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First published online 1 March 2005
doi: 10.1242/jcs.01726

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Signal recognition particle assembly in relation to the function of amplified nucleoli of Xenopus oocytes

John Sommerville^1,*, Craig L. Brumwell^2,, Joan C. Ritland Politz² and Thoru Pederson²

¹ Division of Cell and Molecular Biology, School of Biology, University of St Andrews, KY16 9TS, UK
² Department of Biochemistry and Molecular Pharmacology, and Program in Cell Dynamics, University of Massachusetts Medical School, Worcester, MA 01605, USA

View larger version (54K): [in a new window]   Fig. 1. Expression of endogenous SRP RNA and SRP proteins in Xenopus oocytes. (A) Staged oocytes were collected to give an equal volume of soluble extract and RNA was extracted for RT-PCR and quantitation on 2% agarose gels. RT-PCR products were generated using primers specific for SRP RNA (20 cycles) and SRP54 mRNA (25 cycles), and 5S RNA was separated directly from the total RNA sample. (B) Sedimentation analysis of SRP19 protein present in cytoplasms isolated from stage IV oocytes. Gradient fractions were immunoblotted using a chicken antibody directed against SRP19 protein. Reactive bands were detected in fractions sedimenting at 10-12 S. Positions of sedimentation mass markers run in a parallel gradient are indicated by stars (from left: haemoglobin, Mr 67,000; alcohol dehydrogenase, Mr 180,000; apoferritin, Mr 443,000; microglobulin, Mr 670,000; immunoglobulin M, Mr 960,000: see also Fig. 5). Positions of electrophoretic mass markers are shown (kDa). (C) Immunoblot using anti-SRP19 of the 10-12 S fraction isolated from extracts equivalent to the range of oocyte stages and oocyte numbers described in (A). (D) Relative amounts of product per oocyte were calculated by dividing amounts detected from equal masses of oocytes by the number of oocytes contained in that mass at each stage. Graph shows: SRP RNA (open circles), SRP54 mRNA (open squares), 5 S RNA (black circles) and SRP19 protein (black squares). Intensity of staining with ethidium bromide or recorded on film after ECL was captured using a GeneSnap system and measured using GeneTools (Synoptics Ltd).

View larger version (81K): [in a new window]   Fig. 2. Incorporation of SRP RNA into amplified nucleoli. Recombinant human SRP RNA (A-C) and control RNA antisense to canine SRP RNA (D-F) were labelled with Alexa 488 and injected into the GVs of Xenopus oocytes. After 18 hours, GVs were isolated and spread preparations were counterstained with DAPI. Fields containing nucleoli (No) were located by phase-contrast microscopy (A,D) and generally also contained chromosomes (Ch), Cajal bodies (CB) and B-snurposomes (smaller particles throughout the field). DNA-rich regions of chromosomes (chromomeres) and nucleoli (fibrillar centres) were located by DAPI fluorescence (B,E) and injected RNA was located by Alexa 488 fluorescence (C,F). SRP RNA was targeted specifically to nucleoli (C), whereas control antisense RNA failed to concentrate in any nuclear structures (F), although autofluorescence is seen from a contaminating yolk platelet (YP).

View larger version (83K): [in a new window]   Fig. 3. In situ hybridization of TRITC-labelled PNA probes with amplified nucleoli. Probes antisense to Xenopus SRP RNA (A-D) and sense of yeast SRP RNA (control, E-G) were hybridized to GV spread preparations. Phase-contrast images (A,E) show nucleoli (No), chromosomes (Ch) and Cajal bodies (CB). DAPI images (B,F) show staining of fibrillar centres and chromomeres. TRITC fluorescence shows that the antisense probe hybridizes to nucleoli, although not significantly to the fibrillar centres (C). This differential localization is seen clearly in merged images in which the DAPI image is computer-coloured green (D). Coincidence of signals appears as yellow: the small amount of coincidence in nucleoli can be accounted for by overlapping nucleolar components, whereas complete coincidence is seen with the wide-spectrum autofluorescence of a contaminating yolk platelet (YP). The control probe fails to hybridize to nucleoli, although signal is seen over the yolk platelets (YP) in (G).

View larger version (48K): [in a new window]   Fig. 4. Import of GFP-SRP19 into the germinal vesicle and export back to the cytoplasm. Samples of GFP-SRP19 (20 ng) were injected into the cytoplasm of oocytes, and GVs and cytoplasms were isolated under oil at 1, 2, 5 and 10 hours (h) after injection. (A) Protein was extracted and analysed by immunoblotting using antibodies to GFP. Bands at 47 kDa corresponding to the mass of the fusion protein (lane S, noninjected protein sample) show a steady increase in translocation from cytoplasm (C) to GV (N). (B) Ratios of nuclear to cytoplasmic concentration ([N]/[C]) were calculated from band density scans and mean values from four different injection experiments were plotted against time (open circles). In a separate series of experiments, co-injection of 25 ng of the import receptor importin- shows an enhanced initial rate of nuclear uptake of GFP-SRP19 (filled circles). (C) Export of GFP-SRP19 from the germinal vesicle to the cytoplasm. From 10 hours after injection of GFP-SRP19 into the cytoplasm the nuclear to cytoplasmic concentration ratio ([N]/[C]) starts to fall (white columns), indicating export from the GV. This fall is inhibited by incubating the oocytes in the presence of 50 nM leptomycin B (LMB, grey columns).

View larger version (37K): [in a new window]   Fig. 5. Sedimentation analysis of GFP-SRP19 present in cytoplasm isolated from injected oocytes. The injected oocytes were either immediately enucleated and frozen or incubated for 48 hours before enucleation and freezing. Half of the 48 hour sample was treated with ribonuclease A (RNase) for 30 minutes in buffer lacking Mg2+. Clarified cytoplasms from 25 oocytes taken immediately after injection (open circles), after 48 hours (filled circles) and after 48 hours and treated with RNase (open squares) were loaded on glycerol gradients and centifuged under the same conditions as described in Fig. 1. Gradient fractions were collected and (A) scanned at for fluorescence at 510 nm (FL, arbitrary units). Fractions from parallel gradients loaded with the sedimentation markers described in Fig. 1 were scanned at 280 nm (Mr, open triangles). The predicted sedimentation position of SRP (11 S, Mr 335,000) is indicated by an arrow. Fractions were then precipitated with acetone and (B) analysed by immunoblotting using antibodies to GFP or, in the case of markers, staining of the gel with Coomassie brilliant blue.

View larger version (81K): [in a new window]   Fig. 6. Incorporation of injected GFP-SRP19 into amplified nucleoli. At 1 (A-C), 2 (D-F), 3 (G-I) and 5 (J-L) hours after injecting GFP-SRP19 into the cytoplasm of oocytes, GVs were isolated and spread preparations were stained with DAPI and examined for fluorescence. Although nucleoli (No), chromosomes (Ch) and Cajal bodies (CB) were identified in most fields by phase-contrast (A,D,G,J), and DAPI staining (B, E, H, K) only nucleoli showed GFP fluorescence (C,F,I,L). The fluorescent signal is seen to spread with time from regions around the DAPI-staining fibrillar centres at 2 hours after injection (E, F) to occupy most of the nucleolar space by 5 hours (K,L).

View larger version (56K): [in a new window]   Fig. 7. Location of GFP-SRP19 in partially dissociated nucleoli. At 5 hours after injecting GFP-SRP19 into the cytoplasm of oocytes, GV spreads were prepared in the absence of Mg2+ and either examined unfixed (A-C) or after storing the preparations for 18 hours in 70% ethanol (D-F). Distended nucleolar substructure is evident from the phase-contrast images (A,D), with globular dense fibrillar component organized around individual DAPI-staining fibrillar centres (B,E). The GFP signal remained located at the dense fibrillar component (C,F) and was absent from chromosomes (D-F) and other nuclear structures (not shown).

View larger version (58K): [in a new window]   Fig. 8. Immunostaining of amplified nucleoli with antibodies to SRP19. GV spreads were prepared in low-salt medium without Mg2+. Nucleoli (No), Cajal bodies (CB) and B-snurposomes were identified by phase-contrast (A) and DAPI staining (B), but only the nucleoli were immunostained using chicken anti-SRP19 with FITC-conjugated anti-chicken IgG as a secondary antibody (C). The centres of the stained structures are seen to be relatively free of fluorescence. (D-F) The DAPI staining (D) and the immunostaining (E) are merged (F), with the DAPI computationally coloured red and yellow indicating spatial coincidence of the two signals.

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