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

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• Genetic variants can influence gene expression and regulation.
• MicroRNAs (miRNAs) play crucial roles in post-transcriptional gene silencing by binding to 3'-untranslated regions (3'-UTRs).
• Understanding the interplay between genetic variation, miRNA binding, and gene expression is vital for deciphering complex biological processes.

Purpose of the Study:

• To identify genetic variants associated with allele-specific expression (AE SNPs) that also affect miRNA binding sites in 3'-UTRs.
• To functionally assess the significance of these AE SNPs on miRNA binding and gene regulation using massively parallel reporter assays (MPRA).

Main Methods:

• Leveraged GTEx Analysis V8 data to identify expression quantitative trait loci (eQTLs) in human liver tissue.
• Utilized TargetScanHuman 7.0 and PolymiRTS database to predict miRNA binding sites affected by eQTL-SNPs.
• Selected functional miRNA binding sites based on Ago2-CLIP-seq, CLEAR-CLIP, and eCLIP-seq data.
• Constructed a plasmid library with both main and alternative alleles for selected AE SNPs (102 constructs).
• Performed MPRA by transfecting HepG2 cells and sequencing target DNA and RNA to detect allele-specific expression for 6 SNPs.

Main Results:

• Identified 4394 polymorphic positions in 3'-UTRs representing eQTLs from 629,559 SNP-gene pairs.
• Selected 51 miRNA binding sites affected by eQTL-SNPs with strong functional evidence.
• Successfully detected allele-specific expression for 6 AE SNPs using MPRA in HepG2 cells.

Conclusions:

• AE SNPs impacting miRNA binding sites are prevalent and functionally significant.
• MPRA is a powerful tool for validating the functional impact of genetic variants on gene regulation.
• These findings contribute to understanding the genetic basis of gene expression regulation and its implications in human diseases.