RNA Polymerase II Targets Pre-mRNA Splicing Factors to Transcription Sites In Vivo  Tom Misteli, David L Spector  Molecular Cell  Volume 3, Issue 6, Pages 697-705 (June 1999) DOI: 10.1016/S1097-2765(01)80002-2

Figure 1 Expression of Exogenous RNA Pol II LS in PemHeLa Cells HeLa cells stably expressing the pem gene were either mock transfected or transfected with HA-RNA pol II LS WT or -Δ5 CTD. Transfected cells were grown for 24 hr, and cells were selected for 72 hr in the presence of α-amanitin. (A) Cell lysates were probed for the presence of exogenous RNA pol II LS by Western blotting using an anti HA-epitope antibody. Exogenous RNA pol II LS was detected in transfected cells, and the two forms of RNA pol II LS were expressed at similar levels. (B) Overall transcriptional activity of transfected cells was measured using in situ Br-UTP incorporation. The transcriptional activity of normally growing cells (a) or cells transfected with either the RNA pol II LS WT (b) or -Δ5 (c) was similar. For samples transfected with RNA pol II LS, the Br-UTP incorporation assay was performed in the presence of 2.5 μg/ml α-amanitin. Bar, 15 μm. Molecular Cell 1999 3, 697-705DOI: (10.1016/S1097-2765(01)80002-2)

Figure 2 Pre-mRNA Splicing Factors Are Not Targeted to a Transcription Site in the Absence of the CTD PemHeLa cells were transfected with either RNA pol II-WT or -Δ5 in the absence of pem transcription, transfected cells were selected in α-amanitin, the pem gene was induced for 4 hr by removal of tetracycline, and the cells were fixed. Splicing factors were detected by indirect immunofluorescence using specific antibodies, and the pem RNA was detected by in situ hybridization. The in situ hybridization conditions used are optimized to favor maximal detection of RNA accumulated at the transcription site versus RNA in the nucleoplasm and cytoplasm. (A) In cells expressing RNA pol II LS-WT (a–c), splicing factor SC35 (red) colocalized with pem RNA (green). In cells expressing RNA pol II LS-Δ5 (d–f), the site of pem transcription was free of SC35. Bar, 5 μm. (B) Quantitative measurement of colocalization between SC35 and pem RNA. A test line was drawn so as to include the pem RNA signal (green) and a neighboring speckle (red). In RNA pol II-WT cells, the two peaks coincided; in RNA pol II-Δ5 cells, the two peaks were separated. (C) Quantitative analysis of colocalization. Cells were labeled with antibodies against SC35, U2-B′′, or the m3G-cap on snRNAs. The percentage of cells exhibiting colocalization of splicing factors and pem RNA was determined in mock transfected cells or cells transfected with either RNA pol II LS-WT or -Δ5. Results represent means of five experiments ± SD. Molecular Cell 1999 3, 697-705DOI: (10.1016/S1097-2765(01)80002-2)

Figure 3 Pem RNA Is Not Spliced in the Absence of the CTD (A) Schematic representation of pem gene and in situ hybridization probes used. Exons are denoted as E3–E6, introns as I3–I5. “Intron 5” serves as positive control and recognizes an intron sequence in intron 5. “Exon 4/5” is a splice junction probe and recognizes only RNA containing spliced exon 4 and 5. “Exon 5/4” serves as a negative control. (B) In situ hybridization using specific splice junction probes. HeLa cells expressing pem were transfected with either RNA pol II LS-WT (a–c) or -Δ5 (d–f), selected in the presence of α-amanitin, the pem gene induced, and pem RNA detected by in situ hybridization using oligonucleotide probes. Representative cells are shown, and the values indicate the percentage of such cells in the sample. Bar, 5 μm. Molecular Cell 1999 3, 697-705DOI: (10.1016/S1097-2765(01)80002-2)

Figure 4 Mapping of RNA Pol II–Interacting Domain in SR Proteins PemHeLa cells were transfected with the indicated SR protein construct. ΔRS, deletion of the RS domain; 13, deletion of the second RNA-binding domain; 23, deletion of the first RNA-binding domain; 1, expression of the first RNA-binding domain alone; 2, expression of the second RNA-binding domain alone; NPc, RS domain fused to the nucleoplasmin core domain. Cells were grown for 24 hr (A and B) or additionally treated for 3 hr with 100 μM DRB to prevent phosphorylation of RNA pol II LS (C). Cells were lysed and splicing factors recovered by immunoprecipitation using an anti-T7 tag antibody. The immunocomplex was separated by SDS-PAGE, and the presence of RNA pol II LS was probed by Western blotting using antibody H14. The RS domain of SR proteins is involved in their interaction with RNA pol II LS in a CTD phosphorylation–dependent manner. Molecular Cell 1999 3, 697-705DOI: (10.1016/S1097-2765(01)80002-2)