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First published online 17 August 2004
doi: 10.1242/jcs.01308

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A role for Cajal bodies in the final steps of U2 snRNP biogenesis

Dobrila Nesic^*, Goranka Tanackovic and Angela Krämer

Department of Cell Biology, Faculty of Sciences, University of Geneva, 30, quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland

View larger version (75K): [in a new window]   Fig. 7. Analysis of domains in SF3a66 required for nuclear and subnuclear localization. (A) Schematic representation of recombinant SF3a66 proteins fused to GFP. Amino acids present in, or deleted from the recombinant proteins are given in parentheses after the names of the proteins. Conserved regions are indicated above the diagram and numbered according to the amino acids in full-length SF3a66. (B) GFP-tagged 3a66-N, -C and -Zn were transiently expressed in HeLa cells and visualized by fluorescence in living cells. (C) HeLa cells were transiently transfected with plasmids encoding GFP-SF3a66 proteins as indicated. Fluorescence was monitored after fixation of cells with cold methanol. Cells in (d) were immunostained with anti-p80-coilin (middle panel); the right panel represents the computer-generated overlay. (D) HeLa cells were transiently transfected with GFP-3a66-Zn. Proteins (as indicated) were visualised after fixation of cells with cold methanol. SF3a60 was visualised by immunofluorescence with anti-3a60 and AMCA-coupled anti-rabbit antibodies, p80-coilin with a mouse anti-p80-coilin and Texas Red-coupled anti-mouse antibodies. Arrows point to CBs. Bar, 10 µm.

View larger version (67K): [in a new window]   Fig. 1. Comparison of the localization of endogenous SF3a subunits with other splicing components. (A-E) HeLa cells were permeabilized with Triton X-100, fixed with paraformaldehyde and treated with antibodies against the following proteins: (A) SF3a60 and SF3a66; (B) SF3a66 and SF3a120; (C) SF3a60 and SC35; (D) p80-coilin and SF3a66; (E) SF3a60 and U2 B''. (F) Cells were hybridized with a 2' O-methyl oligoribonucleotide complementary to U2 snRNA and immunostained with an anti-SF3a66 antibody. Images shown in all left panels were obtained after staining with FITC-coupled secondary antibodies. Images shown in middle panels were stained with rhodamine-coupled secondary antibodies. The right panels show computer-generated overlays of the two images. CBs appear in green in panel (D), and red in panels (E) and (F). Arrows in (E) and (F) indicate the absence (left panels) or presence (middle and right panels) of CBs. Bar, 10 µm.

View larger version (66K): [in a new window]   Fig. 2. Analysis of interactions of GFP-tagged SF3a subunits with endogenous subunits and the 15S U2 snRNP. (A-D) HeLa cells were transiently transfected with plasmids encoding full-length GFP-tagged SF3a subunits (FL) or GFP-SF3a60 and GFP-SF3a66 deleted for their zinc finger domains (Zn) as indicated above the figure. SF3a complexes were precipitated from small-scale nuclear extracts prepared 48 hours post-transfection with Protein G-Sepharose-coupled anti-GFP (lanes 2-6). Bound proteins were analysed by western blotting for the presence of SF3a60 (A), SF3a66 (B), SF3a120 (C) and SF3b155 (D). Lane 1 shows the extract prepared from cells overexpressing GFP-3a120-FL as an example for the input of the immunoprecipitation reactions. Lane 7 shows the same extract treated with Protein G-Sepharose in the absence of anti-GFP. Positions of precipitated proteins are indicated on the right. The migration of molecular mass standards (in kDa) is shown on the left.

View larger version (23K): [in a new window]   Fig. 3. Localization of transiently expressed SF3a subunits fused to GFP in live cells. HeLa cells were transiently transfected with plasmids encoding GFP, GFP-ER and SF3a subunits fused to GFP as indicated. Fluorescence was monitored in living cells. The arrows point to bright foci. Bar, 10 µm.

View larger version (106K): [in a new window]   Fig. 4. Presence of transiently expressed GFP-3a60 and GFP-3a66 in nuclear speckles and CBs. (A-D) The localization of transiently expressed GFP-3a60 and GFP-3a66 was analyzed after fixation of HeLa cells in cold methanol. The left panels show the fluorescence of GFP-3a60 (A and B) and GFP-3a66 (C and D). Images in the middle panels were obtained after immunostaining with anti-SC35 (A and C) or anti-p80-coilin (B and D) and rhodamine-coupled secondary antibodies. The right panels represent computer-generated overlays of the images shown on the left and in the middle. Colocalization of GFP-3a60 and GFP-3a66 with p80-coilin in CBs (indicated by arrows) appears in yellow in (B) and (D). CBs stained by GFP-3a60 and GFP-3a66 but not by SC35 appear green in the overlays in (A) and (C). Bar, 10 µm.

View larger version (58K): [in a new window]   Fig. 5. Analysis of domains in SF3a60 required for nuclear and subnuclear localization in live cells. (A) Schematic representation of recombinant SF3a60 proteins fused to GFP. Amino acids present in, or deleted from the recombinant proteins are given in parentheses after the names of the proteins. Conserved regions are indicated above the diagram and numbered according to the amino acids in full-length SF3a60. (B) GFP-3a60 proteins with N- or C-terminal or internal deletions (as indicated) were transiently expressed in HeLa cells. Fluorescence was monitored in live cells. Bar, 10 µm.

View larger version (79K): [in a new window]   Fig. 6. Comparison of the distribution of transiently expressed GFP-3a60 proteins with SC35 and p80-coilin in fixed cells. (A-H) HeLa cells were transiently transfected with plasmids encoding GFP (A), GFP-3a60-FL (B), GFP-3a60-N1 (C), GFP-3a60-C4 (D), GFP-3a60-C3 (E), GFP-3a60-Zn (F and G) and GFP-3a60-SAP (H). Cells were fixed in cold methanol and immunostained with anti-p80-coilin (D and G) or anti-SC35 (E, F and H). The images in (A-C) and the left panels of (D-H) show fluorescence of GFP. The middle panels of (D-H) show immunolocalization of p80-coilin or SC35, and the right panels represent computer-generated overlays of the two images. Bar, 10 µm.