Deciphering the cis-regulatory code of alternative splicing in human cell lines

Alternative splicing (AS) is a key mechanism of gene expression diversity, and dysregulation of AS is implicated in many human diseases. Identifying variants that disrupt splicing and characterizing their impact on isoform abundance across different cell types remains challenging. Here, we perform massively parallel reporter assays (MPRAs) to measure splicing phenotypes of over 87,000 sequence variants in five cell lines of different tissue origin. The MPRA encompasses 2,096 short human exons from 1,733 genes and tens of thousands of single and double variants. Our measurements identify variants that strongly modulate splicing but are currently classified as variants of unknown significance or have not yet been described. We systematically identify putative RNA-binding protein (RBP) binding motifs and quantify effect sizes associated with the disruption of these motifs, suggesting possible mechanisms for variant impact. A comparison across cell lines enables the identification of variants that differentially modulate splicing. Massively parallel splicing reporter assay (MPRA) testing ~87,000 exon and variant sequences cloned into a Citrine–SMN2 minigene backbone. Constructs were transfected into five human cell lines (HEK293, HeLa, K562, MCF7, HMC3) with biological replicates. Sequencing was used to quantify exon inclusion (PSI) across replicates, and processed PSI values are reported.