� Biomic � sciences at the ULg � by mmqkkj

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




                                   Michel Georges

                         Unit of Animal Genomics
                    Faculty of Veterinary Medicine
                                University of Liège
                                           Belgium
Acknowledgments
   Ulg:                                        Wyeth Research:
          Alex Clop                                 James Tobin
          Fabienne Marcq                       INRA:
          Haruko Takeda                             Jacques Bouix
          Dimitri Pirottin                          Elisabeth Laville
          Xavier Tordoir                            Catherine Larzul
          Florian Caiment                           Francis Eychenne
          Françoise Meish                           Dragan Milinkovic
          Samuel Hiard                              Bernard Bibé
          Carole Charlier                           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
miRNA        polar overdominance
biology
                                Hypermuscled Texel:
                                  Patrocles mutation
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 …




        Muscle-specific chalone of the
         TGF superfamily of GDFs.

                     MSTN
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 …




             MSTN
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 …
                 G+6723G-A


     42 Texels


                                                      Strong signature of
                                                       selection
                         90 controls - 11 breeds



                                                      Heterozygous « Qq »
                                                       rams exclude all but the
                                                       g+6723G-A SNP.



                                                      The g+6723A allele is
                                                       virtually Texel-specific.
     F Qq rams




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




      G
miRNA « seeds »




     5’ dominant versus 3’ compensatory sites



     From Lewis et al. (2005) Cell 120: 15-20
… predicted to be a target site for
miR1, miR206 (and miR122).




        G




   Probability to occur « by chance alone »: < 0.01-0.05
Obvious hypothesis …
miR1 and miR206 are conserved in
sheep and strongly expressed in
skeletal muscle …

                                     miR1 and miR206 are
                                 strongly expressed in SM




miR1.1, miR1.2, miR122 and
miR206 are conserved in sheep.
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   Pri-miR   BGHpA   pcDNA3.1-miR
miR1/206 – mutant MSTN interaction
supported by reporter assay
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                                          enrichment
         Texel A allele                                      WT 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 …
          SNPs                         miRNA targets
92,967 SNPs in 21,206 3’UTRs   540 X-targets (Xie et al., 2005)
                               444 L-targets (Lewis et al., 2005)
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

                     CNVs
           SNPs                eQTL




         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
Thank you for your attention

								
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