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

Reviewer #1 (Public Review):

The manuscript has helped address a long-standing mystery in splicing regulation: whether splicing occurs co- or post-transcriptionally. Specifically, the authors (1) uniquely combined smFISH, expansion microscopy, and live cell imaging; (2) revealed the ordering and spatial distribution of splicing steps; and (3) discovered that nascent, not-yet-spliced transcripts move more slowly around the transcription site and undergo splicing as they move through the clouds. Based on the experimental results, the authors suggest that the observation of co-transcriptional splicing in previous literature could be due to the limitation of imaging resolution, meaning that the observed co-transcriptional splicing might actually be post-transcriptional splicing occurring in proximity to the transcription site. Overall, the work presented here clearly provides a comprehensive picture of splicing regulation.

Major points:
1. Linearity of expansion microscopy. For Figure 2B, it would be helpful to display the same sample before and after expansion, just like Supplementary Figure 3, but with a transcription site and "cloud". In the current version, the transcription site looks quite different in the not-expanded (more green dots on the left) and expanded image (more green dots on the top).

2. FISH dot colocalization. What is the colocalization rate of FISH dots in general under experimental conditions? In addition, in Figures 2C and 2G, why do some 3'exon dots not have co-localized 5'exon dots?

3. It would be helpful if the authors uploaded a few examples of live cell imaging movies.

4. It is recommended to double-check the text for errors.

Reviewer #2 (Public Review):

Allison Coté et al. investigated the ordering and spatial distribution of nascent transcripts in several cells using smFISH, expansion microscopy, and live-cell imaging. They find that pre-mRNA splicing occurs post-transcriptionally at the clouds around the transcription start site, termed the transcription site proximal zone. They show that pre-mRNA may undergo continuous splicing when they pass through the zone after transcription. These data suggest a unifying model for explaining previously reported co-transcriptional splicing events and provide a direction for further study of the nature of the slow-moving zone around the transcription start site.

This paper is well-written. The findings are very important, and the data supports the conclusions well. However, some aspects of the image and description need to be clarified and revised.

The authors describe Figure 4E and 4F results in the main text as that "we performed RNA FISH simultaneously with immunofluorescence for SC35, a component of speckles, and saw that this compartmentalized pre-mRNA did indeed appear near nuclear speckles both before (Supplementary Figure 6C) and after (Figure 4E) splicing inhibition." However, no SC35 staining is shown in the Figure 4E. A similar situation happened in describing Figure 4F.