The spatial distributions of pre-mRNAs suggest post-transcriptional splicing of specific introns within endogenous genes

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. Measuring the timing of splicing relative to the transcription of nascent RNA has yielded conflicting interpretations. Biochemical fractionation suggests that RNA is spliced primarily during the process of transcription, but imaging of nascent RNA suggests that splicing happens after the process of transcription has been completed. 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 delay between when an intron is transcribed and when it is spliced out of a pre-mRNA. We show that 4 out of 4 genes we interrogated exhibit some post-transcriptional splicing, and that introns can be spliced in any order. We also show that completely synthesized RNA move slowly through a transcription site proximal zone while they undergo additional splicing and potentially other processing after transcription is completed. In addition, upon leaving this zone, some genes’ transcripts localize to speckles during the process of splicing but some appear to traffic freely through the nucleus without localizing to any other nuclear compartment. Taken together, our observations suggest that the regulation of the timing and localization of splicing is specific to individual introns, as opposed to the previously surmised immediate excision of introns after transcription.