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

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

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

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  1. Polymorphic miRNA–target interactions: a novel source of phenotypic variation. Michel Georges Unit of Animal Genomics Faculty of Veterinary Medicine University of Liège Belgium

  2. Ulg: Alex Clop Fabienne Marcq Haruko Takeda Dimitri Pirottin Xavier Tordoir Florian Caiment Françoise Meish Samuel Hiard Carole Charlier Wyeth Research: James Tobin INRA: Jacques Bouix Elisabeth Laville Catherine Larzul Francis Eychenne Dragan Milinkovic Bernard Bibé Jean-Michel Elsen Acknowledgments • Funding sources: • Ministry of Agriculture of the Walloon Region: BELTEX • French-speaking Community of Belgium: Game ARC • Belgian Government: Molecular Pathogenesis of Genetic Disease PAI • EU: Callimir STREP / Marie-Curie Fellowships

  3. « Double-muscled » Belgian Blues: MSTN KO Hypermuscled Piétrains: IGF2 upregulation Callipyge sheep: Ectopic DLK1 expression polar overdominance miRNA biology Hypermuscled Texel: Patrocles mutation Positional identification of genes influencing muscle mass …

  4. 56 phenotypes measuring body and carcass composition 160 microsatellite markers QTL express QTL mapping in a Romanov x Texel F2 intercross …

  5. A QTL affecting muscle mass maps to OAR2 … Chromosome 2

  6. The three F1 rams are heterozygous Qq for the QTL …

  7. MSTN A QTL affecting muscle mass maps to OAR2 … Muscle-specific chalone of the TGF superfamily of GDFs.

  8. 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.

  9. MASA in offspring of recombinant ram fine-maps QTL to OAR2q …

  10. MSTN A signature of selection fine-maps QTL in vicinity of MSTN gene …

  11. Resequencing the MSTN gene identifies 20 non-coding SNPs … 3 Texels – 7 controls

  12. Strong signature of selection Heterozygous « Qq » rams exclude all but the g+6723G-A SNP. The g+6723A allele is virtually Texel-specific. The g+6723G-A 3’UTRSNP is a strong genetic QTN candidate … G+6723G-A 42 Texels 90 controls - 11 breeds F Qq rams TT TC CC

  13. G+6723G-A reveals one of « Kellis’s octamer motifs » … G

  14. miRNA « seeds » 5’ dominant versus 3’ compensatory sites From Lewis et al. (2005) Cell 120: 15-20

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

  16. Obvious hypothesis … AAAAA AAAAA AAAAA AAAAA

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

  18. Texel sheep have ≈ 3-fold reduction in circulating MSTN levels … … supports translational inhibition.

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

  20. pcDNA3.1-miR pCMV Pri-miR BGHpA miR1/206 – mutant MSTN interaction supported by reporter assay

  21. miR1/206 – mutant MSTN interaction supported by reporter assay

  22. Results: Ago-IP specific target enrichment. Pri-miR-1* Pri-miR-1 IP - Ago IgG - Ago IgG RT + RT - input IP-Ago IP-IgG input IP-Ago IP-IgG enrichment Texel A allele enrichment WT G allele

  23. 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?

  24. Common SNPs affect the target site content of thousands of human genes … SNPs miRNA targets 92,967 SNPs in 21,206 3’UTRs 540 X-targets (Xie et al., 2005) 444 L-targets (Lewis et al., 2005)

  25. Evidence for purifying selection against SNPs altering target site content …

  26. Evidence for purifying selection against SNPs altering target site content …

  27. PATROCLES: the database of polymorphic miRNA-target interactions http://www.patrocles.org

  28. PATROCLES: the database of polymorphic miRNA-target interactions http://www.patrocles.org

  29. PATROCLES: the database of polymorphic miRNA-target interactions http://www.patrocles.org

  30. PATROCLES: the database of polymorphic miRNA-target interactions http://www.patrocles.org

  31. PATROCLES: the database of polymorphic miRNA-target interactions http://www.patrocles.org

  32. CNVs SNPs eQTL PATROCLES: the database of polymorphic miRNA-target interactions http://www.patrocles.org

  33. PATROCLES: the database of polymorphic miRNA-target interactions http://www.patrocles.org

  34. PATROCLES: the database of polymorphic miRNA-target interactions http://www.patrocles.org

  35. PATROCLES: the database of polymorphic miRNA-target interactions http://www.patrocles.org

  36. PATROCLES: the database of polymorphic miRNA-target interactions http://www.patrocles.org

  37. Thank you for your attention

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