SpliceImpactR maps alternative RNA processing events driving protein functional diversity.

Alternative RNA processing is a key regulator of gene expression, shaping transcript and protein diversity essential for cell function. Yet, how distinct alternative RNA processing events alter protein function remains unclear. Here, we introduce SpliceImpactR-available as a Bioconductor package and interactive R Shiny application-an open-source framework that systematically identifies RNA isoform switches across the human transcriptome, including alternative first and last exons, exon skipping, intron retention, hybrid exons, and splice site variants, and predicts their impact on encoded proteins. Applying SpliceImpactR across all annotated human isoforms and 17 350 samples spanning 54 tissues, we find that intron retention and hybrid exons frequently shift protein-coding transcripts into non-coding isoforms. Strikingly, when both isoforms remain protein-coding, 87% of alternative RNA processing events produce substantial changes in protein sequence. Widespread frameshifts introduced by alternative splicing are often rescued by co-regulated downstream exons, uncovering a buffering mechanism that maintains protein integrity. Alternative last exons drive the most extensive structural changes, while alternative first exons emerge as the most efficient mechanism for reshaping tissue-specific protein domain architecture. We further found that alternative RNA processing events are widely co-regulated and undergo gradual, tissue-specific transitions rather than binary on/off switches. In the cerebellum, we found that alternative first exons progressively increase and activate nuclear localization signals, while in colon adenocarcinoma internal splicing events rewire key oncogenic protein interactions. Together, SpliceImpactR delivers a proteome-wide atlas of splicing regulation, establishing a powerful, accessible platform to decode how context-dependent RNA processing drives human proteomic and tissue diversity.