The In Vivo Kinetics of RNA Polymerase II Elongation during Co-Transcriptional Splicing
Figure 2
Co-transcriptional splicing occurs at the site of transcription.
(A) A probe to the first intron or (B) the second intron (purple) was detected in an E3 cell only at the transcription site while the mature mRNA was detected throughout the cell (probe to exon 1, red). Schemes on the right show the probe binding regions and an enlargement of the colocalized signals at the transcription site. (C–H) The recruitment of the core pre-mRNA splicing machinery to active transcription sites was examined in E3 (left column) and E1 (right column) cells. RNA-FISH with an MS2 probe (yellow) shows the active transcription sites together with snRNA probes in red: (C and F) U1 snRNA, (D and G) U2 snRNA, and (E and H) U6 snRNA. Enlargements show the signals at the transcription sites. Plots on the right depict the degree of colocalization of the signals at the transcription site across the blue line. (I) RNA-FISH signal of U1 and U2 snRNAs (middle) at the active transcription site (seen with the MS2 probe, left) of an E3 cell and (J) an E1 cell were measured. The green box shows a random nucleoplasmic area, and the red and blue boxes show the area of the transcription site. The plot shows the correlation of the U1 and U2 snRNA signals at the transcription site (red and blue dots) in comparison to the nucleoplasm (green dots) (bar, 5 µm). (K) The ratio between U1 and U2 snRNA signals measured by RNA-FISH, at the transcription sites of all genes. The data in (I), (J), and (K) show a constant correlation in the intron-containing genes, and no correlation in the intronless E1 gene, meaning that U1 and not U2 is recruited even to an intronless gene.