Hierarchical regulation and molecular functions of co-transcriptional splicing

Eukaryotic gene expression is precisely regulated through the coupling of transcription and RNA splicing, with an estimated 70%-90% of introns undergoing co-transcriptional splicing. Although the mechanistic basis of this coupling remains incompletely understood, emerging evidence establishes co-transcriptional splicing not as a passive byproduct but as an actively regulated process, whose dysregulation leads to specific molecular and functional defects. To help comprehend this critical aspect of gene expression and stimulate future research, the field would benefit from a systematic overview that integrates detection methods, quantitative metrics, regulatory mechanisms, functional consequences, and disease associations. In this review, we aim to achieve this goal by summarizing the evolution of detection technologies and highlighting how they enable quantitative assessment through three key metrics: splicing time, order, and efficiency. We further synthesize evidence that regulation hinges on two fundamental elements: splice site accessibility and spliceosome assembly, with other factors primarily acting through these core aspects. Beyond its role in mRNA maturation, we explore how co-transcriptional splicing influences RNA fate and provides feedback to the chromatin landscape to shape epigenetic states and preserve genomic stability. We further link these regulatory principles to disease mechanisms arising from co-transcriptional splicing defects and highlight potential therapeutic strategies. Finally, we outline outstanding challenges, particularly in characterizing splicing dynamics and heterogeneity, and discuss future directions.