polymorphic mirna–target interactions: a novel source of phenotypic variation
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 PresentationTRANSCRIPT
![Page 1: Polymorphic miRNA–target interactions: a novel source of phenotypic variation](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/1.jpg)
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](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/2.jpg)
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](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/3.jpg)
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
![Page 4: Polymorphic miRNA–target interactions: a novel source of phenotypic variation](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/4.jpg)
QTL mapping in a Romanov x Texel F2 intercross …
56 phenotypes measuring body and carcass composition 160 microsatellite markers QTL express
![Page 5: Polymorphic miRNA–target interactions: a novel source of phenotypic variation](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/5.jpg)
A QTL affecting muscle mass maps to OAR2 …
Chromosome 2
![Page 6: Polymorphic miRNA–target interactions: a novel source of phenotypic variation](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/6.jpg)
The three F1 rams are heterozygous Qq for the QTL …
![Page 7: Polymorphic miRNA–target interactions: a novel source of phenotypic variation](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/7.jpg)
A QTL affecting muscle mass maps to OAR2 …
MSTN
Muscle-specific chalone of theTGF superfamily of GDFs.
![Page 8: Polymorphic miRNA–target interactions: a novel source of phenotypic variation](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/8.jpg)
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.
![Page 9: Polymorphic miRNA–target interactions: a novel source of phenotypic variation](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/9.jpg)
MASA in offspring of recombinant ram fine-maps QTL to OAR2q …
![Page 10: Polymorphic miRNA–target interactions: a novel source of phenotypic variation](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/10.jpg)
A signature of selection fine-maps QTL in vicinity of MSTN gene …
MS
TN
![Page 11: Polymorphic miRNA–target interactions: a novel source of phenotypic variation](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/11.jpg)
Resequencing the MSTN gene identifies 20 non-coding SNPs …
3 Texels – 7 controls
![Page 12: Polymorphic miRNA–target interactions: a novel source of phenotypic variation](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/12.jpg)
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
![Page 13: Polymorphic miRNA–target interactions: a novel source of phenotypic variation](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/13.jpg)
G+6723G-A reveals one of « Kellis’s octamer motifs » …
G
![Page 14: Polymorphic miRNA–target interactions: a novel source of phenotypic variation](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/14.jpg)
miRNA « seeds »
From Lewis et al. (2005) Cell 120: 15-20
5’ dominant versus 3’ compensatory sites
![Page 15: Polymorphic miRNA–target interactions: a novel source of phenotypic variation](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/15.jpg)
… predicted to be a target site for miR1, miR206 (and miR122).
Probability to occur « by chance alone »: < 0.01-0.05
G
![Page 16: Polymorphic miRNA–target interactions: a novel source of phenotypic variation](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/16.jpg)
Obvious hypothesis …
AAAAA
AAAAA
AAAAA
AAAAA
![Page 17: Polymorphic miRNA–target interactions: a novel source of phenotypic variation](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/17.jpg)
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
![Page 18: Polymorphic miRNA–target interactions: a novel source of phenotypic variation](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/18.jpg)
Texel sheep have ≈ 3-fold reduction in circulating MSTN levels …
… supports translational inhibition.
![Page 19: Polymorphic miRNA–target interactions: a novel source of phenotypic variation](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/19.jpg)
mRNA allelic imbalance in GA heterozygotes …
… supports allele-specific mRNA degradation in P-bodies.
![Page 20: Polymorphic miRNA–target interactions: a novel source of phenotypic variation](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/20.jpg)
miR1/206 – mutant MSTN interaction supported by reporter assay
pCMV BGHpAPri-miR pcDNA3.1-miR
![Page 21: Polymorphic miRNA–target interactions: a novel source of phenotypic variation](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/21.jpg)
miR1/206 – mutant MSTN interaction supported by reporter assay
![Page 22: Polymorphic miRNA–target interactions: a novel source of phenotypic variation](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/22.jpg)
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
![Page 23: Polymorphic miRNA–target interactions: a novel source of phenotypic variation](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/23.jpg)
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?
![Page 24: Polymorphic miRNA–target interactions: a novel source of phenotypic variation](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/24.jpg)
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
![Page 25: Polymorphic miRNA–target interactions: a novel source of phenotypic variation](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/25.jpg)
Evidence for purifying selection against SNPs altering target site content …
![Page 26: Polymorphic miRNA–target interactions: a novel source of phenotypic variation](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/26.jpg)
Evidence for purifying selection against SNPs altering target site content …
![Page 27: Polymorphic miRNA–target interactions: a novel source of phenotypic variation](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/27.jpg)
PATROCLES: the database of polymorphic miRNA-target interactions
http://www.patrocles.org
![Page 28: Polymorphic miRNA–target interactions: a novel source of phenotypic variation](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/28.jpg)
PATROCLES: the database of polymorphic miRNA-target interactions
http://www.patrocles.org
![Page 29: Polymorphic miRNA–target interactions: a novel source of phenotypic variation](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/29.jpg)
PATROCLES: the database of polymorphic miRNA-target interactions
http://www.patrocles.org
![Page 30: Polymorphic miRNA–target interactions: a novel source of phenotypic variation](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/30.jpg)
PATROCLES: the database of polymorphic miRNA-target interactions
http://www.patrocles.org
![Page 31: Polymorphic miRNA–target interactions: a novel source of phenotypic variation](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/31.jpg)
PATROCLES: the database of polymorphic miRNA-target interactions
http://www.patrocles.org
![Page 32: Polymorphic miRNA–target interactions: a novel source of phenotypic variation](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/32.jpg)
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](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/33.jpg)
PATROCLES: the database of polymorphic miRNA-target interactions
http://www.patrocles.org
![Page 34: Polymorphic miRNA–target interactions: a novel source of phenotypic variation](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/34.jpg)
PATROCLES: the database of polymorphic miRNA-target interactions
http://www.patrocles.org
![Page 35: Polymorphic miRNA–target interactions: a novel source of phenotypic variation](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/35.jpg)
PATROCLES: the database of polymorphic miRNA-target interactions
http://www.patrocles.org
![Page 36: Polymorphic miRNA–target interactions: a novel source of phenotypic variation](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/36.jpg)
PATROCLES: the database of polymorphic miRNA-target interactions
http://www.patrocles.org
![Page 37: Polymorphic miRNA–target interactions: a novel source of phenotypic variation](https://reader035.vdocument.in/reader035/viewer/2022062305/568158cc550346895dc61696/html5/thumbnails/37.jpg)
Thank you for your attention