Dustin Griesemer, James R Xue, Steven K Reilly, Jacob C Ulirsch, Kalki Kukreja et al.
3' untranslated region (3'UTR) variants are strongly associated with human traits and diseases, yet few have been causally identified. We developed the massively parallel reporter assay for 3'UTRs (MPRAu) to sensitively assay 12,173 3'UTR variants. We applied MPRAu to six human cell lines, focusing on genetic variants associated with genome-wide association studies (GWAS) and human evolutionary adaptation. MPRAu expands our understanding of 3'UTR function, suggesting that simple sequences predominately explain 3'UTR regulatory activity. We adapt MPRAu to uncover diverse molecular mechanisms at base pair resolution, including an adenylate-uridylate (AU)-rich element of LEPR linked to potential metabolic evolutionary adaptations in East Asians. We nominate hundreds of 3'UTR causal variants with genetically fine-mapped phenotype associations. Using endogenous allelic replacements, we characterize one variant that disrupts a miRNA site regulating the viral defense gene TRIM14 and one that alters PILRB abundance, nominating a causal variant underlying transcriptional changes in age-related macular degeneration.
Dustin Griesemer, James R Xue, Steven K Reilly, Jacob C Ulirsch, Kalki Kukreja, Joe R Davis, Masahiro Kanai, David K Yang, John C Butts, Mehmet H Guney, Jeremy Luban, Stephen B Montgomery, Hilary K Finucane, Carl D Novina, Ryan Tewhey, Pardis C Sabeti. Genome-wide functional screen of 3′UTR variants uncovers causal variants for human disease and evolution. 2021-09-16. https://doi.org/10.1016/j.cell.2021.08.025
A secondary MPRAu tiling library tested 5-bp non-overlapping deletions across the 100-bp sequence and all single-nucleotide substitutions within ±10 bp of 80 tamVars, using both reference and alternate sequence backgrounds.
The pooled CMS+GWAS 100-bp reference/alternate 3′UTR library was assayed in primary human mammary epithelial cells (HMEC) using an episomal GFP reporter with a random hexamer barcode; steady-state RNA output was compared with plasmid DNA input across five transfections.
The pooled CMS+GWAS 100-bp reference/alternate 3′UTR library was assayed in SK-N-SH neuroblastoma cells using an episomal GFP reporter with a random hexamer barcode; steady-state RNA output was compared with plasmid DNA input across four transfections.
The study’s separately transfected CMS and GWAS 100-bp reference/alternate 3′UTR libraries were assayed in HEK293FT cells using an episomal GFP reporter with a random hexamer barcode; steady-state RNA output was compared with plasmid DNA input across five transfections.
A secondary MPRAu tiling library tested 5-bp non-overlapping deletions across the 100-bp sequence and all single-nucleotide substitutions within ±10 bp of 80 tamVars, using both reference and alternate sequence backgrounds.
The pooled CMS+GWAS 100-bp reference/alternate 3′UTR library was assayed in GM12878 lymphoblastoid cells using an episomal GFP reporter with a random hexamer barcode; steady-state RNA output was compared with plasmid DNA input across four transfections.
A secondary MPRAu tiling library tested 5-bp non-overlapping deletions across the 100-bp sequence and all single-nucleotide substitutions within ±10 bp of 80 tamVars, using both reference and alternate sequence backgrounds.
The pooled CMS+GWAS 100-bp reference/alternate 3′UTR library was assayed in HepG2 cells using an episomal GFP reporter with a random hexamer barcode; steady-state RNA output was compared with plasmid DNA input across four transfections.
The pooled CMS+GWAS 100-bp reference/alternate 3′UTR library was assayed in K562 cells using an episomal GFP reporter with a random hexamer barcode; steady-state RNA output was compared with plasmid DNA input across four transfections.
A secondary MPRAu tiling library tested 5-bp non-overlapping deletions across the 100-bp sequence and all single-nucleotide substitutions within ±10 bp of 80 tamVars, using both reference and alternate sequence backgrounds.
A secondary MPRAu tiling library tested 5-bp non-overlapping deletions across the 100-bp sequence and all single-nucleotide substitutions within ±10 bp of 80 tamVars, using both reference and alternate sequence backgrounds.
A secondary MPRAu tiling library tested 5-bp non-overlapping deletions across the 100-bp sequence and all single-nucleotide substitutions within ±10 bp of 80 tamVars, using both reference and alternate sequence backgrounds.
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 6 files (ZIP)MPRAu_Methods_ENCODE.pdfSOURCES.txtsupplementary_table_1_mprau_results.xlsxsupplementary_table_2_luciferase_validation.xlsxsupplementary_table_3_barcode_effects.xlsxsupplementary_table_4_finemapping_support.xlsx