SpliceImpactR maps alternative RNA processing events driving protein functional diversity

Alternative RNA processing is a key regulator of gene expression, driving transcript and proteomic diversity essential for cell function. However, the precise impacts of different alternative RNA processing events on protein function remain poorly understood. Here, we introduce SpliceImpactR, an open-source tool that systematically identifies RNA isoform switches—including alternative first and last exons, exon skipping, intron retention, hybrid exons, and splice site variation—across the human transcriptome, and predicts their impact on encoded proteins. We find that intron retention and hybrid exons frequently alter the coding potential of transcripts. Strikingly, when both isoforms remain protein-coding, 87% of alternative RNA processing events result in substantial changes to the protein sequence. Among these, alternative last exons drive the largest changes, frequently disrupting protein-protein interactions. Across human tissues, alternative first exons drive the largest relative changes in proteins, often resulting in tissue-specific protein domains. Notably, frameshifts introduced by alternative splicing are often rescued by co-regulated alternative downstream exons, suggesting a buffering mechanism in isoform regulation. Moreover, we show that alternative splicing exhibits gradual, tissue-specific variation, rather than binary on/off behavior, enabling conserved regulation of protein domains across tissues. Together, our results provide a proteome-wide view of splicing regulation, uncovering widespread, context-dependent impacts of alternative splicing on the human proteome.