Cryptic splicing mediates genetic and therapeutic perturbation of human gene expression levels [Cut&Tag]

Alternative splicing (AS) in human genes is widely viewed as a mechanism for enhancing proteomic diversity. AS can also impact gene expression levels without increasing protein diversity by producing “unproductive” transcripts that are targeted for rapid degradation by nonsense-mediated decay (NMD). However, the relative importance of this regulatory mechanism remains underexplored. To better understand the impact of AS-NMD relative to other regulatory mechanisms, we analyzed population-scale genomic data across eight molecular assays, covering various stages from transcription to cytoplasmic decay. We report threefold more unproductive splicing compared to prior estimates using steady-state RNA. This unproductive splicing compounds across multi-intronic genes, resulting in 15% of all transcript molecules from protein-coding genes being unproductive. Leveraging genetic variation across cell lines, we find that GWAS trait-associated loci explained by AS are as often associated with NMD-induced expression level differences as with differences in protein isoform usage. Employing the splice-switching drug risdiplam to manipulate AS at hundreds of genes, we find that ~3/4 of drug-induced isoforms are targeted by NMD, suggesting that most aberrant splicing influences expression levels. Our findings suggest much of the impact of AS is mediated by NMD-induced changes in gene expression rather than diversification of the proteome.