The “cutting edge” of non-canonical RNA splicing

Splicing, including alternative splicing, is a fundamental post-transcriptional mechanism in eukaryotes that generates functional proteins and transcript diversity. Canonical splicing follows well-defined rules, such as sufficient intron and exon lengths, specific splice junction orientations, consensus dinucleotides at donor and acceptor sites, and mediation by the spliceosome. However, certain splicing events deviate from these canonical rules. This review synthesizes multiple forms of non-canonical splicing within a framework that reflects their increasing deviation from canonical mechanisms, including non-canonical splice sites, non-canonical splicing in lncRNAs, microexons, recursive splicing, trans-splicing, and spliceosome-independent splicing. In addition, this review provides a critical analysis of the current state of research for each form of non-canonical splicing and outlines key directions for future investigation. As a case study, we reanalyzed RNA-seq data from mouse neuronal cells to further examine non-canonical splice sites. These analyses show that non-canonical introns tend to be shorter and that many non-canonical junctions retain at least one canonical donor or acceptor dinucleotide, supporting the view that a substantial subset remains compatible with spliceosome-mediated recognition. Together, this review provides a structured perspective on how canonical splicing rules can be relaxed, repurposed, or bypassed across distinct biological contexts.