Upload
lita
View
32
Download
0
Tags:
Embed Size (px)
DESCRIPTION
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
Citation preview
Polymorphic miRNA–target interactions: a novel source of phenotypic variation.
Michel Georges
Unit of Animal GenomicsFaculty of Veterinary Medicine
University of LiègeBelgium
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
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
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 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 …
MS
TN
Resequencing the MSTN gene identifies 20 non-coding SNPs …
3 Texels – 7 controls
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
G+6723G-A reveals one of « Kellis’s octamer motifs » …
G
miRNA « seeds »
From Lewis et al. (2005) Cell 120: 15-20
5’ dominant versus 3’ compensatory sites
… predicted to be a target site for miR1, miR206 (and miR122).
Probability to occur « by chance alone »: < 0.01-0.05
G
Obvious hypothesis …
AAAAA
AAAAA
AAAAA
AAAAA
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
pCMV BGHpAPri-miR pcDNA3.1-miR
miR1/206 – mutant MSTN interaction supported by reporter assay
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
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 3’UTRs
SNPs
540 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 interactions
http://www.patrocles.org
PATROCLES: the database of polymorphic miRNA-target interactions
http://www.patrocles.org
PATROCLES: the database of polymorphic miRNA-target interactions
http://www.patrocles.org
PATROCLES: the database of polymorphic miRNA-target interactions
http://www.patrocles.org
PATROCLES: the database of polymorphic miRNA-target interactions
http://www.patrocles.org
PATROCLES: the database of polymorphic miRNA-target interactions
http://www.patrocles.org
SNPs
CNVs
eQTL
PATROCLES: the database of polymorphic miRNA-target interactions
http://www.patrocles.org
PATROCLES: the database of polymorphic miRNA-target interactions
http://www.patrocles.org
PATROCLES: the database of polymorphic miRNA-target interactions
http://www.patrocles.org
PATROCLES: the database of polymorphic miRNA-target interactions
http://www.patrocles.org
Thank you for your attention