The review establishes the first systematic framework encompassing "detection–quantification–regulation–function–disease/therapy," providing both an integrated perspective for established researchers and a comprehensive roadmap for newcomers to the field.
1. Detection methods and quantitative metrics
The review categorizes detection technologies into imaging and high-throughput sequencing approaches. Imaging enables direct visualization of splicing at the single-cell and single-molecule level, while sequencing captures nascent RNA at the genome-wide scale. Three key metrics are highlighted: splicing time, splicing order, and splicing efficiency — which collectively characterize the kinetics of co-transcriptional splicing.
2. Molecular mechanisms and functions
The review organizes co-transcriptional splicing regulation into two core themes: substrate (splice site) accessibility and machinery (spliceosome) assembly. The former is influenced by Pol II elongation rate, chromatin state, nascent RNA structure, and RNA modifications. The latter depends on Pol II, chromatin adaptors, RNA-binding proteins, splicing factor modifications, and nuclear spatial organization.
Beyond mRNA maturation, co-transcriptional splicing regulates chromatin structure, restricts R-loop formation, maintains genomic stability, and influences RNA modifications, editing, nuclear retention, degradation, translation, and protein isoform diversity. It serves as a critical hub connecting transcription, RNA processing, chromatin regulation, and protein output.
3. Disease associations and therapeutic implications
Dysregulation of co-transcriptional splicing is linked to hematological disorders, cancers, and neurological diseases. Disease mutations can alter splice site recognition, impair spliceosome assembly, or disrupt splicing–transcription/chromatin feedback, leading to aberrant RNA fate, genomic instability, and cellular dysfunction.
These findings suggest new therapeutic strategies. Beyond correcting aberrant splicing directly, future interventions could target splice site accessibility, spliceosome assembly, chromatin states, or R-loop accumulation via antisense oligonucleotides, small molecules, or epigenetic combination therapies.
4. Conclusions and future perspectives
Co-transcriptional splicing is evolving from an RNA processing step to a critical entry point for understanding gene expression. Integrating live-cell imaging, long-read sequencing, and single-cell and spatial omics will further advance this frontier. However, many mechanisms remain debated and translational applications are still nascent — marking this as a field ripe for exploration.
Author Information
Professor Yajing Hao (China National Center for Bioinformation) and Professor Xiang-Dong Fu (Westlake University) are co-corresponding authors. Rui Niu and Aiming Zhang are co-first authors. Professor Michael G. Rosenfeld (UC San Diego, HHMI) provided valuable advice. This work was supported by the National Key Research and Development Program of China and the National Natural Science Foundation of China.
