The quantitative impact of 3′UTRs on gene expression

Control of gene expression is fundamental to biology, and post-transcriptional regulation is an important component of this process. In mammals, the 3′UTR in particular serves as a major source of regulatory information within the transcript. Here we developed an accurate massively parallel reporter assay (MPRA) system to evaluate the impact of >1400 full-length human 3′UTRs on RNA abundance, stability, translational regulation, and total protein output. We demonstrated that our MPRA is consistent with regulation of the corresponding endogenous transcripts. We used the MPRA datasets to model the relative contributions of RNA abundance and translational efficiency toward total 3′UTR-mediated regulation, revealing an unexpectedly large role for 3′UTR-specified translational control, and providing additional evidence that much of 3′UTR-encoded regulation is mediated by concerted regulation of translation plus decay. We observed relationships between GC content and 3′UTR length and different modes of regulation, and identified sequence motifs corresponding to regulatory RNA-binding proteins associated with mediating 3′UTR-dependent gene expression. We compared regulation from >1400 3′UTRs under control of two dissimilar promoters, which revealed promoter-associated differences in post-transcriptional regulation for certain 3′UTRs. Together, this dataset represents a comprehensive characterization of 3′UTR-mediated quantitative regulation.