Figures & data
Figure 1. Spliceosome depletion induces cell cycle defects. (A) HeLa cells were transfected with the indicated siRNA pools (4 siRNAs per pool). Taxol (200 nM) was added at 24 h after transfection. Samples were collected after 42 h in taxol. DNA content histograms obtained by flow cytometry using propidium iodide (PI) staining are shown. A sample histogram of an asynchronously growing culture is shown as comparison (left). (B) Cell cycle analysis after spliceosome depletion. Cells were transfected with the individual indicated siRNAs, grown asynchronously, collected 48 h after transfection and analyzed by flow cytometry. The DNA content histograms obtained by PI staining are shown.
Figure 2. Spliceosome depletion induces interphase delays. (A-B) HeLa cells transfected with the indicated siRNAs were synchronized in G1 by thymidine block and subsequently released into medium containing taxol. Samples were taken at the indicated time points after release and subjected to cell cycle profile analysis by flow cytometry using propidium iodide (PI) and anti-MPM2 staining. Overlaid DNA content histograms are shown. (C) Quantification of mitotic cells (MPM2+) for the experiment in (B). (D) Representative flow cytometry profiles of cells stained with PI and MPM2 at 12 h after release. The percentage of mitotic cells (MPM2+) is indicated. (E) Western blots of total lysates of cells in (B) blotted with the indicated antibodies. (F) Western blots of total lysates of cells in (B) at the time of release (t = 0 h) blotted with the indicated antibodies.
Figure 3. Spliceosome depletion results in increased DNA damage and checkpoint-dependent G2 arrest. (A) Analysis of DNA damage by immunostaining. HeLa cells were transfected with the indicated siRNAs. Cells were fixed at 48 h after transfection and stained with antibodies against γ-H2AX and DAPI. Sample micrographs are shown. (B) Quantification of the percentage of γ-H2AX-positive cells in (A). (C) Quantification of nuclear γ-H2AX intensity in (A). Each dot represents one nucleus. (D) HeLa cells were transfected with Control (siControl) or siSNRPB-3 siRNA together with siRNAs against the indicated DNA damage checkpoint components. Western blots of total lysates blotted with the indicated antibodies are shown. (E) Quantification of mitotic entry. HeLa cells transfected with the indicated combinations of siRNAs were incubated with taxol at 24 h after siRNA transfection. Samples were collected after 15 h in taxol, and the cell cycle profile was analyzed by flow cytometry using Propidium Iodide (PI) and anti-MPM2 staining. The percentage of mitotic cells (MPM2+) is indicated.
Figure 4. Spliceosome depletion also results in mitotic defects. (A) HeLa H2B-GFP cells were transfected with the indicated siRNAs (1 nM), and live cell imaging was performed starting at 24 h after siRNA transfection. Cumulative frequency of cells entering mitosis during the time lapse is shown. (B) Quantification of the mitotic phenotypes observed in (A). (C) Representative micrographs of the time lapse imaging in (A). Nuclear envelope breakdown (NEBD) is set as time 0 h. Arrow points to a misaligned chromosome. Arrowhead marks a lagging chromosome.
Figure 5. Different degrees of spliceosome depletion cause different cell cycle phenotypes. (A) Cell cycle analysis of HeLa cells transfected with the indicated siRNAs (5 nM). Top panel: Overlaid DNA content histograms obtained by flow cytometry using PI staining in the absence (Log) or presence of taxol (Tax)(15 h). Bottom panel: Quantification of mitotic cells in the taxol samples by PI and anti-MPM2 staining. Percentage of mitotic cells is indicated. (B) Western blots of total lysates from HeLa cells transfected with the indicated siRNAs at the indicated concentrations. Samples were taken at 48 h after transfection and blotted with the indicated antibodies. (C-D) HeLa cells were transfected with the indicated siRNAs at the indicated concentrations. Samples were taken at 48 h after transfection and analyzed by Western blotting (C) or flow cytometry using PI (D).
Figure 6. Graded requirement for the spliceosome during the cell cycle. Schematic drawing of a dosage model to explain the varying cell cycle phenotypes caused by spliceosome inactivation. In this model, spliceosome is required for pre-mRNA splicing and subsequent expression of multiple proteins at all stages of the cell cycle. Depletion of spliceosome components results in cell cycle defects whose severity depends upon the efficiency of spliceosome depletion. Mild spliceosome depletion results in defects in the later stages of the cell cycle (e.g. mitosis or G2), whereas severe spliceosome depletion elicits a block in G1.
