Post-transcriptional splicing can occur in a slow-moving zone around the gene

Splicing is the stepwise molecular process by which introns are removed from pre-mRNA and exons are joined together to form mature mRNA sequences. The ordering and spatial distribution of these steps remain controversial, with opposing models suggesting splicing occurs either during or after transcription. We used single molecule RNA FISH, expansion microscopy, and live cell imaging to reveal the spatiotemporal distribution of nascent transcripts in mammalian cells. At super-resolution levels, we found that pre-mRNA formed clouds around the transcription site. These clouds indicate the existence of a transcription site proximal zone through which RNA move more slowly than in the nucleoplasm. Full length pre-mRNA undergo continuous splicing as they move through this zone following transcription, suggesting a model in which splicing can occur post-transcriptionally but still within the proximity of the transcription site, thus seeming co-transcriptional by most assays. These results may unify conflicting reports of co-transcriptional versus post-transcriptional splicing. To corroborate the use of dispersed RNA FISH signal to measure post-transcriptional splicing, we used nascent RNA sequencing. We isolated nascent RNA through metabolic labelling and cellular fractionation, then calculated a "splicing index" on a per-intron basis (see Drexler et al, 2019).