At least one intron from every tested endogenous gene is post-transcriptionally spliced.

A. Schematic depicting three categories of splicing.

B. Schematic depicting RNA FISH method and translation of RNA FISH images into dispersal graphs. Computed spots are calculated by fitting a gaussian to the distribution of intensity signals and pinpointing the center of that gaussian at sub-pixel resolution. Gene depicted is FKBP5.

C. Example images of dispersed (intron 4) and not dispersed (introns 1 and 3) transcription sites, via RNA FISH. Each image is a max merge of 30 optical Z sections, 0.3µm step distance.

D. Graphs indicating dispersal distance or the distance of each detected pre-mRNA to its nearest transcription site. Leftmost graphs of CPS1, EEF2, and TM4SF1 data are from HeLa cells, FKBP5 data is from A549 cells after 8 hours in dexamethasone, rightmost graphs of EEF2 and TM4SF1 data are from CRL-2097. All scale bars represent 5um unless otherwise noted.

Transcripts dwell at the site of transcription after transcription is completed.

A. Gene and probe position diagram for CPS1 pre-mRNA and mRNA.

B. Example images of RNA FISH for CPS1 before and after expansion microscopy for individual mRNA and transcription sites (in HeLa cells, different representative cells shown in each image).

C. Example images of 5’, 3’, and middle intron CPS1 RNA FISH after expansion.

D. Schematic depicting simulated RNA FISH signal for three potential scenarios: a slow moving zone around the site of transcription (I), free diffusion away from the transcription site (II), or co-transcriptional splicing of tethered transcripts (III).

E. Schematic depicting RNA FISH signal for diffusion or a slow moving proximal zone for completed (but not necessarily spliced) transcripts

F. Line scans of fluorescence intensity (arbitrary units) from transcription sites like in C.

G. Example images of co-IF (polymerase II) and RNA FISH for CPS1 after expansion. Quantification of images like that in G, and others, representing distance from each RNA spot to the nearest polymerase II IF spot.

H. Graph depicting area (in pixels) of polygons drawn around individual transcription sites after expansion.

I. Distance from 5’ to 3’ of expanded and unexpanded mRNA and transcription sites, as detected by CPS1 RNA FISH. This distance was calculated using nearest neighbors with replacement. All scale bars represent 5 µm unless otherwise noted. All expanded images and calculations are shown in expanded space scale (reduce by 4.65X to yield original space scale).

Per probe set dispersal and percent of splicing that occurs distally post-transcriptional

Percent of intermediate products that splice in one order versus the other

Live cell imaging corroborates a slow moving zone around the site of transcription

A-B. Representative images from two cells of a transcriptional burst of TFF1 labeled with MS2-GFP. Each image is taken one second apart. Shown are the raw, computationally identified transcription site images, as well as the binarized image used to calculate eccentricity.

C. Plot of eccentricity for each transcription site image, alongside representative images that show both eccentric and non-eccentric sites.

D. Graphs of eccentricity measured in individual transcription sites tracked over time

Some transcripts localize to a speckle proximal compartment after splicing inhibition.

A. Schematic of possibilities for RNA movement post-transcription.

B. Example images of introns of specified genes before and after pladienolide B treatment (treatment status as indicated on figure). Outline represents nucleus.

C. Description of 3 response types to pladienolide B.

D. EEF2 RNA FISH exon and intron images with pladeinolide B treatment. Scale bar = 5um.

E. EEF2 RNA FISH intron only images with pladeinolide B treatment. Scale bar = 5um.

F. RPL13A, GAPDH, and EEF2 RNA FISH intron images with pladeinolide B treatment. Scale bar = 5um.

G. Costaining of RNA FISH for specified intron and SC35 IF in the same cell treated with pladeinolide B. Scale bar = 5um.

H. Quantification of speckle decile from previously published data (TSA Seq 2.0), compared with distance from nearest speckle calculated based on RNA FISH of specified genes without pladeinolide B treatment.

Intron dispersal varies with transcription level and is therefore not an inherent property of each intron.

A. Gene and probe diagram for FKBP5 and schematic of dexamethasone treatment schedule.

B. Quantification of FKBP5 RNA FISH exon and intron spots over time of treatment in dexamethasone (in A549 cells).

C. Dispersal graphs (as quantified from RNA FISH) of FKBP5 introns 1, 8, and 9 over time in dexamethasone (in A549 cells).

D. Graph showing the percentage of intron intensity that is distally post-transcriptional for 3 FKBP5 introns over time in dexamethasone