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First published online 3 April 2007
doi: 10.1242/jcs.001529

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A regulatory role for CRM1 in the multi-directional trafficking of splicing snRNPs in the mammalian nucleus

Division of Pathology and Neuroscience, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK

View larger version (31K): [in this window] [in a new window]   Fig. 1. Incorporation of FP-tagged Sm proteins into snRNPs for FRET analyses. (A) Assembly of Sm subcomplexes D1/D2 and E/F/G onto snRNA forms a stable subcomplex. Subsequent binding of the D3/B complex completes the Sm core domain and places SmD1 and SmB next to each other (adapted from Kambach et al., 1999b, with permission from Elsevier). (B) Transfection of cells with different combinations of FP-tagged Sm proteins is predicted to produce complexes containing YFP-SmB next to CFP-SmD1, resulting in FRET interaction, or YFP-SmB and CFP-SmB in separate complexes, producing no FRET and acting as a negative control.

View larger version (13K): [in this window] [in a new window]   Fig. 2. FRET detection of fully assembled Sm cores in speckles and CBs. (A) HeLa cells were transfected with plasmids to express YFP-SmB and CFP-SmD1. The YFP (acceptor) fluorescence was bleached in a region of interest. A transient (1 second) increase in fluorescence of the donor was seen, resulting from the decrease in the intensity of acceptor fluorescence. (B) FRET efficiencies obtained in different regions of the nucleus in HeLa cells expressing YFP-SmB and CFP-SmD1 for 24 or 48 hours. No significant difference was seen in the efficiency calculated for each region using a one-way analysis of variance (ANOVA) test. The negative control of YFP-SmB and CFP-SmB gave a small negative value, probably a result of some inadvertent bleaching of the CFP donor by the 532 nm laser. A positive control using a single vector with YFP and CFP (pYFPCFP) separated by seven amino acids gives a FRET efficiency of 0.43 (data not shown). Efficiencies of less than 0.05 are generally regarded as not significant.

View larger version (123K): [in this window] [in a new window]   Fig. 3. Mature PA-GFP-Sm-tagged snRNPs show unrestricted exchange between nuclear structures. Repeated activation of a region of interest (circled) containing a CB (A,B) or a speckle (C) results in rapid accumulation of PA-GFP-SmB in both speckles (arrowheads) and CBs (arrows) throughout the entire nucleus. In each panel, the left-hand image shows the cell before any laser activation, the central image is immediately after the first activation event and the right-hand image is immediately after the tenth activation event. Bar, 22 µm.

View larger version (54K): [in this window] [in a new window]   Fig. 4. Mature snRNPs do not show a measurable export from the nucleus to the cytoplasm. (A) HeLa cells stably expressing YFP-SmB were bleached repeatedly using a region of interest including a region of the cytoplasm of one cell and a region of the nucleus of its neighbour (rectangle in first image). The cells were imaged following each bleach event. In the cell undergoing bleaching of the nucleus, the entire nucleus was rapidly depleted of YFP-SmB. By contrast, the cell undergoing cytoplasmic bleaching lost virtually none of its nuclear signal. (B) Intensity of YFP-SmB signal in the nucleus (outside the bleached region) for the two bleached cells against time.

View larger version (41K): [in this window] [in a new window]   Fig. 5. Recently imported snRNPs are unable to accumulate in speckles. Repeated activation of a region (circled) containing a CB results in rapid accumulation of signal in distant CBs (arrows). No accumulation in speckles can be detected. The left-hand image shows the cell before any laser activation, the central image is immediately after the first activation event and the right-hand image is immediately after the tenth activation event. Bar, 22 µm.

View larger version (90K): [in this window] [in a new window]   Fig. 6. At early timepoints, YFP-SmB can exit the nucleus into the cytoplasm. (A,B) HeLa cells expressing YFP-SmB for 24 hours were bleached repeatedly using a region of interest including a region of the cytoplasm of one cell and a region of the nucleus of its neighbour (rectangle in first image). The cells were imaged following each bleach event. In the cell undergoing bleaching of the nucleus, the entire nucleus was rapidly depleted of YFP-SmB. In the cell undergoing cytoplasmic bleaching, the nucleus also lost a significant amount of the nuclear signal. Bar, 22 µm. (C) Intensity of YFP-SmB signal in the nucleus (outside the bleached region) for the two bleached cells in A against time. (D) Intensity of YFP-SmB in a CB and a region of the nucleoplasm in a cell undergoing cytoplasmic bleaching demonstrates that signal is lost equally from the CB and the nucleoplasm.

View larger version (65K): [in this window] [in a new window]   Fig. 7. CRM1 localizes to CBs and is lost rapidly from them following treatment with LMB. In untreated HeLa cells, Crm1 (A) is found in CBs (arrows in A,B,D) where it colocalizes with snRNPs, detected with anti-Sm antibodies (B). Treatment of HeLa cells with LMB causes the loss of CRM1 from CBs (arrowheads in E,F,H). At timepoints up to 4 hours, snRNPs, detected by anti-Sm (F,H) or anti-TMG (J,L) antibodies, are still clearly seen in CBs (arrowheads and arrows). The CB marker protein, coilin, is also clearly seen in CBs after 4 hours of LMB treatment (I,L). Images A-H are deconvolved sections of HeLa cell nuclei; images I-L are three-dimensional projections of deconvolved serial sections of HeLa cell nuclei taken at 0.2 µm z-intervals. Bars, 13 µm.

View larger version (21K): [in this window] [in a new window]   Fig. 8. CRM1 is required for the retention of snRNPs in CBs. The loss of PA-GFP-Sm from CBs was measured in HeLa cells before and after treatment for 2 hours with LMB. (A) Average intensity of CBs over time, corrected for bleaching of the sample and expressed as a percentage of the pre-bleach intensity of each CB. The loss of PA-GFP-SmB from CBs is more rapid in cells treated with LMB. (B) Mean half-lives of loss of PA-GFP-SmB from CBs under control and LMB-treated conditions.

View larger version (30K): [in this window] [in a new window]   Fig. 9. LMB does not affect the ability of speckles to accumulate snRNPs. HeLa cells expressing PA-GFP-SmB were treated with LMB for 4 hours. A region of the nucleus containing a CB (circle in A, arrow in B) was repeatedly activated with a 406 nm laser and short time-lapse sequences were taken between each bleach event to monitor the spread of PA-GFP-SmB in the nucleus. The signal rapidly accumulated in speckles throughout the nucleus (arrowheads in C). Image A shows the cell before any laser activation, image B is immediately after the first activation event and image C is immediately after the tenth activation event. Bars, 11 µm.

View larger version (49K): [in this window] [in a new window]   Fig. 10. CRM1 interacts with newly imported snRNPs in vivo. (A) Immunoprecipitations from lysates of cells expressing YFP-SmB (i) or YFP (ii) using anti-GFP antibodies. Detection of products using anti-GFP demonstrates efficient precipitation of YFP-SmB and YFP (GFP Beads lane). Duplicate blots probed with anti-CRM1 demonstrate co-immunoprecipitation of endogenous CRM1 with GFP-SmD1 (iii) or YFP-SmB (iv) after 24 hours of expression (GFP Beads lanes), but not with GFP-SmD1 (v) or YFP-SmB (vi) expressed for 48 hours or YFP alone (vii) (GFP Beads lanes). (B) Immunoprecipitations from lysates of cells expressing CRM1-GFP using anti-GFP antibodies. Detection of products using anti-GFP demonstrates efficient precipitation of CRM1-GFP (i) (GFP Beads lane). Duplicate blot probed with anti-SMN (ii) or Y12 anti-Sm (iii) demonstrates co-immunoprecipitation of endogenous SMN and Sm proteins with GFP-CRM1, whereas the use of anti-U2B'' (iv) demonstrates no co-immunoprecipitation of this marker of mature snRNPs. (C) Immunoprecipitations from cells expressing GFP-SMN or UIA-GFP using anti-GFP antibodies. GFP-SMN (i) co-immunoprecipitates endogenous CRM1, whereas U1A-GFP (ii) does not.