Evidence for Positive Selection on a Number of MicroRNA Regulatory Interactions during Recent Human Evolution
Figure 3
rs683 modulates endogenous TYRP1 targeting by miR-155 in SK-MEL-19 cells.
(A) Genotyping the rs683 locus in SK-MEL-19 cells. The region around rs683 was amplified from SK-MEL-19 genomic DNA and sequenced. Sequence traces (shown) revealed rs683 heterozygosity at the TYRP1 locus, as indicated. (B) Ectopic miR-155 expression reduces TYRP1 protein levels. TYRP1 levels in the skin-derived melanoma cell line, SK-Mel-19 were analyzed by performing immunoblotting on cell lysates from miR-CTL transfected or cells transfected with increasing amounts of miR-155 as indicated. Densitometric quantitation of TYRP1 levels is indicated as protein levels relative to the mock transfectants. (C) miR-155 reduces TYRP1 mRNA levels in SK-Mel19. SK-MEL19 cells were mock transfected or transfected with the indicated miR-CTL or miR-155. mRNA was extracted and TYRP1 levels assessed by qPCR. Results are plotted relative to miR-CTL-treated cells. Bars are the mean ± standard deviation of triplicate experiments. The differences in expression between miR-155 transfected cells and either mock or miR-CTL are statistically significant (P<0.01, Students t test). (D) Targeting of the derived allele by miR-155. SK-Mel-19 cells were transfected with increasing amounts of miR-155 as indicated. mRNA was then extracted and expression of the ancestral (blue) versus the derived allele (red) assessed by allele-specific TaqMan SNP qPCR. Results are plotted as the expression level of each TYRP1 allele relative to controls (log2 transformed). Note that the transcripts carrying the derived allele were suppressed by miR-155 greater that 2-fold, whereas transcripts carrying the ancestral allele were only modestly affected.