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Nuclear PtdIns(4,5)P2 assembles in a mitotically regulated particle involved in pre-mRNA splicing

Shona L. Osborne1, Claire L. Thomas1, Steve Gschmeissner2 and Giampietro Schiavo1,*

1 Molecular Neuropathobiology Laboratory, Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London, WC2A 3PX, UK
2 Electron Microscopy Unit, Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London, WC2A 3PX, UK



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  		Fig. 1. Monoclonal antibody 2C11 recognises PtdIns(4,5)P2 inserted in a membrane bilayer. (A) 2C11 recognises PtdIns(4,5)P2 but not DNA or RNA on dot-blot. Serial dilutions of PtdIns(4,5)P2 (0.0125-0.4 µg), DNA and total PC12 cell RNA (0.03-1 µg) were spotted on membranes and probed with 2C11 antibody. (B) 2C11 specifically interacts with PtdIns(4,5)P2-containing liposomes. Liposomes containing radioactive PC as a tracer and either PtdIns (empty bars) or PtdIns(4,5)P2 (filled bars) were incubated with immobilised anti-IgM or 2C11. Radioactivity associated with beads was measured and results expressed as a percentage of the total. Error bars represent s.d. based on three experiments. (C,D) Cryosections of extruded liposomes containing PtdIns(4,5)P2 (C) and PtdIns (D) were probed with 2C11 followed by gold-conjugated secondary antibodies. 34% of the total PtdIns(4,5)P2-containing liposomes (n=170) were labelled with one or more gold particles compared with 0% (n=96) in the case of PtdIns-containing liposomes. Bars, 50 nm.

 


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  		Fig. 2. PtdIns(4,5)P2 is localised in electron-dense particles in HeLa cell nuclei. Detergent-permeabilised (A,D-I) or unpermeabilised (B,C) HeLa cells were incubated with 2C11 followed by a fluorescent (A and D-I) or a 10 nm gold (B,C)-conjugated secondary antibody. PtdIns(4,5)P2 signal was associated with electron dense areas in the nucleus (B), resembling IGCs (arrow), and in the nucleolus (C), where both the fibrillar centres (open triangles) and portions of the dense granular components (filled triangles) are labelled. n, nucleus; nu, nucleolus. Fluorescent images were acquired with a CCD camera-equipped Zeiss microscope. Pre-incubation of 2C11 with liposomes containing PtdIns(4,5)P2 (G) or with an excess of the soluble headgroup of PtdIns(4,5)P2 (GroPIns(4,5)P2, (H) abolished the nuclear staining, while PtdIns (D) or isomers of PtdIns(4,5)P2 (E,F) had no effect. Pre-incubation of cells with neomycin, which binds to several phosphoinositides, also abolished staining (I). Bars, 10 µm (A,D-I); 200 nm (B,C).

 


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  		Fig. 3. The localisation of the nuclear structures containing PtdIns(4,5)P2 is cell-cycle dependent. Synchronised NIH-3T3 cells were fixed at different stages of mitosis and the distributions of DNA and PtdIns(4,5)P2 were visualised with Hoechst 33342 (blue, A,D,G,J,M), and 2C11 antibody (red, B,E,H,K,N), respectively. Pseudo-colour merged images emphasise the lack of co-localisation between PtdIns(4,5)P2 and DNA (merge, C,F,I,L,O). Fluorescent images were acquired with a CCD camera-equipped Zeiss microscope. Bars, 10 µm. Cells in telophase were prepared for electron microscopy and labelled with 2C11 (P). The dotted line in (P) outlines the plasma membrane (pm). Bar, 200 nm.

 


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  		Fig. 4. PtdIns(4,5)P2 co-localises with SC-35 in interphase cells. Detergent-permeabilised HeLa cells were co-stained with Cy3-2C11 (B,E,H,J,K) and either anti-SC-35 (A), anti-Sm (D) or anti- RNA Pol IIo (H5, G) antibodies. The yellow pseudo-colour (merge; C,F,I) shows the extent of co-localisation between the two antigens. RNase (K), but not DNase I (J) treatment abolishes PtdIns(4,5)P2 staining. Confocal microscopy images corresponding to the projection of a series of 0.4 µm sections are shown. Bars, 10 µm.

 


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  		Fig. 5. PtdIns(4,5)P2 co-localises with RNA Pol IIo and SC-35 in mitotic cells. Synchronised HeLa cells in late telophase were co-stained with Cy3-2C11 (B,E) or 2C11 (H) and either anti-RNA Pol IIo (H5, A), anti-SC-35 (D) or anti-Sm (G). The yellow pseudocolour (merge; C,F,I) shows the extent of co-localisation between the antigens. Confocal microscopy images corresponding to the projection of a selected series of 0.4 µm sections are shown. Bars, 10 µm.

 


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  		Fig. 6. Nuclear PtdIns(4,5)P2 associates with RNA Pol IIo and snRNP components. (A) Immunoprecipitates from HeLa nuclear extracts were analysed by SDS-PAGE. Coomassie blue staining shows that several proteins are recovered by 2C11, but not IgM beads. This association is attenuated by pre-incubation with an excess of GroPIns(4,5)P2 but not GroPIns. Closed circles indicate the position of the antibody bands. Total refers to one tenth of the starting material. (B) Samples were analysed by western blotting with antibodies against the hyperphosphorylated (RNA Pol IIo, arrow) or the unphosphorylated (RNA Pol IIa, *) forms of the large subunit of RNA Pol II (Kim et al., 1997), Sm proteins or hnRNP A1. IgM and 2C11 immunoprecipitates (P) were loaded together with half of the input (start) and half of the supernatant from the immunoprecipitation (S). RNA Pol IIo and Sm proteins are found in the 2C11 but not in the IgM immunoprecipitate, whilst RNA Pol IIa and hnRNP A1 remain in the supernatant. (C) Analysis of [32P]-labelled RNAs associated with the PtdIns(4,5)P2 immunoprecipitate. Bands corresponding to the U1-U6 snRNAs are recovered by 2C11, but not by the control anti-IgM antibody. For comparison, an immunoprecipitation with the antibody Y12 (anti-snRNP) are included. Total refers to one tenth of the input RNA.

 


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  		Fig. 7. Immunodepletion of HeLa nuclear extract with 2C11 causes a specific inhibition of pre-mRNA splicing. (A) In vitro splicing reaction with a ß-globin RNA probe. Splicing is inhibited by pre-incubation of nuclear extract with 2C11 beads. This effect is blocked by pre-treatment of the antibody with GroPIns(4,5)P2 but not GroPIns. A schematic representation of the starting probe, intermediates and product of the splicing reaction is shown on the right. (B) The beads used in the immunodepletion experiment presented in (A) and an equivalent amount of untreated 2C11 beads were analysed by western blotting using anti-RNA Pol IIo (H5). (C) Splicing efficiency was quantified and expressed as the percentage of processed RNA in the samples versus the total, taking the mock-treated sample as 100%. Bars represent the s.e. of six experiments. (D) The inhibition of splicing seen on treatment of the nuclear extract with immobilised 2C11 is partially rescued by pre-treatment of 2C11 beads with GroPIns(4,5)P2 but not GroPIns. Results are expressed as in (C) and bars represent the s.e. of four experiments.

 


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  		Fig. 8. PtdIns(4,5)P2-associated factors are required for splicing. (A) 2C11 but not IgM depleted HeLa nuclear extracts are impaired in their ability to support the splicing of {delta}-crystallin mRNA. The inhibition is blocked by pre-incubation of the 2C11 beads with GroPIns(4,5)P2 but not GroPIns. The inhibition of splicing seen is comparable to that observed using an anti-snRNP antibody (Y12). A schematic representation of the start, intermediates and product of the splicing reaction is shown on the right. (B) Immunoprecipitated material can be eluted from 2C11 beads by incubation with an excess of PtdIns(4,5)P2 or GroPIns(4,5)P2, as seen by western blotting using anti-RNA Pol IIo (H5; lower panel; P, pellet; S, supernatant). Re-addition of these eluates to the depleted splicing reaction partially restores the splicing activity, whereas re-addition of control eluates or the lipids alone have no effect.

 


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  		Fig. 9. PtdIns(4,5)P2 associates with splicing complexes. Splicing reactions were carried out prior to immunoprecipitation. In these conditions, intermediates and products of the splicing reaction associate with anti-PtdIns(4,5)P2, but not control beads. Pre-incubation of the antibody with GroPIns(4,5)P2 but not GroPIns inhibits the recovery. A positive control immunoprecipitation with Y12 is shown for comparison.

 

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© The Company of Biologists Ltd 2001