Genetic variants affecting RNA stability influence complex traits and disease risk II

Gene expression is jointly modulated by transcriptional regulation and mRNA stability, yet the latter is often overlooked in studies on genetic variants. Leveraging metabolic labeling data (Bru/BruChase-seq) and a new computational pipeline, RNAtracker, we distinguished allele-specific RNA stability (asRS) from allele-specific RNA transcription (asRT) events. Our analysis identified >5,000 asRS variants, revealing comparable impact of stability on allelic imbalance as transcriptional regulation. This study highlights RNA stability as a critical, yet understudied mechanism linking genetic variation and disease. Overall design: Synthetic DNA oligos containing the variants of interest and their flanking sequences (164 nucleotides total) were cloned into the 3'UTR of the eGFP gene. The expression of this reporter gene was driven by the CMV early enhancer/chicken beta actin (CAG) promoter. These oligos were then electroporated into HeLa cells. Following electroporation (24 h), total RNA was extracted for sequencing targeting the tested variant regions. Specifically, the test sequences were amplified from both the plasmid library and mRNA to generate DNA-seq and RNA-seq libraries. Three biological replicates were collected for each experiment and a high correlation was observed between replicates (R=0.84). Sequencing data of the plasmid DNA and mRNA were compared to identify sites associated with significant expression differences between the two alleles using MPRAnalyze. FDR = 0.1 and |ln(Fold Change)| = 0.1 were required to call significance.