Jessica D. West, Hannah J. Smith, Luyen Tien Vu, Elizabeth A. Fogarty, Kenneth A. Matreyek et al.
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.
Jessica D. West, Hannah J. Smith, Luyen Tien Vu, Elizabeth A. Fogarty, Kenneth A. Matreyek, Douglas M. Fowler, Andrew Grimson. The quantitative impact of 3′UTRs on gene expression. 2025-06-20. https://doi.org/10.1093/nar/gkaf568
More than 1,400 full-length human 3′UTRs were integrated as single-copy barcoded GFP reporters in a HEK293T Bxb1 landing pad driven by the moderate PGK promoter. The experiment measures relative protein output by GFP-bin sorting, steady-state RNA abundance by RNA/DNA barcode ratios, and translational distribution by polysome profiling.
More than 1,400 full-length human 3′UTRs were integrated as single-copy barcoded GFP reporters in a HEK293T Bxb1 landing pad driven by the strong CAG promoter. The experiment measures relative protein output by GFP-bin sorting, steady-state RNA abundance by RNA/DNA barcode ratios, and translational distribution by polysome profiling.
The 173-element pilot library was integrated into a HEK293T Tet-Off Bxb1 landing pad, pulsed without doxycycline for 4 hours, and then transcriptionally shut off with doxycycline. Barcode RNA was measured at 0, 1, 2, 3, 4, 8, and 20 hours alongside DNA and total-RNA reference libraries to quantify relative decay behavior.
The pilot library of 173 full-length human 3′UTRs was integrated as single-copy Bxb1 GFP reporters in a HEK293T Tet-On landing pad and sorted into five GFP bins in two independent runs. Barcode CPMs were used to summarize protein output, RNA/DNA abundance, and polysome-associated translation for each 3′UTR.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
Download all 24 files (ZIP)gkaf568_supplemental_file.pdfgkaf568fig1.gifgkaf568fig1.jpggkaf568fig2.gifgkaf568fig2.jpggkaf568fig3.gifgkaf568fig3.jpggkaf568fig4.gifgkaf568fig4.jpggkaf568fig5.gifgkaf568fig5.jpggkaf568fig6.gifgkaf568fig6.jpggkaf568figgra1.gifgkaf568figgra1.jpggkaf_supp_page-04.pnggkaf_supp_page-05.pngGSE270252_family.soft.gzGSE270252_utrome_barcode_counts.tsv.gzGSE270253_barcode_3UTR_linkages.tsv.gzGSE270253_family.soft.gzGSE270254_family.soft.gzPMC12207410_supplementaryFiles.zipsource_notes.txt
