FIGURE 4.

CBC depletion reduces snRNP interactions with active transcription sites. (A) Diagram of the E3 construct stably integrated into U2OS cells. Expression is driven by a minimal CMV promoter (P) under control of the tetracycline response element and induced in the presence of doxycycline (dox) by the transactivator rtTA. The gene contains three β-globin exons (E1–E3) and a CFP coding sequence, 18 MS2 repeats, a polyadenylation cleavage signal, and a transcription terminator (T). (B) Imaging of nascent RNA at the E3 transcription unit in fixed cells by RNA FISH (Cy3-labeled probe to the MS2 sequence repeats; red) together with the stably integrated U1 snRNP (U1-70K-GFP; green), U2AF65-GFP, or U5 snRNP proteins (Prp8-GFP). Scale bars, 5 μm. (C) FRAP curves show recovery of U1-70K-GFP, U2AF65-GFP, and Prp8-GFP fluorescence at bleached spots placed in nucleoplasm, speckles, or at the E3 transcription site visualized by MS2BP-mCherry. Three independent experiments were performed; in each 10–20 cells were tested. Error bars represent the SEM. For U1-70K-GFP and Prp8-GFP, the differences between recovery curves in control and CBC knockdown cells are statistically significant, as established using the Mann–Whitney test.