The in vivo kinetics of RNA polymerase II elongation during co-transcriptional splicing

Abstract

RNA processing events that take place on the transcribed pre-mRNA include capping, splicing, editing, 3' processing, and polyadenylation. Most of these processes occur co-transcriptionally while the RNA polymerase II (Pol II) enzyme is engaged in transcriptional elongation. How Pol II elongation rates are influenced by splicing is not well understood. We generated a family of inducible gene constructs containing increasing numbers of introns and exons, which were stably integrated in human cells to serve as actively transcribing gene loci. By monitoring the association of the transcription and splicing machineries on these genes in vivo, we showed that only U1 snRNP localized to the intronless gene, consistent with a splicing-independent role for U1 snRNP in transcription. In contrast, all snRNPs accumulated on intron-containing genes, and increasing the number of introns increased the amount of spliceosome components recruited. This indicates that nascent RNA can assemble multiple spliceosomes simultaneously. Kinetic measurements of Pol II elongation in vivo, Pol II ChIP, as well as use of Spliceostatin and Meayamycin splicing inhibitors showed that polymerase elongation rates were uncoupled from ongoing splicing. This study shows that transcription elongation kinetics proceed independently of splicing at the model genes studied here. Surprisingly, retention of polyadenylated mRNA was detected at the transcription site after transcription termination. This suggests that the polymerase is released from chromatin prior to the completion of splicing, and the pre-mRNA is post-transcriptionally processed while still tethered to chromatin near the gene end.

Figure 1. Generating a cell system with comparable genes containing increasing numbers of introns.

(A) Gene constructs containing increasing numbers of introns and exons of the human β-globin mini-gene were generated and termed E1, E3, E4, and E6. Common to all the genes are: Tet-inducible promoter (orange; Tet responsive element (TRE), minimal promoter (Pmin)) that is induced in the presence of rtTA (yellow) and doxycycline (dox); the genes consist of the β-globin intron and exon sequences (exon 1, purple; exon 2, green; exon 3, pink) in frame with a CFP-SKL peroxisomal protein (blue), and a series of 18 MS2 repeats in the 3′UTR (cyan) for the in vivo labeling of the mRNA via binding of YFP-MS2 proteins (green stars). The 3′-end of the gene contains a polyA site. Intron sizes, the total size of the gene, the MS2 repeat region, and the 3′UTR are marked. (B) RT-PCR showing the production of the correctly spliced E3, E4, and E6 mRNAs. GAPDH was used as a control. Sizes(nt) are marked above the bands. (C) E3 cell induced with dox for 12 h (activation times are similar in all experiments unless indicated otherwise) showing the gene locus marked by RFP-LacI (red), the transcribed mRNA at the transcription site and throughout the cell labeled with YFP-MS2 (yellow), and the CFP-SKL protein product in cytoplasmic peroxisomes (cyan). (D) Actively transcribing transcription sites (yellow, YFP-MS2 tagged mRNA) and gene locus (red, RFP-LacI) show the recruitment of endogenous RNA Pol II (purple) by immunofluorescence with the H14 Ab that detects initiating polymerases. Enlargements show the overlay at the transcription site. CFP-peroxisomes are seen in cyan. (E) RNA-FISH showing mRNAs detected throughout an E3 cell with fluorescent probes to the first exon (red), the CFP exon (exon 3, purple), and the MS2 repeats (green). Scheme shows the probe binding sites and the enlargement of the transcription site (bar, 5 µm).

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.

Figure 3. Comparison of transcriptional kinetics on active E1, E3, and E4 transcription sites.

(A) An active transcription site (YFP-MS2, bottom cell) was photobleached and the fluorescence recovery was tracked over time (frames on right). These experiments were performed on a 3D FRAP system (XZ and YZ representations are seen under and to the right of the original image, respectively). (B) Scheme of the elongation assay showing bleaching of the YFP-MS2 proteins (yellow) bound to the mRNA and the recovery of fluorescence as new polymerases elongate through the MS2 region. (C) Recovery curves of the YFP-MS2 FRAP measurements performed on E1, E3, and E4 transcription sites. Average of n>10 experiments for each cell type with SDE.

Figure 4. Increased spliceosome recruitment and change in transcriptional kinetics in relation to increasing intron numbers.

(A) Quantification of the RNA-FISH signals showing the ratio of the second intron to the last exon (CFP region) on the different transcription sites (all normalized to the E3 ratio). (B) A 3D representation of the deconvolved images of an RNA-FISH experiment in which MS2 mRNA (in yellow) and U5 snRNA (in red) were detected. The signals at the transcription site were measured for both channels. The elongated form of the signal is due to the 3D projection of the images. (C) Quantification of the RNA-FISH signal showing the ratio between transcription site associated U5 (blue dots) or U6 (red dots) snRNAs in comparison to exon 1 on the E3, E4, and E6 genes. Each dot represents a transcription site ratio. (D) FRAP recovery curves of YFP-MS2 on E3 versus E6 genes with standard error bars (n>10).

Figure 5. Dynamics of E6 mRNA are affected by active splicing and not by polymerase kinetics.

(A) FRAP recovery curves of transfected GFP-Pol II recruited to the E3 and E6 genes. (B) ChIP analysis of Pol II distribution along the E3 (blue bars) and E6 (red bars) genes. Black boxes under the schemes indicate primer positions. The data were normalized to the promoter by setting the promoter value at 100%. A t-test for each primer set showed no significant differences (p>0.05). (C) Inhibition of splicing by Spliceostatin (SSA) for 9 h showed that unspliced pre-mRNA (intron probe, magenta) was distributed throughout the cell and in speckles (RNA-FISH on E6 cells) and was retained in the nucleus (bottom), whereas in untreated cells pre-mRNA was detected only at the site of transcription (intron probe, magenta; exon probe, red) (bar, 5 µm). (D) SSA treatment (6 h) caused the nuclear retention of intron-containing E3 mRNA (middle) versus the regular nucleo-cytoplasmatic dispersal of E1 intronless mRNA (right). Left, untreated E3 cell. RNA-FISH was performed to the MS2 region of the mRNAs (yellow). (E) FRAP recovery curves of YFP-MS2 at the transcription sites of untreated and SSA-treated E3 and E6 cells. (F) ChIP analysis of Pol II distribution along the E6 gene in untreated (red bars) and SSA-treated (green bars) cells. Boxes under the schemes indicate primer positions. The data were normalized to the promoter by setting the promoter value at 100%. A t-test for each primer set showed no significant differences (p>0.05).

Figure 6. RNA splicing leads to retention of the pre-mRNA on the transcription site.

(A) RNA FISH in an E3 cell with a polyT probe (red) and an MS2 probe (yellow) showing that polyadenylated mRNAs were formed and were present on the transcription sites. (B) Recruitment of GFP-PABP2 (pseudocolored red) to the transcription site labeled with YFP-MS2 (yellow). Plots on the right in (A) and (B) depict the degree of colocalization of the signals at the transcription site across the line (bar, 5 µm). (C) The ratio of the RNA-FISH signal of polyA to the last exon on the E3, E4, and E6 active transcription sites, showing no aberrant accumulation of transcripts without polyA signal. (D) Half-time of release of mRNA from transcription sites following actinomycin D treatment (time measured from ActD addition) in E3 (n  =  7) and E4 (n  =  7) cells compared to E6 cells (n  =  8). Inset shows two sample time-series of transcription site inactivation and the exponential fit from which half-times of clearance were calculated (blue, E3; red, E6). (E) The ratio between the polymerase signal and the first exon on the E3, E4, E6, and SSA-treated E6 genes. Each red dot represents the ratio of intensities measured on a single transcription site. The scheme at the top shows the polymerase antibody (red) and the mRNA (FISH exon 1, yellow). (F) YFP-MS2 FRAP data and the fitted simulation of the E3 and E6 genes. The calculated residuals of the fit are presented in the inset. (G) RNA-FISH ratio of intron 2 to the last exon at the transcription site in E1, E3, E4, and E6 cells. The experimental data points (pink, from Figure 4A) were compared to the simulation output (red) of the same experiment. Simulations were performed with either short (50 sec, E3 kinetics; E1, E3, E4, E6) or long (10 min, E6 kinetics; E6 rightmost point) transcript retention times.