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

Abstract

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.

Two classes of active transcription sites and their roles in developmental regulation

“…6 A ). This interpretation is consistent with previous studies that used smFISH to investigate transcriptional progression ( 7 , 37 – 39 ). Moreover, alternative explanations seem unlikely.…”

RNA velocity unraveled

“…In actuality, mRNA molecules are polymers, and their production often takes considerable time [ 159 ]; on an even finer level of detail, transcriptional elongation is neither infinitely fast nor constant, and exhibits pauses or “pile-ups,” which may themselves have a regulatory role [ 160 ]. Adding yet more complexity, splicing may occur co-transcriptionally or post-transcriptionally [ 161 ], with deterministic or stochastic intron removal order [ 80 ], making the categories of “spliced” and “unspliced” molecules even less tenable. Certain models of elongation can be solved [ 61 , 162 ] and simulated [ 163 ], but rapidly become mathematically intractable.…”

RNA, Genome Output and Input

“…Intriguingly, a recent preprint applying RNA-FISH combined with expansion microscopy revealed that transcripts after completion of synthesis and chromatin dissociation remain locally restricted within sub-micron distances from gene loci for some time (Coté et al, 2020). The absence of gradients of decreasing RNA concentration from the encoding gene contrasts the notion of immediate free diffusion or transport away from genes after transcription termination and 3' RNA end processing.…”