Emma J. K. Kowal, Yuta Sakai, Michael P. McGurk, Zoe J. Pasetsky, Christopher B. Burge
Spliceosomal introns are a ubiquitous feature of eukaryotic genes, whose presence often boosts the expression of their host gene, a phenomenon known as intron-mediated enhancement (IME). IME has been noted across diverse genes and organisms but remains mysterious in many respects. For example, how does intron sequence affect the magnitude of IME? In this study, we performed a massively parallel reporter assay (MPRA) to assess the effect of varying intron sequence on gene expression in a high-throughput manner, in human cells, using tens of thousands of synthetic introns with natural splice sites and randomized internal sequence. We observe that most random introns splice efficiently and enhance gene expression as well as or better than fully natural introns. Nearly all introns stimulate gene expression ∼eight-fold above an intronless control, at both mRNA and protein levels, suggesting that the primary mechanism acts to increase mRNA levels. IME strength is positively associated with splicing efficiency and with the intronic content of poly-uridine stretches, which we confirm using reporter experiments. In sum, this work assesses the IME of a diverse library of introns and uncovers sequence-dependent aspects, but suggests that enhancement of gene expression is a general property of splicing, largely independent of intron sequence.
Emma J. K. Kowal, Yuta Sakai, Michael P. McGurk, Zoe J. Pasetsky, Christopher B. Burge. Sequence-dependent and -independent effects of intron-mediated enhancement learned from thousands of random introns. 2025-02-24. https://doi.org/10.1093/nar/gkaf097
A dual-reporter library of 19,342 sequence-mapped synthetic introns with natural splice sites and randomized internal sequence was integrated as single copies into HILO-RMCE HEK293T A2 cells. Ten biological RNA-seq replicates measured spliced GFP, unspliced GFP, and dTomato barcode counts.
A smaller validation library tested 30 parent random introns in original, U4-deleted, and U4-added sequence forms, for 90 designed intron sequences, together with intronless controls. The pool was transiently transfected into HEK293T A2 cells and assayed by two-replicate RNA-seq of dTomato, spliced GFP, and unspliced GFP reporter reads.
The integrated random-intron reporter library was sorted into GFP/dTomato-high green and GFP/dTomato-low red populations through three successive FACS stages in two independent trajectories. RNA-seq of the twelve sorted bins provides stage-specific spliced GFP, unspliced GFP, and dTomato counts for the same trusted intron elements.
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.
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