Transcript
Page 1: 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 GenomicsFaculty of Veterinary Medicine

University of LiègeBelgium

Page 2: Polymorphic miRNA–target interactions: a novel source of phenotypic variation

Acknowledgments 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

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

Page 3: Polymorphic miRNA–target interactions: a novel source of phenotypic variation

Positional identification of genes influencing muscle mass …

« Double-muscled » Belgian Blues: MSTN KO

Hypermuscled Piétrains: IGF2 upregulation

Callipyge sheep:Ectopic DLK1 expression

polar overdominance

Hypermuscled Texel:Patrocles mutation

miRNAbiology

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QTL mapping in a Romanov x Texel F2 intercross …

56 phenotypes measuring body and carcass composition 160 microsatellite markers QTL express

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A QTL affecting muscle mass maps to OAR2 …

Chromosome 2

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The three F1 rams are heterozygous Qq for the QTL …

Page 7: Polymorphic miRNA–target interactions: a novel source of phenotypic variation

A QTL affecting muscle mass maps to OAR2 …

MSTN

Muscle-specific chalone of theTGF superfamily of GDFs.

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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.

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

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

MS

TN

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Resequencing the MSTN gene identifies 20 non-coding SNPs …

3 Texels – 7 controls

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The g+6723G-A 3’UTR 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

contr

ols

-

11

bre

eds

F Qq rams

G+6723G-A

TT TC CC

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

G

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miRNA « seeds »

From Lewis et al. (2005) Cell 120: 15-20

5’ dominant versus 3’ compensatory sites

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… predicted to be a target site for miR1, miR206 (and miR122).

Probability to occur « by chance alone »: < 0.01-0.05

G

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Obvious hypothesis …

AAAAA

AAAAA

AAAAA

AAAAA

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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

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Texel sheep have ≈ 3-fold reduction in circulating MSTN levels …

… supports translational inhibition.

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mRNA allelic imbalance in GA heterozygotes …

… supports allele-specific mRNA degradation in P-bodies.

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miR1/206 – mutant MSTN interaction supported by reporter assay

pCMV BGHpAPri-miR pcDNA3.1-miR

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miR1/206 – mutant MSTN interaction supported by reporter assay

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Results: Ago-IP specific target enrichment.

RT +

RT -

input IP-Ago

Pri-miR-1 Pri-miR-1*

IP - Ago IgG - Ago IgG

IP-IgG input IP-Ago IP-IgG

enrichmentTexel A allele

enrichmentWT G allele

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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?

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Common SNPs affect the target site content of thousands of human genes …

92,967 SNPs in 21,206 3’UTRs

SNPs

540 X-targets (Xie et al., 2005)444 L-targets (Lewis et al., 2005)

miRNA targets

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Evidence for purifying selection against SNPs altering target site content …

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Evidence for purifying selection against SNPs altering target site content …

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PATROCLES: the database of polymorphic miRNA-target interactions

http://www.patrocles.org

Page 28: Polymorphic miRNA–target interactions: a novel source of phenotypic variation

PATROCLES: the database of polymorphic miRNA-target interactions

http://www.patrocles.org

Page 29: Polymorphic miRNA–target interactions: a novel source of phenotypic variation

PATROCLES: the database of polymorphic miRNA-target interactions

http://www.patrocles.org

Page 30: Polymorphic miRNA–target interactions: a novel source of phenotypic variation

PATROCLES: the database of polymorphic miRNA-target interactions

http://www.patrocles.org

Page 31: Polymorphic miRNA–target interactions: a novel source of phenotypic variation

PATROCLES: the database of polymorphic miRNA-target interactions

http://www.patrocles.org

Page 32: Polymorphic miRNA–target interactions: a novel source of phenotypic variation

PATROCLES: the database of polymorphic miRNA-target interactions

http://www.patrocles.org

SNPs

CNVs

eQTL

Page 33: Polymorphic miRNA–target interactions: a novel source of phenotypic variation

PATROCLES: the database of polymorphic miRNA-target interactions

http://www.patrocles.org

Page 34: Polymorphic miRNA–target interactions: a novel source of phenotypic variation

PATROCLES: the database of polymorphic miRNA-target interactions

http://www.patrocles.org

Page 35: Polymorphic miRNA–target interactions: a novel source of phenotypic variation

PATROCLES: the database of polymorphic miRNA-target interactions

http://www.patrocles.org

Page 36: Polymorphic miRNA–target interactions: a novel source of phenotypic variation

PATROCLES: the database of polymorphic miRNA-target interactions

http://www.patrocles.org

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Thank you for your attention


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