The Search for and Functional Analysis of Genetic Variants in microRNA-Binding Sites using Massively Parallel Reporter Assay.

ABSTRACT

A large-scale search for the genetic variants with a bias in the representation of alleles in transcriptome data (AE SNPs) and the binding sites in microRNA 3'-UTRs was performed and their functional significance was assessed using massively parallel reporter assay (MPRA). Of the 629,559 associated "SNP-gene" pairs (eQTLs) discovered in the human liver tissue according to the GTEx Analysis V8 data, 4394 polymorphic positions in the 3'-UTRs of the genes, which represent the eQTLs for these genes were selected. The TargetScanHuman 7.0 algorithm and PolymiRTS database were searched for the potential microRNA-binding sites. Of the predicted microRNA sites affected by eQTL-SNPs, we selected 51 sites with the best evidence of functionality according to Ago2-CLIP-seq, CLEAR-CLIP, and eCLIP-seq for RNA-binding proteins. For MPRA, a library of the plasmids carrying the main and alternative alleles for each AE SNP (in total, 102 constructs) was created. Allele-specific expression for 6 SNPs was detected by transfection of the HepG2 cell line with the constructed plasmid library and sequencing of target DNA and RNA sequences using the Illumina (MiSeq) platform.