A Phospholipase C-Dependent Inositol Polyphosphate Kinase Pathway Required for Efficient Messenger RNA Export

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Science  02 Jul 1999:
Vol. 285, Issue 5424, pp. 96-100
DOI: 10.1126/science.285.5424.96


In order to identify additional factors required for nuclear export of messenger RNA, a genetic screen was conducted with a yeast mutant deficient in a factor Gle1p, which associates with the nuclear pore complex (NPC). The three genes identified encode phospholipase C and two potential inositol polyphosphate kinases. Together, these constitute a signaling pathway from phosphatidylinositol 4,5-bisphosphate to inositol hexakisphosphate (IP6). The common downstream effects of mutations in each component were deficiencies in IP6 synthesis and messenger RNA export, indicating a role for IP6 in GLE1function and messenger RNA export.

Spatial and temporal activation of inositol signaling pathways modulates protein machines that enable eukaryotic cells to sense changes in their environment. An essential component within this circuitry is phosphatidylinositol 4,5-bisphosphate (PIP2), which is hydrolyzed by phosphatidylinositol (PI)-specific phospholipase C to produce messenger molecules including inositol 1,4,5-trisphosphate (IP3) (1–3). IP3regulates the release of intracellular calcium (1), and its modification by multiple dephosphorylation or phosphorylation reactions indicates that IP3function may not limited to calcium signaling (3,4). IP3 is a precursor to more phosphorylated inositols, such as IP5 and IP6, whose signaling functions have not been fully elucidated (5).

In eukaryotic cells, transcription and translation are both spatially and temporally segregated by the nuclear envelope (NE). These processes are bridged by the NPC, an essential protein machine that spans the NE and allows the selective nucleocytoplasmic exchange of proteins, RNAs, and ions. Nuclear export of mRNAs requires a series of events: pre-mRNA processing, ribonucleoprotein targeting to the NPC, and energy-dependent translocation through the portal (6). The processing and transport of mRNA are tightly linked, such that splicing, polyadenylation, and capping all affect the export process (7). Several essential requirements for mRNA export have been identified, including NPC-associated proteins, shuttling heterogenous nuclear ribonucleoprotein (hnRNP) complexes, and the small guanosine triphosphate–binding protein Ran (8). Here, we show that phospholipase C and two proteins that influence the generation of IP6 are required for efficient NPC-mediated export of mRNA.

Gle1p (also known as Rss1p) is an essential NPC-associated factor required for mRNA export in both yeast and human cells (9, 10). To identify additional factors required for Gle1p-mediated export of mRNA, we conducted a genetic screen in the yeast Saccharomyces cerevisiae for mutations that were lethal in combination with the temperature-sensitivegle1-2(P380L) mutant (11). The mutants were classified into four complementation groups (Fig. 1A). Mutations of GLE1 are synthetically lethal in combination with a nup100 null allele (9), and with mutations in genes encoding various nucleoporins (12). Therefore, each complementation group was tested for rescue of the synthetic lethal phenotype with a panel of centromeric plasmids that each expressed a given nucleoporin gene. NUP100 rescued the largest group; however, none of the plasmids could complement the other three groups. These groups were therefore initially designated gsl (forGLE1 synthetic lethal).

Figure 1

Isolation of genes encoding regulators of mRNA export in a synthetic lethal screen with the gle1-2 (P380L)mutant. (A) Complementation analysis of gle1synthetic lethal alleles. Mutant strains were mated in all pairwise combinations and assayed for nonsectoring diploids, resulting in four groups. Temperature sensitivity was assayed at 37°C. The open reading frame (ORF) that complemented the group is designated as registered with the Stanford Genome Database (SGD); n.d., not determined, asGSL3 has not been identified. (B) Inhibition of nuclear export of poly(A)+ RNA in gsl1,gsl2, and gsl3 mutants. In situ hybridization with a digoxigenin-coupled oligo(dT) probe was conducted with wild-type and mutant cells after incubation at 37°C for 1 hour (22). Probe localization was detected with a fluorescein isothiocyanate (FITC)–coupled antibody to digoxigenin. Compared to the diffuse cytoplasmic staining in wild-type (WT) cells, marked nuclear accumulation was observed in the mutants. Right panel, coincident DAPI (4′,6′-diamidino-2-phenylindole) staining.

Because Gle1p is specifically required for the export of polyadenylated [poly(A)+] mRNA (9, 12), we used in situ transport assays to test the behavior of the gslmutants. After shift to the restrictive growth temperature, thegsl1Δ, gsl2-6, and gsl3-3 mutants all showed an inhibition of poly(A)+ RNA export (Fig. 1B). Over the same time course, no perturbations were observed in nuclear protein import, protein export, or in the morphology of NPCs or the NE (13). Thus, the gsl mutants are genetically and functionally linked to GLE1, and they have specific defects in mRNA export.

The wild-type GSL1 and GSL2 genes were identified by complementation of the gsl1-4 andgsl2-2 phenotypes with a centromeric yeast genomic library. The minimal complementing DNA fragment forGSL1 contained an open reading frame (YDR315C) predicted to encode a 33-kD protein. To test whether GSL1 was required for cell growth, one copy of the gene in a diploid strain was replaced with the kanamycin resistance gene by homologous recombination. Sporulation and dissection of the heterozygous GSL1/gsl1null (Δ) strain resulted in the recovery of four viable spores per tetrad. The gsl1Δ cells grew similarly to wild-type cells at 23°C, but doubling ceased after 10 hours at 37°C.

The gsl2 group was complemented by PLC1, which encodes the yeast PI-specific phospholipase C that cleaves PIP2 to produce diacylglycerol and IP3(14). Cells lacking PLC1 are viable at 23°C, although they grow slowly, and are inviable at 34°C. To elucidate the role of Plc1p in inositol phosphate (IP) production, we measured the amounts of soluble IPs in wild-type and plc1Δcells by steady-state radiolabeling and high-performance liquid chromatography (HPLC) analysis (Fig. 2). Inositol, glycerophosphoinositol (groPI), inositol monophosphate (IP1), as well as a major peak of IP6(representing ∼3% of the free inositol), were detected in extracts of wild-type cells (Fig. 2A). In contrast, plc1Δ cells fail to produce detectable amounts of IP6 (Fig. 2B), demonstrating that Plc1p activity is required for its production.

Figure 2

Requirement of Plc1p for production of IP6. (A and B) HPLC analysis of soluble extracts prepared after steady-state [3H]inositol labeling of (A) wild-type or (B) plc1Δ yeast strains (23). Cells were incubated in complete minimal medium containing [3H]inositol (50 μCi/ml) and harvested during the logarithmic growth phase as described (23). Soluble fractions from approximately 5 × 107 cells were resolved by Partisphere strong-anion exchange (SAX) (4.6 mm by 125 mm) HPLC using a linear gradient from 10 mM to 1.7 M ammonium phosphate (pH 3.5) over 25 min, followed by further elution with 1.7 M ammonium phosphate for 25 min. Peaks for inositol (2 min), groPI (4 min), IP1 (7 to 10 min), and IP6 (45 min) were detected in wild-type extracts. (C) Pulse-chase analysis of cells overexpressing Plc1p. Log phase wild-type cells (1 × 109) harboring a galactose-inducible PLC1plasmid (provided by J. Thorner) were grown in complete minimal medium supplemented with 2% raffinose. Expression of Plc1p was induced for 8 hours by addition of galactose (2%) after which 100 μCi/ml [3H]inositol was added for 4 min. Cells were centrifuged, washed, and resuspended in medium supplemented with 1 mM inositol, but with no [3H]inositol. Samples (2 × 108 cells) were taken at the indicated time points, and amounts of soluble inositol phosphates were measured. Individual IP isomers were assigned on the basis of co-elution with known IP standards and sensitivity or resistance to various inositol polyphosphate phosphatase enzymes.

We used a pulse-chase labeling strategy to further define the pathway of Plc1p-dependent IP6 production in cells overexpressing Plc1p (Fig. 2C). Overproduction of Plc1p resulted in accumulation of multiple inositol polyphosphates. Single major isomers that co-eluted with standards of IP3, inositol 1,4,5,6-tetrakisphosphate (IP4), inositol 1,3,4,5,6-pentakisphosphate (IP5), and IP6 were observed. At the earliest time point (t = 0 min), amounts of IPs were such that IP3 > IP4 > IP5 >>> IP6. As the chase time progressed, the relative amounts changed such that IP3 = IP4 = IP5≥ IP6 (t = 20 min), and at later times IP6 > IP5 ≫ IP4 = IP3 (t = 80 min). In addition, a bisphosphorylate isomer was detected at later chase times (designated IP2x because it eluted after inositol 1,4-bisphosphate). At steady-state, cells overexpressing Plc1p had 25 times more IP6 than wild-type cells, such that the amount of IP6 was similar to that of free inositol (as above). These results indicate that activation of Plc1p results in the production of IP3, which appears to be rapidly and sequentially phosphorylated to produce IP6.

The gsl1 and gsl3 strains also failed to produce IP6 (Fig. 3). Compared to wild-type cells (Fig. 3A), the gsl1Δ mutant had increased amounts of IP3, IP4, and particularly IP5, but no detectable IP6 (Fig. 3B). This mutant had an unidentified IP3 isomer (IP3x) and a compound that appeared to be diphosphoinositol tetrakisphosphate (PP-IP4) because it eluted between IP5 and IP6 (15). Thus, Gsl1p may be either an IP5 kinase or a regulator thereof. In contrast, at 23°C, the gsl3-3 mutant showed a loss of IP5 and IP6, and had increased amounts of IP2x, IP3, and IP4 (Fig. 3C). Shifting the gsl3-3 mutant to 37°C resulted in the disappearance of IP4, the appearance of an unidentified IP4 isomer (IP4x), and increases in the amounts of IP2x and IP3 (Fig. 3D). These results indicate a possible block at the IP4 kinase step with failure to produce IP5 in gsl3-3 cells at 23°C. At 37°C, the gsl3-3 mutation appears to reduce IP3 kinase activity. The gsl3-1 mutant grown at 23°C showed a profile similar to that of the gsl3-3 mutant at 37°C. Thus, Gsl3p may be either a dual-function IP3 or IP4 kinase, or a regulator thereof.

Figure 3

Requirement of GSL1 andGSL3 for production of IP6. Extracts were prepared from steady-state [3H]inositol-labeled cells and were separated by HPLC (Fig. 2). The cells [(A) wild-type, 23°C; (B) gsl1Δ, 23°C; (C)gsl3-3, 23°C; and (D) gsl3-3, 37°C] were labeled over eight doublings at 23°C in complete minimal medium containing 20 μCi of inositol per milliliter and harvested as in Fig. 2. The gsl3-3 cells (D) were temperature-shifted to 37°C for 6 hours before harvest. Individual IP isomers were assigned as in Fig. 2.

To directly test if GSL1 encodes an intrinsic IP5-kinase, we analyzed recombinant Gsl1p for activity. Initially, we purified [3H]-labeled IP5 from radiolabeled gsl1Δ cells as a substrate (Fig. 4A). Incubation with recombinant Gsl1p resulted in a specific conversion of IP5 to IP6. The IP5 isomer that accumulates ingsl1Δ and in Plc1p-overexpressing cells appears to be inositol 1,3,4,5,6-pentakisphosphate, on the basis of its HPLC elution profile, and this isomer appears to be the precursor to IP6in plants, animals, yeast, and slime molds (3, 1619). Recombinant Gsl1p was incubated with [γ-32P]-labeled adenosine triphosphate and pure inositol 1,3,4,5,6-pentakisphosphate. A single [32P]-labeled peak that co-eluted with IP6was observed (Fig. 4B). Therefore, Gsl1p is an IP52-kinase, which we designate Ipk1p (for inositol polyphosphate kinase).

Figure 4

Intrinsic IP5 2-kinase activity of Gsl1p/Ipk1p. The coding sequence of GSL1/IPK1 was fused in frame to glutathione S-transferase (GST), expressed in bacteria, and purified. (A) [3H]IP5substrate assay. The [3H]IP5 was purified from ipk1Δ cells, and a fraction containing 1500 counts per minute was incubated with either 100 ng of GST only (top) or 1 ng of GST-Ipk1p (bottom) in 50 mM HEPES (pH 7.5), 50 mM NaCl, 10 mM MgCl2, and 2.5 mM adenosine triphosphate at 37°C for 30 min. Reactions were resolved by HPLC (Fig. 2). A conversion of IP5 to IP6 of ∼40% was observed in GST-Ipk1p reactions. Conversion was time and dose-dependent. (B) Assays were done as above in 50 mM Hepes (pH 7.5), 50 mM NaCl, 10 mM MgCl2, 20 μM I(1,3,4,5,6)P5 (Matreya, Pleasant Gap, Pennsylvania), and 100,000 cpm [γ-32P]ATP at 37°C for 30 min, and separated by HPLC. Approximately 10% of the original radioactivity was converted to [32P]IP6.

To further examine connections between Gle1p and the IP6pathway, we conducted genetic suppression analysis (as above). Overexpression of PLC1 in a temperature-sensitivegle1-4 mutant rescued growth at 30°C; however rescue was not observed in a gle1-4 mutant at 37°C or in agle1-4 ipk1Δ background. Overexpression ofGLE1 did not rescue the gsl2-6/plc1 mutant and resulted in lethality at all temperatures tested. These results support the functional linkage of GLE1 and PLC1, and suggest that IP6 production is required for the rescue of the gle1 mutant.

To localize the production of IP6 in the cell, protein A–tagged Ipk1p was expressed in ipk1Δ cells and analyzed by indirect immunofluorescence microscopy (Fig. 5A). Staining for Ipk1p was concentrated in the nucleus and in a punctate pattern at the nuclear periphery that is typical of yeast NPCs and NE. The tagged protein was functionally similar to Ipk1p, and nuclear localization was confirmed by subcellular fractionation analysis (20).

Figure 5

Localization of Ipk1p at or near the NPC and NE (20). (A). The intracellular distribution of Ipk1p was determined by indirect immunofluorescence microscopy of yeastipk1Δ cells expressing a protein A–Ipk1p fusion protein (with an NH2-terminal fusion of the five tandem immunoglobulin G–binding domains from Staphylococcus aureusprotein A to full-length Ipk1p, on a LEU2/2 μ plasmid and under the control of the GLE1 promoter). Yeast were fixed for 5 min and prepared as described (22). Cells were probed with a rabbit anti-mouse antibody (Cappel Laboratories; 1:200 dilution) and with a FITC-conjugated goat anti-rabbit antibody (Cappel Laboratories; 1:200). (B) Coincident staining of the nuclei with DAPI. Bar, 5 μm.

Our results indicate a requirement for Plc1p-dependent generation of IP6 to support mRNA export from the nucleus. Each of the three gsl complementation groups correspond to genes that influence IP6 production. Ipk1p encodes an IP52-kinase whose amino acid sequence shows no similarity to other known inositol kinases. In plants, animals, and yeast, the major route of IP6 synthesis occurs via such an IP5 2-kinase (3, 1619). Gsl3p may function as or regulate a dual-function IP3 or IP4 kinase similar to an activity purified from budding and fission yeast (17). Stimuli that activate IP6production may coincidentally enhance mRNA export. IP6accumulates under stress conditions (17), and Gle1p is required for the export of certain heat-shock mRNAs (12). We predict IP6 acts as a positive regulator of Gle1p-mediated mRNA export. IP6 may induce conformational changes in the NPC that facilitate translocation of hnRNP complexes (6). Alternatively, it may facilitate removal of an export inhibitor (8). Precedent exists for direct modulation of protein activity through IP6 binding; the binding of IP6 to mammalian synaptotagmin may alter its interaction with the clathrin assembly protein AP2 and thereby inhibit synaptic vesicle trafficking (21). NPC proteins, hnRNP components, and Gle1p are candidates for IP6-binding proteins in the RNA export pathway.

  • * To whom correspondence should be addressed. E-mail: yorkj{at} (J.D.Y.) or swente{at}


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