Polymorphic miRNAtarget interactions: a novel source of phenotypic variation.
Michel Georges Unit of Animal Genomics Faculty of Veterinary Medicine University of Lige Belgium Acknowledgments Ulg: Wyeth Research: INRA: Funding sources: Alex Clop
Fabienne Marcq Haruko Takeda Dimitri Pirottin Xavier Tordoir Florian Caiment Franoise Meish Samuel Hiard Carole Charlier Wyeth Research: James Tobin INRA: Jacques Bouix Elisabeth Laville Catherine Larzul Francis Eychenne Dragan Milinkovic Bernard Bib Jean-Michel Elsen 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 Positional identification of genes influencing muscle mass
Double-muscled Belgian Blues: MSTN KO Hypermuscled Pitrains: IGF2 upregulation Callipyge sheep: Ectopic DLK1 expression polar overdominance Hypermuscled Texel: Patrocles mutation miRNA biology QTL mapping in a Romanov x Texel F2 intercross
56 phenotypes measuring body and carcass composition 160 microsatellite markers QTL 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
MSTN Muscle-specific chalone of the TGF 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 Texels 90 controls breeds F Qq rams TT TC CC G+6723G-A reveals one of Kelliss octamer motifs miRNA seeds 5 dominant versus 3 compensatory sites
From Lewis et al. (2005) Cell 120: 15-20 predicted to be a target site for miR1, miR206 (and miR122).
Probability to occur by chance alone : < Obvious hypothesis AAAAA AAAAA AAAAA AAAAA miR1 and miR206 are conserved in sheep and strongly expressed in skeletal muscle
strongly expressed in SM miR1.1, miR1.2, miR122 and miR206 are conserved in sheep. 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
pCMV BGHpA Pri-miR pcDNA3.1-miR miR1/206 mutant MSTN interaction supported by reporter assay 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 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
miRNA targets 92,967 SNPs in 21,206 3UTRs 540 X-targets (Xie et al., 2005) 444 L-targets (Lewis et al., 2005) 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 interactions
PATROCLES: the database of polymorphic miRNA-target interactions
PATROCLES: the database of polymorphic miRNA-target interactions
PATROCLES: the database of polymorphic miRNA-target interactions
PATROCLES: the database of polymorphic miRNA-target interactions
PATROCLES: the database of polymorphic miRNA-target interactions
SNPs CNVs eQTL PATROCLES: the database of polymorphic miRNA-target interactions
PATROCLES: the database of polymorphic miRNA-target interactions
PATROCLES: the database of polymorphic miRNA-target interactions
PATROCLES: the database of polymorphic miRNA-target interactions
Thank you for your attention