Polymorphic miRNA–target interactions: a novel source of phenotypic variation

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Polymorphic miRNA–target interactions: a novel source of phenotypic variation. Michel Georges Unit of Animal Genomics Faculty of Veterinary Medicine University of Liège Belgium. Ulg: Alex Clop Fabienne Marcq Haruko Takeda Dimitri Pirottin Xavier Tordoir Florian Caiment Françoise Meish - PowerPoint PPT Presentation

Text of Polymorphic miRNA–target interactions: a novel source of phenotypic variation

  • Polymorphic miRNAtarget interactions: a novel source of phenotypic variation.Michel Georges

    Unit of Animal GenomicsFaculty of Veterinary MedicineUniversity of LigeBelgium

  • AcknowledgmentsUlg:Alex ClopFabienne MarcqHaruko TakedaDimitri PirottinXavier TordoirFlorian CaimentFranoise MeishSamuel HiardCarole Charlier

    Wyeth Research:James TobinINRA:Jacques BouixElisabeth LavilleCatherine LarzulFrancis EychenneDragan MilinkovicBernard BibJean-Michel ElsenFunding sources:Ministry of Agriculture of the Walloon Region: BELTEXFrench-speaking Community of Belgium: Game ARCBelgian Government: Molecular Pathogenesis of Genetic Disease PAIEU: Callimir STREP / Marie-Curie Fellowships

  • Positional identification of genes influencing muscle mass

  • QTL mapping in a Romanov x Texel F2 intercross 56 phenotypes measuring body and carcass composition160 microsatellite markersQTL express

  • A QTL affecting muscle mass maps to OAR2 Chromosome 2

  • The three F1 rams are heterozygous Qq for the QTL

  • A QTL affecting muscle mass maps to OAR2 Muscle-specific chalone of theTGF superfamily of GDFs.

  • Texel sheep produce normal levels of normal MSTN mRNA

    Sequencing the MSTN ORF from gDNA & cDNA:no polymorphism.

    Northern blot and qRT-PCR:no obvious qualitative or quantitative difference.

  • MASA in offspring of recombinant ram fine-maps QTL to OAR2q

  • A signature of selection fine-maps QTL in vicinity of MSTN gene

  • Resequencing the MSTN gene identifies 20 non-coding SNPs 3 Texels 7 controls

  • The g+6723G-A 3UTR SNP is a strong genetic QTN candidate

    Strong signature of selection

    Heterozygous Qq rams exclude all but the g+6723G-A SNP.

    The g+6723A allele is virtually Texel-specific.

    42 Texels90 controls - 11 breedsF Qq ramsG+6723G-ATTTCCC

  • G+6723G-A reveals one of Kelliss octamer motifs G

  • miRNA seedsFrom Lewis et al. (2005) Cell 120: 15-205 dominant versus 3 compensatory sites

  • predicted to be a target site for miR1, miR206 (and miR122).Probability to occur by chance alone : < 0.01-0.05G

  • Obvious hypothesis AAAAAAAAAAAAAAAAAAAA

  • miR1 and miR206 are conserved in sheep and strongly expressed in skeletal muscle miR1.1, miR1.2, miR122 andmiR206 are conserved in sheep.miR1 and miR206 are strongly expressed in SM

  • Texel sheep have 3-fold reduction in circulating MSTN levels supports translational inhibition.

  • mRNA allelic imbalance in GA heterozygotes supports allele-specific mRNA degradation in P-bodies.

  • miR1/206 mutant MSTN interaction supported by reporter assay

  • miR1/206 mutant MSTN interaction supported by reporter assay

  • Results: Ago-IP specific target enrichment. RT +RT -inputIP-AgoPri-miR-1Pri-miR-1*IP - Ago IgG - Ago IgGIP-IgGinputIP-AgoIP-IgGenrichmentTexel A alleleenrichmentWT G allele

  • Conclusions:Very strong evidence that g+6723G-A is the QTN, creating a MSTN hypomorph by revealing an illegitimate miRNA target site

    Polymorphic miRNA-target interactions: common mediators of phenotypic variation?

  • Common SNPs affect the target site content of thousands of human genes 92,967 SNPs in 21,206 3UTRsSNPs540 X-targets (Xie et al., 2005)444 L-targets (Lewis et al., 2005)miRNA targets

  • Evidence for purifying selection against SNPs altering target site content

  • Evidence for purifying selection against SNPs altering target site content

  • PATROCLES: the database of polymorphic miRNA-target interactionshttp://www.patrocles.org

  • PATROCLES: the database of polymorphic miRNA-target interactionshttp://www.patrocles.org

  • PATROCLES: the database of polymorphic miRNA-target interactionshttp://www.patrocles.org

  • PATROCLES: the database of polymorphic miRNA-target interactionshttp://www.patrocles.org

  • PATROCLES: the database of polymorphic miRNA-target interactionshttp://www.patrocles.org

  • PATROCLES: the database of polymorphic miRNA-target interactionshttp://www.patrocles.org

  • PATROCLES: the database of polymorphic miRNA-target interactionshttp://www.patrocles.org

  • PATROCLES: the database of polymorphic miRNA-target interactionshttp://www.patrocles.org

  • PATROCLES: the database of polymorphic miRNA-target interactionshttp://www.patrocles.org

  • PATROCLES: the database of polymorphic miRNA-target interactionshttp://www.patrocles.org

  • Thank you for your attention