pre-mRNA spatial distributions suggest that splicing can occur post-transcriptionally

Splicing is the molecular process by which introns are removed from pre-mRNA and exons are joined together to form the sequence of the mature mRNA. While the biochemical steps of splicing have been largely worked out, the ordering and spatial distribution of these steps, especially in relation to the transcriptional process itself, remain controversial. Here, we use single molecule RNA FISH together with expansion microscopy to measure the spatial distribution of nascent and partially spliced transcripts in mammalian cells, allowing us to infer the order in which an intron is transcribed and spliced out of the pre-mRNA. We show that 4 out of 4 genes we interrogated exhibit clear post-transcriptional splicing of at least one intron, and that introns can be spliced in any order. Expansion microscopy further revealed the presence of a transcription site proximal zone in which the motion of RNA is slower than in the nucleoplasm. Full length pre-mRNA undergo continuous splicing as they move through this zone after transcription. Upon leaving this zone, some genes9 transcripts localize to speckles during the process of splicing while others appear to traffic freely through the nucleus without localizing to any other nuclear compartment. Our results suggest a model in which transcription and splicing are largely independent, and the existence of a slow moving zone around the transcription site allows for the unification of co-transcriptional and post-transcriptional models of splicing.