1 Molecular Neuropathobiology Laboratory, Imperial Cancer Research Fund, 44 Lincolns Inn Fields, London, WC2A 3PX, UK
2 Electron Microscopy Unit, Imperial Cancer Research Fund, 44 Lincolns Inn Fields, London, WC2A 3PX, UK
*Author for correspondence (e-mail: g.schiavo{at}icrf.icnet.uk)
Accepted April 9, 2001
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SUMMARY |
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Key words: Mitosis, Phosphatidylinositol(4,5)-bisphosphate, RNA polymerase II
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INTRODUCTION |
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Several roles have been ascribed to nuclear phosphoinositides. Nuclear PtdIns(4,5)P2, like cytoplasmic PtdIns(4,5)P2, has been suggested to be the target of PtdIns(4,5)P2-specific phospholipases. The resultant production of DAG is in turn required for the activation of a sub-set of protein kinase C (PKC) isoforms with a nuclear localisation (DSantos et al., 1998). Nuclear targets whose functions are modulated by PKC-mediated phosphorylation include DNA polymerases, topoisomerases, histones and nuclear envelope proteins (DSantos et al., 1998; Irvine, 2000). The concomitant production of the second messenger InsP3 appears to participate in nuclear calcium homeostasis, which has been implicated in several physiological processes such as DNA synthesis, modulation of gene transcription, apoptosis and chromatin condensation.
A more direct link has recently been demonstrated between PtdIns(4,5)P2 and the process of chromatin remodelling. PtdIns(4,5)P2 is able to stabilise the association of the SWI/SNF-like BAF complex with chromatin and the nuclear matrix (Zhao et al., 1998). PtdIns(4,5)P2 could also influence DNA template availability via the inhibition of histone-mediated repression on RNA polymerase II activity (Yu et al., 1998). In support of this, chromatin has been shown to bind phospholipids via histones and non-histone chromosomal-associated proteins (Manzoli et al., 1977).
PtdIns(4,5)P2 and some enzymes involved in its synthesis have been co-localised in the nucleus with components of small nuclear ribonucleoprotein particles (snRNPs) (Boronenkov et al., 1998), which are involved in pre-mRNA processing. Interestingly, genetic evidence has implicated nuclear phosphoinositides and their hydrolysis products in the export of mRNA via the nuclear pore complex (York et al., 1999). This novel regulatory pathway involves the generation of several inositol polyphosphates, which appear to have distinct functions (York et al., 1999; Odom et al., 2000; Saiardi et al., 2000). In addition, inositol hexakisphosphate has recently been demonstrated to act as an essential cofactor in DNA repair by non-homologous end joining (Hanakahi et al., 2000).
Here, we demonstrate that detergent-resistant nuclear PtdIns(4,5)P2 is associated with electron-dense structures, whose morphology and distribution are cell-cycle dependent and resemble that of interchromatin granule clusters (IGCs). Elements of the transcriptional and pre-mRNA processing machinery interact with this pool of nuclear PtdIns(4,5)P2, and PtdIns(4,5)P2 immunoprecipitates contain intermediates and products of the splicing reaction. Immunodepletion and add-back experiments demonstrate that PtdIns(4,5)P2 and interacting factors are essential, but not sufficient, for pre-mRNA splicing. These findings suggest that PtdIns(4,5)P2 is a component of the pre-mRNA processing machinery.
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MATERIALS AND METHODS |
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Salmon sperm DNA or total cellular RNA were spotted on a nylon Hybond N plus membrane (Amersham Pharmacia Biotech) and PtdIns(4,5)P2 on nitrocellulose (Schleicher and Schuell). Nucleic acids were crosslinked to the membrane by heating for 2 hours at 80°C. Filters were blocked for 1 hour at room temperature with 1% ovalbumin, 1% polyvinylpyrrolidone in PBS and then incubated with 2C11 (1:500) in the same buffer. HRP-conjugated anti-mouse secondary antibodies (1:2000, Dako) were applied in 3% polyvinylpyrrolidone in PBS. Blots were developed using ECL Plus (Amersham Pharmacia Biotech).
Immunofluorescence and electron microscopy analysis
HeLa and NIH-3T3 were synchronised by treatment with nocodazole (100 ng/ml) overnight, tapped off and after washing, plated on poly-L-lysine coated coverslips. After paraformaldehyde fixation (3.7% in PBS) for 10 minutes, coverslips were incubated with 50 mM NH4Cl for 15 minutes and then blocked using PBS containing 2% bovine serum albumin (BSA), 0.25% gelatin, 0.2% glycine and 0.2% Triton X-100 for 1 hour. The primary antibody was appropriately diluted (2C11 1:200; anti-Sm 1:2000; anti-SC35 1:2000; H5 1:1000) in PBS with 1% BSA, 0.25% gelatin and 0.2% Triton X-100 and incubated for 1 hour. Cells were washed with 0.2% gelatin in PBS and the fluorescent secondary antibody (1:200, Molecular Probes) applied for 20 minutes in the same buffer as the primary antibody. Cy3-2C11 was prepared by incubating 2C11 with N-hydroxysuccinimidyl-Cy3 ester (Amersham-Pharmacia Biotech) in 100 mM Hepes-NaOH, pH 8.0. The ratio between dye and 2C11 was optimised for each reaction. For co-localisation experiments using two monoclonal antibodies, an additional blocking step with an excess of unlabelled primary antibody (30-fold) was performed following incubation with the secondary antibody and prior to application of Cy3-2C11.
In competition experiments, 2C11 was pre-incubated with liposomes containing 95% (mole/mole) PC and 5% mole/mole of different phosphoinosidites (Echelon) in PBS for 1 hour at room temperature. Where indicated, neomycin (1 mM) was added to the blocking solution. RNase A (1 mg/ml; 15 minutes) and DNase I (100 µg/ml; 2 hours) treatments were carried out post-fixation in PBS containing 5 mM MgCl2, 4% Tween-20 prior to blocking.
Cryosections of HeLa cells and extruded liposomes (Duzgunes and Wilschut, 1993) containing 90% PC plus 10% PtdIns, or 94% PC, 2% PtdIns(4,5)P2 and 4% PtdIns in 20 mM Hepes-KOH, pH 7.4, 0.1 mM DTT were labelled as previously described (Slot and Geuze, 1985). 2C11 antibody was used at 1:10 dilution and followed by 10 nm gold-conjugated rabbit anti-mouse IgM (1:100, British Biocell). Sections were examined and photographed with a JEOL 1010 TEM.
Immunoprecipitation
HeLa nuclear extracts (Dignam et al., 1983) were pre-cleared by incubation with 20 µl protein G-sepharose beads for 1 hour at 4°C, before the addition of either 20 µl of anti-IgM conjugated or 20 µl 2C11-conjugated protein G-sepharose beads. Samples were incubated for 2 hours at 4°C and beads were collected by centrifugation for 1 minute at 1200 g at 4°C. Immunoprecipitates were washed four times in 20 mM Hepes-NaOH pH 7.9, 100 mM KCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.25% NP-40 and then prepared for SDS-PAGE. Proteins were either stained with Coomassie blue or transferred to nitrocellulose and analysed by western blotting with appropriate antibodies. For analysis of associated RNAs, HeLa nuclear extracts were prepared using the Dignam method with modifications (Abmayr et al., 1988) from cells labelled overnight with 1 mCi [-32P]-orthophosphate per 150 mm dish. Associated [32P]-labelled RNAs were phenol/chloroform extracted following proteinase K treatment and separated on a 6% acrylamide/7 M urea denaturing gel. snRNAs were identified according to molecular weight and by comparison with parallel immunoprecipitations using the Y12 anti-snRNP antibody (Lerner et al., 1981).
Splicing assays
Splicing assays were carried out in a final volume of 20 µl, containing 30% HeLa nuclear extract, 0.8 U/ml RNasin, 0.4 mM ATP, 20 mM creatine phosphate, 3 mM MgCl2, 0.6% polyvinyl alcohol and 3 ng RNA probe. Uniformly radiolabelled ß-globin (Krainer et al., 1984), Ad-2 (Pellizzoni et al., 1998) or -crystallin (sp14-15) (Pellizzoni et al., 1998) transcripts were prepared using [
-32P]-CTP (Amersham Pharmacia Biotech) and the Riboprobe in vitro transcription system (Promega). After incubation for 3 hours (ß-globin) or 1 hour at 30°C (
-crystallin, Ad-2), the RNA was purified by phenol/chloroform extraction and ethanol precipitation and analysed by gel electrophoresis on a 6% (ß-globin,
-crystallin) or 10% (Ad-2) acrylamide, 7 M urea denaturing gel. For immunodepleted samples, the reaction mix was incubated for 1 hour at 4°C with protein G-sepharose beads alone or conjugated with anti-IgM or 2C11 prior to the addition of the RNA probe. For the antibody competition, 2C11 beads were pre-incubated with 250 µM GroPIns or GroPIns(4,5)P2 in PBS for 30 minutes at room temperature. Beads were washed once with splicing buffer prior to use. Quantitation was performed using a Phoshorimager (Molecular Dynamics) for splicing reactions and using NIH Image for western blots.
For the elution and add-back experiments, immunoprecipitations were carried out in 19 µl splicing reaction containing 40% nuclear extract for 90 minutes at 4°C. Immunoprecipitated material was eluted by incubating beads for 15 minutes at 4°C with 5 µl elution buffer (14 mM Hepes-NaOH, pH 7.9, 40 mM KCl, 3 mM MgCl2, 0.4 mM ATP, 20 mM creatine phosphate, 0.6% polyvinyl alcohol) containing 300 µM PtdIns, di-butyl PtdIns(4,5)P2 or GroPIns(4,5)P2. The supernatant or 5 µl of the lipids alone was added to the depleted reaction mix to give a final concentration of 30% nuclear extract. 3 ng -crystallin RNA was used per reaction.
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RESULTS |
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Pre-incubation of the 2C11 antibody with an excess of liposomes containing different phosphoinositides showed that the nuclear staining is abolished by PtdIns(4,5)P2, but not by any other lipid, including PtdIns(3,4,5)P3 and the PtdIns(4,5)P2 isomers PtdIns(3,4)P2 and PtdIns(3,5)P2 (Fig. 2D-G). PtdIns(4,5)P2-treated samples present a cytoplasmic dotted staining (Fig. 2G). This was never observed in control preparations and could be due to the non-specific binding of antibody-PtdIns(4,5)P2 liposome aggregates to cytoplasmic structures. Similar competition experiments were therefore performed using soluble phosphoinositide head-groups. L--glycerophospho-D-myo-inositol(4,5)bisphosphate (GroPIns(4,5)P2) (Fig. 2H) but not the unphosphorylated GroPIns, totally abolished the nuclear signal and did not result in the additional cytoplasmic staining. InsP3 can compete 2C11 binding (S.L.O. and G.S., unpublished). However neomycin, an aminoglycoside antibiotic that binds to several phosphoinositides (Gabev et al., 1989) but not InsP3 (Arbuzova et al., 2000), abolished 2C11 immunolabelling (Fig. 2I). Together, these results strongly suggest that the nuclear antigen recognised by 2C11 is PtdIns(4,5)P2.
The localisation of detergent-resistant PtdIns(4,5)P2 is cell-cycle dependent
Biochemical studies investigating the cell-cycle regulation of phosphoinositides have suggested that they vary during S-phase and are important for the progression of mitosis (Uno et al., 1988; Imoto et al., 1994; York and Majerus, 1994). As shown in Fig. 3, the distribution of the detergent-resistant PtdIns(4,5)P2 changes dramatically during mitosis. Upon nuclear membrane disassembly, detergent-resistant PtdIns(4,5)P2 immunoreactivity shifts to the cytoplasm, where it remains during chromosome partitioning (Fig. 3I,L). During the later stages of mitosis, PtdIns(4,5)P2 staining undergoes a remarkable concentration into a limited number of very bright structures that remain cytoplasmic even when the DNA has re-localised to the newly formed nuclei of the two daughter cells (Fig. 3M-O). As shown by immuno-electron microscopy (Fig. 3P), these mitotic structures appear morphologically indistinguishable from those observed in interphase, lacking any apparent lipid bilayer morphology and with no visible connection to the plasma membrane. PtdIns(4,5)P2 immunoreactivity does not overlap with the DNA. This is particularly evident during chromosome condensation, when PtdIns(4,5)P2 staining is clearly excluded from the area occupied by the genetic material (Fig. 3D-F)
Nuclear PtdIns(4,5)P2 is associated with a sub-class of nuclear bodies
To identify the nature of the PtdIns(4,5)P2-containing compartment, an immunofluorescence screen was performed using several nuclear markers. Only a limited overlap was observed between the interphase distribution of PtdIns(4,5)P2 and that of a large number of antigens tested, including various transcription factors, such as the polycomb family member Ring 1 and PML (Lamond and Earnshaw, 1998; Matera, 1999). The 2C11 staining pattern is reminiscent of that seen for a number of nuclear antigens involved in pre-mRNA processing and associated with interchromatin granule clusters (IGCs) and perichromatin fibrils (PFs) (Spector, 1993b; Lewis and Tollervey, 2000). IGCs lack active transcription foci, are resistant to nuclease treatment and have been suggested to act as storage compartments for splicing factors (Spector, 1993a; Fakan, 1994; Mintz et al., 1999). By contrast, PFs, which are often associated with the periphery of IGCs, contain nascent transcripts and are sensitive to RNase degradation (Spector, 1993a; Fakan, 1994).
We tested the co-localisation of nuclear PtdIns(4,5)P2 with the splicing factor SC-35, a classic marker of IGCs (Spector et al., 1991) and PFs, and the common snRNP components, Sm proteins. SC-35 and PtdIns(4,5)P2 signals co-localise in interphase cells (Fig. 4A-C), whereas Sm antigens only partially overlap with PtdIns(4,5)P2 immunoreactivity as expected from their wider distribution not only in IGCs and PFs but also in the nucleoplasm and coiled bodies (Spector et al., 1983) (Fig. 4D-F). A partial co-localisation was also observed with the hyperphosphorylated form of the largest subunit of RNA polymerase II (Kim et al., 1997) (H5, Fig. 4G-I). The C-terminal domain of this subunit is present as an unphosphorylated (RNA Pol IIa) or as a number of hyperphosphorylated forms (RNA Pol IIo) (Corden and Patturajan, 1997; Bentley, 1999; Hirose and Manley, 2000). Recent work has shown that RNA Pol IIo associates with IGCs and shuttles between these structures and sites of active transcription (Bregman et al., 1995). PtdIns(4,5)P2 is not present in coiled bodies as demonstrated by the lack of co-localisation with p80-coilin (S.L.O. et al., unpublished).
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The distribution of PtdIns(4,5)P2 and RNA Pol IIo exactly overlap during mitosis, this being particularly evident in late telophase (Fig. 5A-C). These PtdIns(4,5)P2-containing particles also contain the majority of SC-35 (Fig. 5D-F), a feature that identifies them as mitotic interchromatin granules (Spector et al., 1991; Ferreira et al., 1994). However, only a minor fraction of Sm proteins are associated with these structures, the majority localise to an area occupied by the newly formed nuclei of the two daughter cells (Ferreira et al., 1994) (Fig. 5G-I). These results suggest that PtdIns(4,5)P2-containing particles undergo dynamic changes in composition during the cell-cycle.
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Extraction of RNA present in 2C11 immunoprecipitates from [32P]-labelled HeLa cell nuclear extracts, revealed radioactive bands corresponding to the U1-U6 small nuclear RNAs (snRNAs) in the anti-PtdIns(4,5)P2, but not in the control immunoprecipitate (Fig. 6C). The extent of tRNA recovery in the 2C11 immunoprecipitate varied between experiments and was not competed by pre-incubation of 2C11 with GroPIns(4,5)P2.
Together, these results demonstrate that a pool of nuclear PtdIns(4,5)P2 is associated in a detergent resistant manner with a multi-subunit complex comprising both protein and nucleic acid components of the transcriptional and pre-mRNA splicing machinery.
PtdIns(4,5)P2-containing nuclear structures are essential for pre-mRNA splicing
Based on its composition, we asked whether the pool of proteins associated with nuclear PtdIns(4,5)P2 have an active involvement in pre-mRNA splicing. We tested this hypothesis using an in vitro splicing assay combined with an immunodepletion approach. Three different RNA probes were tested: ß-globin (Krainer et al., 1984) (Fig. 7), -crystallin (Pellizzoni et al., 1998) (Fig. 8) and adenovirus 2 major late pre-mRNA (Ad-2) (Pellizzoni et al., 1998). In all cases, immunodepletion of HeLa cell nuclear extract with anti-PtdIns(4,5)P2 antibody beads inhibited the splicing reaction, whereas immunodepletion with protein G beads or anti-IgM beads had no significant effect. In Fig. 7A, this inhibition is seen as a decrease in the amount of product and splicing intermediate. Pre-incubation of the antibody with the GroPIns(4,5)P2, but not GroPIns (Fig. 7A,D) prevents the 2C11-mediated inhibition. Parallel western blotting of the immunoprecipitates with anti-RNA Pol IIo (H5, Fig. 7B) show that RNA Pol IIo is associated with the 2C11 immunoprecipitate only and that this association is competed by pre-incubation of the beads with GroPIns(4,5)P2 to an extent similar to that seen in the splicing reaction (Fig. 7, compare B,D). The incomplete rescue observed by pre-incubation of the antibody with the soluble headgroup of PtdIns(4,5)P2 (Fig. 7D) could be explained by the absence of an excess of free competitor during the immunodepletion.
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The above immunodepletion experiments are carried out in the absence of an RNA substrate. If the splicing reaction is performed prior to immunoprecipitation and the associated RNA analysed, we find that splicing intermediates and the spliced product specifically associate with 2C11-conjugated beads to a similar extent as they do with anti-snRNP antibody beads (Y12, Fig. 9). Pre-incubation of the 2C11 beads with GroPIns(4,5)P2 but not GroPIns is again able to compete the immunoprecipitation.
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DISCUSSION |
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A hyperphosphorylated form of the largest subunit of RNA Pol II also associates with PtdIns(4,5)P2. The C-terminal domain of this subunit is essential for the assembly of the spliceosome (Misteli and Spector, 1999), truncation inhibits splicing in vivo (McCracken et al., 1997) and its hyperphosphorylation activates splicing in vitro (Hirose et al., 1999), in keeping with the idea that RNA Pol II plays an active role in coupling transcription and pre-mRNA processing (Hirose and Manley, 2000). Although we cannot exclude a role for PtdIns(4,5)P2 in the former process, we find that the nuclear localisation of PtdIns(4,5)P2 and its turnover are independent of active transcription. Inhibiting transcription with the drugs -amanitin (Haaf and Ward, 1996) or DRB (Zandomeni et al., 1986) causes a re-distribution of PtdIns(4,5)P2 into larger, rounder nuclear foci. This phenomenon has been previously described for Sm proteins, SC-35 (Spector et al., 1983) and a subset of nuclear phosphatidylinositol phosphate kinases (Boronenkov et al., 1998). Importantly, in these conditions the nuclear levels of PtdIns(4,5)P2 do not differ between treated and untreated cells (S.L.O. and G.S., unpublished).
During mitosis, when cells are transcriptionally inactive, PtdIns(4,5)P2 assumes a peripheral distribution similar to that observed for RNA Pol II and certain splicing factors (Spector et al., 1991; Ferreira et al., 1994; Kim et al., 1997). In the late stages of telophase and cytokinesis, PtdIns(4,5)P2 concentrates in discrete structures that remain peripheral despite the reformation of the daughter cell nuclei. These structures also contain RNA Pol IIo and SC-35 but not Sm proteins. These PtdIns(4,5)P2-containing complexes thus appear to undergo dynamic changes in composition through the cell-cycle. At the completion of mitosis, it is not clear whether these proteolipid complexes are disassembled before retrieval via the nuclear pore (Nakielny and Dreyfuss, 1999) or if they are transported back through fenestrations still present in the partially assembled nuclear envelope. Direct analysis of PtdIns(4,5)P2-containing structures in living mitotic cells will provide insights into this transport mechanism.
Bearing in mind the multiple functions of RNA Pol II and that, in addition to their role in splicing, Sm and Sm-like proteins have been implicated in other aspects of mRNA processing such as decapping and decay (Bouveret et al., 2000; Tharun et al., 2000), these PtdIns(4,5)P2-containing structures might therefore function as central stations for the maturation and quality control of newly formed RNA. Two alternative, but not mutually exclusive roles can be proposed for the phosphoinositide moiety in these complexes. The first possibility is that PtdIns(4,5)P2 binds nuclear cytoskeletal proteins (Pederson, 2000; Rando et al., 2000). This hypothesis envisages PtdIns(4,5)P2 as a structural interface between the enzymatic core of the spliceosome and cytoskeletal components, such as protein 4.1 which has been described to functionally interact with the splicing apparatus (Lallena et al., 1998).
Recently, PtdIns(4,5)P2 has been shown to block the exit of the SWI/SNF-like BAF chromatin remodelling complex from digitonin-permeabilised nuclei (Zhao et al., 1998). This effect is likely to be mediated via interactions with ß-actin and actin-related proteins that are intrinsic components of this complex (Zhao et al., 1998; Rando et al., 2000). This finding highlights possible similarities between the chromatin remodelling and splicing machineries and suggests an underlying mechanism whereby PtdIns(4,5)P2 functions as a direct modulator of various nuclear multi-subunit protein complexes by coupling them to the actin treadmill. The balance between monomeric and polymeric forms of actin appears to act as a regulator of several nuclear functions, as recently demonstrated for serum response factor-dependent gene transcription (Sotiropoulos et al., 1999).
Alternatively, PtdIns(4,5)P2 could serve as a substrate for nuclear phosphoinositide-modifying enzymes. Hydrolysis of nuclear PtdIns(4,5)P2 by phospholipase C might provide a localised release of DAG and InsP3 (DSantos et al., 1998; Irvine, 2000). Accordingly, the phosphatidylinositol-specific phospholipase C isoforms ß1b and 4 have been localised to the nucleus (Irvine, 2000). In addition to the regulation of Ca2+ release from internal stores, InsP3 is the precursor of inositol polyphosphates, which have been demonstrated to be essential for RNA transport (York et al., 1999; Odom et al., 2000; Saiardi et al., 2000) and DNA double-strand break repair by non-homologous end joining (Hanakahi et al., 2000). Inositol polyphosphates may therefore act as a high turnover switch of the activity of these molecular machineries, whose activation would be restricted to specific nuclear sub-domains and dependent upon the phosphorylation state of the inositol ring.
This work provides the first direct evidence that the splicing machinery is engaged in a proteolipid complex with PtdIns(4,5)P2 that is responsible for the majority of splicing activity in vitro. These findings constitute the basis for future investigations into the molecular mechanisms responsible for the assembly, mitotic trafficking and dynamics of these nuclear PtdIns(4,5)P2-containing complexes and highlight the emerging role of phosphoinositides in nuclear physiology.
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ACKNOWLEDGMENTS |
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