1. Polymorphic miRNA–target interactions: a novel source of phenotypic variation.
Michel Georges
Unit of Animal Genomics
Faculty of Veterinary Medicine
University of Liège
Belgium

2. Acknowledgments Ulg: Wyeth Research: INRA: Funding sources: 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

3. 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
miRNA
biology

4. QTL mapping in a Romanov x Texel F2 intercross …
56 phenotypes measuring body and carcass composition
160 microsatellite markers
QTL express

5. A QTL affecting muscle mass maps to OAR2 …
Chromosome 2

6. The three F1 rams are heterozygous Qq for the QTL …

7. A QTL affecting muscle mass maps to OAR2 …
MSTN
Muscle-specific chalone of the
TGF superfamily of GDFs.

8. 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.

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

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

11. Resequencing the MSTN gene identifies 20 non-coding SNPs …
3 Texels – 7 controls

12. 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 controls breeds
F Qq rams TT TC CC

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

14. miRNA « seeds » 5’ dominant versus 3’ compensatory sites
From Lewis et al. (2005) Cell 120: 15-20

15. … predicted to be a target site for miR1, miR206 (and miR122).
Probability to occur « by chance alone »: <

16. Obvious hypothesis …
AAAAA
AAAAA
AAAAA
AAAAA

17. 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.

18. Texel sheep have ≈ 3-fold reduction in circulating MSTN levels …
… supports translational inhibition.

19. mRNA allelic imbalance in GA heterozygotes …
… supports allele-specific mRNA degradation in P-bodies.

20. miR1/206 – mutant MSTN interaction supported by reporter assay
pCMV
BGHpA
Pri-miR
pcDNA3.1-miR

21. miR1/206 – mutant MSTN interaction supported by reporter assay

22. 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

23. 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?

24. Common SNPs affect the target site content of thousands of human genes …
miRNA targets
92,967 SNPs in 21,206 3’UTRs
540 X-targets (Xie et al., 2005)
444 L-targets (Lewis et al., 2005)

25. Evidence for purifying selection against SNPs altering target site content …

26. Evidence for purifying selection against SNPs altering target site content …

27. PATROCLES: the database of polymorphic miRNA-target interactions

28. PATROCLES: the database of polymorphic miRNA-target interactions

29. PATROCLES: the database of polymorphic miRNA-target interactions

30. PATROCLES: the database of polymorphic miRNA-target interactions

31. PATROCLES: the database of polymorphic miRNA-target interactions

32. PATROCLES: the database of polymorphic miRNA-target interactions
SNPs
CNVs
eQTL

33. PATROCLES: the database of polymorphic miRNA-target interactions

34. PATROCLES: the database of polymorphic miRNA-target interactions

35. PATROCLES: the database of polymorphic miRNA-target interactions

36. PATROCLES: the database of polymorphic miRNA-target interactions

37. Thank you for your attention