Single nucleotide polymorphisms (SNPs) in microRNAs (miRNAs) or their target sites (miR-SNPs) within the 3′-UTR of mRNAs are increasingly thought to play a major role in pathological dysregulation of gene expression. Here, we studied the functional role of miR-SNPs on miRNA-mediated post-transcriptional regulation of gene expression. First, analyses were performed on a SNP located in the miR-155 target site within the 3′-UTR of the Angiotensin II type 1 receptor (AGTR1; rs5186, A > C) mRNA. Second, a SNP in the 3′-UTR of the muscle RAS oncogene homolog (MRAS; rs9818870, C > T) mRNA was studied which is located outside of binding sites of miR-195 and miR-135. Using these SNPs we investigated their effects on local RNA structure, on local structural accessibility and on functional miRNA binding, respectively. Systematic computational RNA folding analyses of the allelic mRNAs in either case predicted significant changes of local RNA structure in the vicinity of the cognate miRNA binding sites. Consistently, experimental in vitro probing of RNA showing differential cleavage patterns and reporter gene-based assays indicated functional differences of miRNA-mediated regulation of the two AGTR1 and MRAS alleles. In conclusion, we describe a novel model explaining the functional influence of 3′-UTR-located SNPs on miRNA-mediated control of gene expression via SNP-related changes of local RNA structure in non-coding regions of mRNA. This concept substantially extends the meaning of disease-related SNPs identified in non protein-coding transcribed sequences within or close to miRNA binding sites.
Genome-wide association studies (GWAS) have identified chromosomal loci that affect risk of coronary heart disease (CHD) independent of classical risk factors. One such association signal has been identified at 6q23.2 in both Caucasians and East Asians. The lead CHD-associated polymorphism in this region, rs12190287, resides in the 3′ untranslated region (3′-UTR) of TCF21, a basic-helix-loop-helix transcription factor, and is predicted to alter the seed binding sequence for miR-224. Allelic imbalance studies in circulating leukocytes and human coronary artery smooth muscle cells (HCASMC) showed significant imbalance of the TCF21 transcript that correlated with genotype at rs12190287, consistent with this variant contributing to allele-specific expression differences. 3′ UTR reporter gene transfection studies in HCASMC showed that the disease-associated C allele has reduced expression compared to the protective G allele. Kinetic analyses in vitro revealed faster RNA-RNA complex formation and greater binding of miR-224 with the TCF21 C allelic transcript. In addition, in vitro probing with Pb2+ and RNase T1 revealed structural differences between the TCF21 variants in proximity of the rs12190287 variant, which are predicted to provide greater access to the C allele for miR-224 binding. miR-224 and TCF21 expression levels were anti-correlated in HCASMC, and miR-224 modulates the transcriptional response of TCF21 to transforming growth factor-β (TGF-β) and platelet derived growth factor (PDGF) signaling in an allele-specific manner. Lastly, miR-224 and TCF21 were localized in human coronary artery lesions and anti-correlated during atherosclerosis. Together, these data suggest that miR-224 interaction with the TCF21 transcript contributes to allelic imbalance of this gene, thus partly explaining the genetic risk for coronary heart disease associated at 6q23.2. These studies implicating rs12190287 in the miRNA-dependent regulation of TCF21, in conjunction with previous studies showing that this variant modulates transcriptional regulation through activator protein 1 (AP-1), suggests a unique bimodal level of complexity previously unreported for disease-associated variants.
Introduction: Genome-wide association studies (GWAS) have identified chromosomal loci that affect risk of coronary heart disease (CHD) independent of classical risk factors. One such association signal has been identified at 6q23.2 in both Caucasians and East Asians. The lead CHD-associated variant in this region, rs12190287, resides in the 3’ untranslated region of the basic-helix-loop-helix transcription factor, TCF21 , and is predicted to alter the seed binding site for miR-224. Allelic imbalance studies demonstrated significant imbalance of the TCF21 transcript that correlated with genotype at rs12190287. Hypothesis: We hypothesize that genetic variation at rs12190287 contributes to allele-specific TCF21 expression imbalance via miR-224 regulation, thus altering CHD risk. Methods and Results: Reporter assays in human coronary artery smooth muscle cells (HCASMC) showed that the disease-associated C allele is selectively targeted by miR-224, leading to reduced expression compared to the protective G allele. Kinetic analyses in vitro revealed faster RNA-RNA complex formation and greater binding of miR-224 with the TCF21 C allelic transcript. In addition, in vitro probing with Pb 2+ and RNase T1 revealed structural differences between the TCF21 variants in proximity of the rs12190287 variant, which are predicted to provide greater access to the C allele for miR-224 binding. miR-224 and TCF21 expression levels were anti-correlated in HCASMC, and miR-224 modulates the transcriptional response of TCF21 to transforming growth factor- and platelet derived growth factor signaling in an allele-specific manner. Lastly, miR-224 and TCF21 were localized in human coronary artery lesions and anti-correlated during atherosclerosis in vivo . Conclusions: Together, these data suggest that miR-224 interaction with the TCF21 transcript contributes to allelic imbalance of this gene, thus partly explaining the genetic risk for CHD at 6q23.2. These studies implicating rs12190287 in the miRNA-dependent regulation of TCF21 , in conjunction with previous studies showing that this variant modulates transcriptional regulation through AP-1, suggests a unique bimodal level of complexity previously unreported for disease-associated variants.
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