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
