Multiplexed Assays of Human Disease-relevant Mutations Reveal UTR Dimer Composition as a Major Determinant of RNA Stability

UTRs contain crucial regulatory elements for RNA stability, translation and localization and their integrity is indispensable for gene expression. It has been estimated that ~3.7% of disease-associated genetic variants are located in UTRs. However, functional interpretation of UTR variants is largely incomplete because efficient experimental assessment and computational prediction are both lacking. To systematically assess the effect of UTR variants on RNA stability, we established an unbiased massively parallel reporter assay with 6,555 UTR variants reported in human disease databases. We found that there are common as well as distinct stability regulation elements in each cell line and that, among RNA-binding protein-binding sites, microRNA seed sequences, structural cues and small k-mers, TA-dimers are the most prominent destabilizing sequence. Overall design: We performed time course assays to examine the relative stability of 6,555 UTR variants collected from the Human Gene Mutation Database (HGMD) and the public archive of interpretations of clinically relevant variants (ClinVar). UTRs fused with GFP coding regions were in vitro transcribed and transfected into human embryonic kidney cells HEK293T or neuroblastoma cells SH-SY5Y. The relative abundance of wild-type (WT) and mutant (mt) alleles was monitored over a time course, and determined by amplicon sequencing. We estimated the decay constant and the half-life (t1/2) of each UTR by its relative abundance over time. We defined stability-altering variants as those with significantly changed decay constant compared to their WT counterpart, determined by weighted linear regression.