Assaying 3-Prime Untranslated Region Variants in a Massively Parallel Fashion

Abstract

Rapid advances in human genome sequencing technologies over the past decade have unraveled thousands of loci in the genome containing variants that cause inherited disease or have been placed under positive selective pressure to protect against acquired diseases such as infectious agents, climate change, and diet restrictions. Although genic regions, including 3'-Untranslated Regions (3’- UTRs), are highly enriched in disease and selection-associated loci, linkage disequilibrium and the lengthy timescale of low-throughput validation hinder functional follow-up of each locus. To address this challenge, we developed MPRAu, a tool for identifying functional 3'-UTR variants by combining the Massively Parallel Reporter Assay with Polysome Profiling. Interestingly, we find that under standard growth conditions in the HEK293 cell line, short (101bp) 3'- UTR segments have surprisingly little impact on translation. Furthermore, out of 9,754 variants tested (one-seventh of 3’-UTR variants with a minor allele frequency greater than 5%), we identify a shortlist of 115 variants with significant allelic differences in steady-state RNA expression, including some implicated in sodium transport, leptin signaling, and creatinine synthesis. Though functional variation identified by MPRA is concordant with known regulatory mechanisms, the vast majority of identified variants fall outside of predicted motifs. This unbiased approach provides concrete leads as well as a means to quickly identify a wealth of causal variants within untranslated regions of the genome.