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snRNP protein expression enhances the formation of Cajal bodies containing p80-coilin and SMN

School of Life Sciences, University of Dundee, MSI/WTB Complex, Dow Street, Dundee, DD1 5EH, UK

View larger version (58K): [in a new window]   Fig. 1. (i) YFP-SmB colocalizes with endogenous Sm proteins in cells from line YFP-SmBE1. Maximum intensity projections of deconvolved serial sections through cells from line YFPSmBE1 in interphase (A,B) and telophase (C,D). The YFPSmB protein (A,C) colocalizes with endogenous Sm proteins (B,D) in Cajal bodies (arrowheads) and speckles (arrows). Bar, 10 µm. (ii) YFP and CFP-SmB are expressed as single fusion proteins at levels lower than endogenous SmB. SDS-PAGE analysis of whole cell lysates from lines YFPSmBE1, CFPSmBE8.8, parental HeLa cells and HeLa cells transiently transfected with a plasmid encoding YFP. An antibody to G/YFP (left-hand panel) showed a single band at 30 kDa in cells expressing YFP alone and single bands at the size predicted for YFPSmB or CFPSmB (58 kDa) in cells from lines YFPSmB E1 and CFPSmBE8.8, respectively. Antibody Y12 to endogenous Sm proteins (right-hand panel) gave a band at 28/29 kDa in all cell lines, representing endogenous SmB/SmB’. In cells from lines YFPSmBE1 and CFPSmBE8.8, additional, less intense bands were detected at 58 kDa, representing YFPSmB and CFPSmB, respectively.

View larger version (45K): [in a new window]   Fig. 2. (i) Three dimensional projections of parental HeLa cells fused to cells from line YFPSmBE1 fixed 1,2,3,5,7,9 and 16 hours after heterokaryon formation (A-G). YFPSmB is shown in grayscale in the top panels, the color panels show YFPSmB (green) overlain with Y12 (anti-Sm) staining (red) and anti-p80-colin staining (blue). YFPSmB is seen in the Cajal body by 1hour after fusion (arrows). Accumulation of YFPSmB in speckles is first seen 3 hours after fusion (arrowheads) and increases with time until, by 16 hours after fusion, cells from line YFPSmBE1 are indistinguishable from cells from the parental HeLa line (G). At the 16 hour timepoint, Cajal bodies are still clearly seen in all cells. Parental HeLa cells fused to HeLa cells expressing GFP-ASF/SF2 (H) show accumulation of GFP-ASF/SF2 in speckles (arrowheads) 2 hours after fusion, with no prior accumulation in Cajal bodies seen. Immunodetection of p80-coilin in these cells (color panel, blue channel) demonstrates that Cajal bodies are present in these cells (arrows). Bar, 10 µm. (ii) Quantification of the proportion of YFPSmB in different nuclear compartments over time. The proportion of YFPSmB signal detected in the Cajal body (red) and speckle (blue) compartments are expressed as a percentage of total nuclear fluorescence for early time points after fusion (1-2 hours), timepoints where speckles are beginning to be visible (4-5 hours), and in unfused cells from line EYFPSmBE1.

View larger version (53K): [in a new window]   Fig. 3. (i) Three-dimensional projections of cells from line YFPSmBE1 fused to cells from line CFPSmBE8.8. A to C shows one cell from each line fused together, fixed two hours after fusion, YFPSmB has accumulated in the Cajal bodies of the nucleus from line CFPSmBE8.8 (arrows in A), while CFPSmB has accumulated in the Cajal bodies of the nucleus from line YFPSmBE1 (arrowheads in B). D to F shows three cells fused together, fixed 16 hours after fusion. Each of the nuclei shows a pattern of Cajal bodies (arrows) and speckles (arrowheads) for both YFPSmB (D) and CFPSmB (E). (ii) Conventional fluorescence images of cells from line YFPSmBE1 injected with the expression vector for ECFPSmD1, fixed after 1hour (A,D), 2 hours (B,E) and 16 hours (C,F). YFPSmB shows a speckled pattern in cells at all time points (D-F), while CFPSmD1 appears first in Cajal bodies after 1 hour (arrows in A), in nucleoli after 2 hours (arrowhead in B), finally showing a speckled pattern after 16 hours (arrows in C and F). Bar, 10 µm.

View larger version (81K): [in a new window]   Fig. 4. (i) Maximum intensity projections of deconvolved serial sections through a cell from line HeLa (D). Immunodetection with antibodies to p80-coilin (A) and SMN (B) demonstrate that, in addition to large Cajal bodies containing both p80-coilin and SMN (arrows in A and B), these cells also contain smaller nuclear bodies containing p80-coilin in the absence of SMN (arrowhead in A) and containing SMN in the absence of p80-coilin (arrowhead in B). Bar, 10 µm. (ii) Single confocal sections of a cell from line HeLa (D) transfected with an expression plasmid encoding YFPSmB and fixed after 16 hours (A-D). The prominent Cajal body containing newly imported snRNP (arrow in B) also contains endogenous p80-coilin (arrow in A) and SMN (arrow in C). Additional bodies containing p80-coilin, but not new snRNP nor SMN, are also seen (arrowheads in A,D, see also inset magnified view in A-D). Bar, 10 µm. Maximum intensity projections of deconvolved serial sections through heterokaryons of cells from line HeLa (D) fused to a cell from line YFPSmBE1 fixed 1 hour after fusion (E-H). YFPSmB has accumulated in Cajal bodies of nuclei from line HeLa (D) (arrows in F). These bodies also contain endogenous p80-coilin (arrow in E) and SMN (arrow in G). Numerous additional bodies containing p80-coilin but neither new snRNP nor SMN are also seen (arrowheads in E,H). Bar, 10 µm.

View larger version (55K): [in a new window]   Fig. 5. Maximum intensity projections of deconvolved serial sections through heterokaryons of cells from line YFPSmBE1 and primary human fibroblasts (DFSF1), stained with antibodies to p80-coilin and SMN. Cells co-cultured without fusion show prominent Cajal bodies in nuclei from line YFPSmBE1 (arrows in A-C), containing p80-coilin (A), YFPSmB (B) and SMN (C). By contrast, DFSF1 nuclei show fine punctate staining with p80-coilin (A) and SMN (C), with no co-localization of the two into obvious Cajal bodies (overlay in D). In cells fixed 2 hours after PEG fusion (E-H), Cajal bodies are seen in nuclei from both cell types. YFPSmB is seen in nuclear bodies in DFSF1 nuclei (arrows in F). These bodies also contain both p80-coilin (arrows in E) and SMN (arrows in G). In cells fixed 16 hours after fusion, YFPSmB is seen in speckles in DFSF1 nuclei fused to YFPSmBE1 nuclei (arrowheads in J). Cajal bodies (arrows in I-K) containing YFPSmB (J), p80-coilin (I) and SMN (K) are also present in DFSF1 nuclei at this time point (overlay in L). Labeling of heterokaryons fixed 2 hours after PEG fusion with antibodies to the trimethyl cap of snRNAs (M) confirms that the nuclear bodies seen in DFSF1 cell nuclei at this time point contain snRNA (arrow in M) in addition to YFPSmB (arrow in N) and p80-coilin (arrow in O). Bar, 10 µm. (ii) Western blot analysis of total cell lysates from DFSF1 cells and HeLa cells. The intensity of signals seen for -tubulin confirms that proteins from similar numbers of cells have been loaded in each lane. The core snRNP, Sm proteins, the U2 snRNP-specific protein U2B'', the U1 snRNP-specific proteinU1A, and the SMN protein are both much more abundant in HeLa cells than in DFSF1 cells.

View larger version (64K): [in a new window]   Fig. 6. Maximum intensity projections of deconvolved serial sections through control DFSF1 cells (A-D) and DFSF1 cells injected with expression vectors encoding GFPCoilin (E-H), GFPSMN (I-L) or YFPSmB (M-T). Control cells show weak, fine punctate staining with anti-p80-coilin (A) and anti-SMN (B) with no co-localization between the two (overlay in D). Endogenous Sm proteins show a speckled localisation (C). Over-expression of GFPCoilin leads to an increase in nucleoplasmic coilin (E, anti-p80 coilin; F, GFP-coilin; H, overlay), but not to formation of Cajal bodies. Endogenous snRNP proteins, detected with Y12, show a speckled nuclear localization (G). Overexpression of GFPSMN leads to fine punctate localization of GFPSMN, predominantly in the cytoplasm (J). No co-localization with p80-coilin (I) or endogenous Sm proteins (K) is seen. Over-expression of YFPSmB for 2 hours results in the formation of nuclear Cajal bodies (arrows in M-O) containing p80-coilin (M), SMN (N) and newly imported snRNP (O). In cells expressing YFPSmB for 16 hours, p80-coilin shows a fine punctate pattern (Q). SMN is seen in small nuclear bodies (arrows in R,T), but no longer shows co-localization with YFPSmB (S,T).

View larger version (62K): [in a new window]   Fig. 7. (i) Confocal sections of HeLa cells injected with expression vectors encoding YFPSmB or GFPASF/SF2 in the presence or absence of LMB and incubated for 8 hours. In cells injected with YFPSmB in the presence of LMB (A-D), expressed YFPSmB does not accumulate in nuclear bodies or speckles (C). Nuclear speckles are still seen using antibodies to endogenous snRNPs (A). p80-coilin is found predominantly in the nucleolus (arrows in B). Control cells, injected with pYFPSmB in the absence of LMB (E-H) show a speckled pattern of YFPSmB (G), with endogenous Sm proteins in speckles and Cajal bodies (E) and p80-coilin in Cajal bodies (F). Cells injected with pGFPASF/SF2 in the presence of LMB (I-L) show the uptake of GFPASF/SF2 into speckles (K). p80-coilin is seen in the nucleoli (arrows in J,L). Bar, 10 µm. (ii) Immonoprecipitation of snRNPs from cells transfected in the presence (A) or absence (B) of LMB using anti-TMG antibodies, detected using anti-GFP antibodies (top panels). In the presence of LMB, GFP-SmD1 is not incorporated into snRNPs (compare lower band in bound versus unbound fraction). In the absence of LMB, GFP-SmD1 is seen in the bound, snRNP, fraction. The lower panels show 3D projections of serial confocal sections through cells treated with LMB (A) and control cells (B). In LMB-treated cells, GFP-SmD1 signal is diffuse, whereas in control cells (B), GFPSmD1 is seen in Cajal bodies and nucleoli.

View larger version (80K): [in a new window]   Fig. 8. (i) Confocal sections of HeLa cells transfected with YFPSmB and treated with LMB 16 hours after transfection (A-H) or cultured in the absence of LMB (I-L). YFPSmB is seen in nuclear bodies in cells treated with LMB (arrows in C,G), but not in speckles. These nuclear bodies contain SMN (arrows in F), but do not contain p80-coilin, which is seen in the nucleolus (arrowheads in A and E). In cells incubated in the absence of LMB, YFPSmB (K) is seen to co-localize with endogenous Sm proteins (J) in Cajal bodies (arrows in I-L) and speckles (arrowheads in I-L). Bar, 10 µm. (ii) Immonoprecipitation of snRNPs from cells transfected with GFPSmD1, incubated overnight, then incubated for 16 hours in the presence (A) or absence (B) of LMB using anti-TMG antibodies, detected using anti-GFP antibodies (top panels). GFPSmD1 (lower band) is seen in the bound fraction in LMB treated cells and in control cells. The lower panels show 3D projections of serial confocal sections through cells treated with LMB (A) and control cells (B). In LMB-treated cells, GFP-SmD1 signal shows localizations characteristic of early time points after transfection, with diffuse signal and accumulation in Cajal bodies but not speckles. In control cells (B), GFPSmD1 shows a speckled localization.