A, Yu JK, Carninci P, Martinez-Morales JR, Crollius HR, Kozmik Z, Weirauch MT, Garcia-Fernàndez J, Lister R, Lenhard B, Holland PWH, Escriva H, Gómez-Skarmeta JL, Irimia M

    2018 - Nature

    Request for full article30464347

    Inconsistencies and Limitations of Current MicroRNA Target Identification Methods.

    Mockly S, Seitz H

    2019 - Methods Mol Biol, 1970:291-314

    Request for full article30963499

    Editorial: miRNA Regulatory Pathways in Metazoans. Advances From in vivo and ex vivo Studies.

    Amar L, Seitz H

    2019 - Front Genet, 10:147

    Request for full article30886628
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    Publications of the team

  • Measuring the contribution of individual miRNA/target interactions to an in vivo phenotype

    The bantam miRNA was discovered by a genetic screen in Drosophila: homozygous mutants die at the early pupal stage, heterozygotes are smaller than wild-type, and over-expressing flies are larger than wild-type; hypomorphs are partially female-sterile (Hipfner et al., 2002). The first proposed target for bantam is the proapoptotic gene hid, whose 3´ UTR contains several seed matches for bantam (Brennecke et al., 2003). Many more targets were proposed (target prediction programs predict ≈ 70 targets with phylogenetically conserved sites for bantam), some of which are measurably repressed by bantam at the protein or mRNA level, but rigorous in vivo validation of the involvement of any target in the growth, lethality or sterility phenotype is still lacking. For example, bantam-mediated regulation of the enabled gene is very clear when assessed by reporter assay (Becam et al., 2011), but genetic ablation of the bantam binding site in the endogenous enabled gene does not appear to perturb the enabled expression pattern or to trigger any particular phenotype (Bassett et al., 2014).

    We will thus focus on a pure in vivo strategy to asses the contribution of individual targets to the bantam phenotype. Using genome editing, we prepared mutant flies where the bantam seed has been mutated into another hexamer. We are currently preparing mutant flies where bantam binding sites on the hid gene are mutated in a compensatory manner, in order to isolate the contribution of the bantam/hid interaction to the global bantam phenotype (similarly to the strategy described in Ecsedi et al., 2015).

    figure 1
    Mutating the bantam miRNA in vivo, we can verify the macroscopic phenotypes controlled by that miRNA. Mutating its binding sites on an individual target, we can evaluate the contribution of that particular interaction to the global phenotype. Combining the two compensatory mutations in the same fly allows us to confirm the importance of that interaction on the in vivo phenotype.

    Identifying miR-34 targets controlling cell proliferation in mammals

    The miR-34 miRNA family has been under intense scrutiny since it was proposed to control cell proliferation both in humans and in mice (He et al., 2007). Many targets were proposed to be involved in miR-34- dependent control of cell proliferation, based on ex vivo studies, but in vivo evidence is still lacking (Concepcion et al., 2012).

    Using a high-throughput screen, we will mutate every candidate miR-34 binding site and measure the effect of such mutation on mammalian cell proliferation. Our goal will be the formal identification of miR-34 direct and indirect targets that affect most strongly cell proliferation, and to provide a precise measurement of their contribution to the phenotype.

    figure 2
    Using a high-throughput screen, we will identify the individual miR-34/target interactions that exert the strongest effect on cell proliferation. Amplification and attenuation mechanisms in the downstream regulatory networks will be identified through the measurement of indirect target expression perturbation.
  • Education and previous positions

    2017: 5 year activity evaluation by the IGH (team “seniorized”, made permanent)
    2011-2016: Junior group leader at the IGH
    2009: HDR at the university Toulouse III Paul Sabatier
    2005-2009: Postdoctoral fellow in Prof. Phillip Zamore’s laboratory, University of Massachusetts Medical School, Worcester (MA, USA)
    2001-2004: PhD student in Dr. Jérôme Cavaillé’s laboratory (LBME, CNRS and université Toulouse III Paul Sabatier)
    1997-2001: Undegraduate student at the École normale supérieure (rue d’Ulm, Paris)

    Lab funding

    2012-2016 : ATIP-Avenir (CNRS and Inserm, co-sponsored by Sanofi)
    2010-2012 : CDA (Human Frontier Science Program)

    Expertise activities

    Referee for scientific journals in the domains of RNA biology, bio-informatics, genomics and molecular genetics (Current Biology, EMBO Reports, Genome Research, Nucleic Acids Research, RNA Biology, ...).
    Referee for national and international grant agencies (ERC, SNF, HFSP, ANR, …).
    Member of Cefic Long-range research initiative’s “Governance leadership team”.
    Correction and authorship of notes for ANSES’ “Bulletin de veille scientifique”.