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

Abstract

UTRs contain crucial regulatory elements for RNA stability, translation and localization, so 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 means of experimental or computational assessment are lacking. To systematically evaluate the effects of UTR variants on RNA stability, we established a massively parallel reporter assay on 6,555 UTR variants reported in human disease databases. We examined the RNA degradation patterns mediated by the UTR library in multiple cell lines, and then applied LASSO regression to model the influential regulators of RNA stability. We found that TA dinucleotides are the most prominent destabilizing element. Gain of TA dinucleotide outlined mutant UTRs with reduced stability. Studies on endogenous transcripts indicate that high TA-dinucleotide ratios in UTRs promote RNA degradation. Conversely, elevated GC content and protein binding on TA dinucleotides protect high-TA RNA from degradation. Further analysis reveals polarized roles of TA-dinucleotide-binding proteins in RNA protection and degradation. Furthermore, the TA-dinucleotide ratio of both UTRs is a common characteristic of genes in innate immune response pathways, implying that the global transcriptomic regulon involves stability coordination via UTRs. We also demonstrate that stability-altering UTRs are associated with changes in biobank-based health indices, providing evidence that UTR-mediated RNA stability contributes to establishing robust gene networks and potentially enabling disease-associated UTR variants to be classified for precision medicine.