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J. Biol. Chem., Vol. 277, Issue 22, 19855-19860, May 31, 2002

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The Protein Phosphatase-1 Regulator NIPP1 Is Also a Splicing Factor Involved in a Late Step of Spliceosome Assembly^*

Monique Beullens and Mathieu Bollen^§

From the Afdeling Biochemie, Faculteit Geneeskunde, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium

Received for publication, January 28, 2002, and in revised form, March 14, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

NIPP1 is a ubiquitous regulator of protein phosphatase-1 (PP1) and is targeted to the splicing factor storage sites (speckles) in the nucleus by its forkhead-associated domain. We show here that NIPP1 is also a component of the spliceosomes in HeLa cell-splicing extracts and that the interaction with the spliceosomes requires a functional forkhead-associated domain. The in vitro splicing of -globin pre-mRNA was not affected by exogenous wild type NIPP1 but was blocked by mutants that lacked residues 225-329. The inhibition by these dominant negative mutants resulted from a block in a late phase of spliceosome assembly, i.e. at the transition between the B-complex and the C-complex. Site-directed mutagenesis furthermore showed that this spliceosomal function of NIPP1 was unrelated to its ability to bind PP1 or RNA. Our data suggest that NIPP1 can function independently as a splicing factor and a phosphatase regulator.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Nuclear inhibitor of protein phosphatase-1 (NIPP1)¹ is one of four ancient regulators of protein phosphatase-1 (PP1) and is ubiquitously expressed in multicellular eukaryotes (1). In vitro, NIPP1 acts as an inhibitor of PP1 toward a variety of substrates, but the inhibitory potency is controlled by multisite phosphorylation and by the binding of RNA (2, 3). NIPP1 is targeted to nuclear storage sites for splicing factors, known as speckles or splicing factor compartments (4, 5). The association of NIPP1 with the speckles is mediated by its forkhead-associated (FHA) domain, which specifically interacts with phosphorylated forms of the essential splicing factors CDC5L (6) and SAP155.² Because CDC5L and SAP155 are also in vitro substrates for PP1, it has been speculated that NIPP1 may target these splicing factors for dephosphorylation by associated PP1. Such a function for PP1 is potentially important because toxins that inhibit protein serine/threonine phosphatases, including PP1, inhibit pre-mRNA splicing (7).

Pre-mRNA splicing is catalyzed by spliceosomes, consisting of small nuclear ribonucleoprotein particles, i.e. the U1, U2, U4, U5, and U6 snRNPs and a large number of non-snRNP-associated splicing factors (8). Spliceosome assembly involves the ordered recruitment of snRNPs and splicing factors as well as multiple rearrangements between spliceosomal components. The first step is the ATP-independent formation of the E-complex and includes the binding of the U1 snRNA to the 5'-splice site and of non-snRNP splicing factors to the polypyrimidine tract and the 3'-splice site. The U2 snRNP first binds loosely to the pre-mRNA in the E-complex, but during the transition to the A-complex, this snRNP is firmly bound to the intronic branch-point sequence in an ATP-dependent manner. The tight association of the U4/U6.U5 tri-snRNP results in the formation of the B-complex, which is converted to the catalytically active C-complex by intraspliceosomal rearrangements. These rearrangements include the expulsion of the U1 and U4 snRNPs and the base pairing of the U2 and U6 snRNAs.

NIPP1 is tightly associated with splicing factors in the speckles (4, 5), but it has not yet been investigated whether NIPP1 is also a component of the spliceosomes and contributes to the control of pre-mRNA splicing. We report here that NIPP1 is indeed recruited to the spliceosomes in HeLa cell nuclear extracts and has an essential role in a late step of spliceosome assembly. Unexpectedly, this function of NIPP1 appears to be unrelated to its ability to regulate PP1.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Rabbit polyclonal antibodies against synthetic peptides of Xenopus laevis SAP155 (¹⁰⁹KIANREDEYKQQRRKMI¹²⁵), human CDC5L (⁷⁸⁸YADLLLEKETLKSKF⁸⁰²), and human NIPP1 (³⁴¹PGKKPTPSLLI³⁵¹) coupled to keyhole limpet hemocyanin were affinity-purified on the bovine serum albumin-coupled peptides linked to CNBr-activated Sepharose 4B (Amersham Biosciences). HeLa cell nuclear extracts were obtained from the Computer Cell Culture Center. Okadaic acid and microcystin LR were purchased from Calbiochem and Protein A-TSK-Sepharose^TM was obtained from Affiland. Phosphorylase b was purified from rabbit skeletal muscle and was phosphorylated in the presence of [-³²P]ATP by purified phosphorylase kinase (9).

Preparation of Recombinant Proteins-- Polyhistidine-tagged NIPP1 fragments and NIPP1 mutants were prepared as described previously (3, 6). Inhibitor-2 was purified by chromatography on blue Sepharose of heat-treated lysates of BL21(DE3) cells that had been transformed with the pET8d-inhibitor 2 plasmid (10).

Splicing Assays-- A capped -globin pre-mRNA fragment, comprising exon 1 through the BamHI site in exon 2, was synthesized in the presence of [-³²P]GTP. This primary transcript was used as a substrate for splicing in HeLa cell nuclear extracts (11). The splicing mixture contained (final concentrations) nuclear extract (20%), 20 fmol of pre-mRNA (in 15 µl of assay mixture), 0.5 mM ATP, 20 mM creatine phosphate, 3.2 mM MgCl₂, 32 mM Hepes, 60 mM KCl, 0.12 mM EDTA, 12% glycerol, 0.6 mM dithiothreitol, and 2.6% polyvinyl alcohol. The splicing products were separated by denaturing PAGE (9%) and visualized by autoradiography.

Immunoprecipitations during the splicing reactions were done as described by Murray et al. (12), except that (de)phosphorylation reactions during the immunoprecipitation were prevented by the addition of 8 mM EDTA and 1 µM microcystin. After the last wash step the ³²P in the pellet was determined (Fig. 1A), and the pelleted RNA was phenol-extracted and subjected to denaturing PAGE (Fig. 1B).

Electrophoretic Analysis of Splicing Complexes-- At the indicated times during splicing of -globin pre-mRNA in HeLa cell nuclear extracts, aliquots were diluted 1.4-fold by the addition of heparin (1 mg/ml finally) and glycerol (10% finally) and stored on ice. After the experiment, the samples were loaded on a native, composite gel that contained 3.5% polyacrylamide, 0.5% agarose, 10% glycerol, 3 mM Tris-base, 25 mM boric acid, and 0.3 mM EDTA. The running buffer contained 3 mM Tris-base, 25 mM boric acid, and 0.3 mM EDTA. The gels (8 × 7.5 × 0.075 cm) were prerun for 60 min at 60 V and run for 3 h at 175 V with cooling.

Protein Phosphatase Assays-- The phosphorylase phosphatase activities were determined as described before (9) except that the liberated ³²P_i was extracted as a phosphomolybdate complex in isobutanol/toluol (13). These assays were done in identical conditions as the splicing assays (see above) except that phosphorylase a was added to the splicing extracts instead of -globin pre-mRNA.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

NIPP1 Is a Component of the Spliceosomes-- To determine whether NIPP1 is physically associated with the spliceosomes, we performed immunoprecipitations from in vitro splicing reactions at various times after the addition of ³²P-labeled -globin pre-mRNA. The fraction of labeled RNA that was precipitated by the NIPP1 antibodies changed with time but was maximal at 20 min when it amounted to about 4% of the total input (Fig. 1A). Immunoprecipitation of the established splicing factors SAP155 (Fig. 1A) and CDC5L (not shown) at this time point resulted in the co-precipitation of 10 and 1% of the added RNA, respectively. Importantly, only very low levels of RNA were precipitated when no primary antibodies were added (Fig. 1A). Also, very little RNA was precipitated with the NIPP1 antibodies when spliceosome assembly was blocked at the transition between the E-complex and the A-complex by the omission of ATP (0 min in Fig. 1A). This finding strongly suggested that NIPP1 did not bind directly to the -globin pre-mRNA and that NIPP1 was recruited by the A-complex at the earliest. By contrast, the SAP155 antibodies already co-precipitated a considerable fraction of the RNA before the addition of ATP. This finding is in accordance with the report that the U2 snRNP, which includes SAP155, is first recruited by the E-complex (14).

View larger version (36K): [in this window] [in a new window]   Fig. 1.   NIPP1 is a component of the spliceosomes. A shows the co-immunoprecipitation of 32P-labeled -globin pre-mRNA and NIPP1 (left panel) or SAP155 (right panel) from in vitro splicing reactions. The immunoprecipitations (IP) were done in control conditions (), after the addition of 10 µM NIPP1-(1-351)(), or in the presence of 10 µM NIPP1-(1-142) (). The open symbols represent the corresponding control immunoprecipitations, i.e. without addition of the primary antibodies. For the zero time point, samples were taken after a preincubation for 15 min at 4 °C but before the addition of ATP (0.5 mM), creatine phosphate (20 mM), and MgCl2 (3.2 mM). B, denaturing PAGE (9%) of the phenol-extracted RNA that was co-immunoprecipitated with NIPP1 or SAP155 at 80 min after the addition of ATP. The splicing substrates and products that were visualized by autoradiography are (from top to bottom) the lariat-exon 2, pre-mRNA, mRNA, the lariat, and exon 1, and these RNA species are identified schematically at the left.

To further explore the specificity of interaction of NIPP1 with the spliceosomes, we have examined how the addition of recombinant NIPP1 or its splicing factor binding FHA domain (residues 1-142) interfered with the precipitation of RNA by NIPP1 or SAP155 antibodies (Fig. 1A). The amount of RNA that co-immunoprecipitated with NIPP1 was doubled by the addition of 10 µM recombinant NIPP1, indicating that not all spliceosomal binding sites had been occupied by the endogenous NIPP1. On the other hand, the addition of 10 µM FHA domain of NIPP1 nearly completely abolished the co-precipitation of full-length NIPP1 and RNA. This effect was not detected (not shown) with an FHA domain that was mutated (S68A, R69A, V70A, H71A) in its phosphate-binding loop (6). Because this mutation also abolished the ability of the FHA domain to interact with phosphorylated CDC5L (6) and SAP155,² these data suggested that the FHA domain of NIPP1 can compete with the endogenous NIPP1 for binding to the spliceosomes via phosphorylated modules. Unexpectedly, the FHA domain also decreased the recruitment of SAP155 to the spliceosomes, in particular after the addition of ATP (Fig. 1A). Because SAP155 and the FHA domain of NIPP1 can directly interact with each other,² this result can possibly be explained by depletion of the pool of SAP155 that is available for uptake in the spliceosomes. An alternative explanation is that full-length NIPP1 is required for keeping SAP155 tightly associated with the spliceosomes (see "Discussion").

We have further analyzed the RNA that co-immunoprecipitated with NIPP1 and SAP155 by denaturing PAGE (Fig. 1B). After an incubation for 80 min under splicing conditions, immunoprecipitation of either of these proteins resulted in a co-precipitation of pre-mRNA, splicing intermediates (exon 1, lariat-exon 2) as well as splicing products (mRNA, lariat), which is further proof of the association of these proteins with functional spliceosomes. Fig. 1B also shows that the addition of a large excess of NIPP1 did not affect the splicing of -globin pre-mRNA, while the addition of the FHA domain blocked splicing completely.

NIPP1 Is Involved in Spliceosome Assembly-- Because the FHA domain (NIPP1-(1-142)) competed with the endogenous NIPP1 for binding to the spliceosomes (Fig. 1A) and also blocked in vitro splicing (Fig. 1B), this suggested that the C-terminal two-thirds of NIPP1 were required for in vitro splicing. To further delineate the essential fragment, we have tested the effects of various NIPP1 mutants on -globin pre-mRNA splicing (Fig. 2). These experiments revealed that a much larger fragment of NIPP1 (NIPP1-(1-224)) also behaved as a dominant negative mutant and blocked splicing completely. The splicing inhibition by both NIPP1-(1-142) and NIPP1-(1-224) was lost by mutation of the phosphate-binding loop in the FHA domain, indicating that the effects of these NIPP1 fragments were dependent on the presence of a functional FHA domain. Accordingly, NIPP1 fragments (NIPP1-(143-351) and NIPP1-(143-224)) that lacked the FHA domain had no effect on pre-mRNA splicing.

View larger version (78K): [in this window] [in a new window]   Fig. 2.   Effect of NIPP1 mutants on in vitro splicing. Splicing of -globin pre-mRNA in nuclear extracts was carried out in the absence (control) or presence of the indicated NIPP1 mutants. The "FHA mutant" refers to a combination of the mutations S68A, R69A, V70A, and H71A. After 120 min, the RNA was phenol-extracted and subjected to denaturing PAGE (9%). The figure shows an autoradiogram of the gel.

Collectively, the previous data indicated that NIPP1 is an essential spliceosomal component and that both the FHA domain (residues 1-142) and the C-terminal third (residues 225-351) of NIPP1 are required for its function in pre-mRNA splicing in nuclear extracts. The last 22 residues of NIPP1 constitute a binding site for both RNA (15) and PP1 (3). However, a mutant that lacked these residues was not inhibitory for splicing (Fig. 2), which confined the C-terminal fragment of NIPP1 that is essential for splicing to residues 225-329. Also, full-length NIPP1 (not shown) or NIPP1-(1-329) (Fig. 2) did not become inhibitory to splicing after mutation (V201A, F203A) of a PP1-binding motif in the central domain of NIPP1. On the other hand, the latter mutant, like the FHA domain (Fig. 1A), inhibited the co-precipitation of wild type NIPP1 and RNA (not shown). Because the V201A + F203A mutant of NIPP1-(1-329) can bind neither PP1 (3) nor RNA (15), these results indicated that the function of NIPP1 in splicing was unrelated to its ability to interact with PP1 or RNA.

The fragments of NIPP1 that blocked splicing did not cause an accumulation of splicing intermediates (Fig. 2), suggesting that they interfered with spliceosome assembly rather than with the splicing process itself. We have therefore used native gel electrophoresis to separate the spliceosomal A-, B-, and C-complexes and to study the effect of the inhibitory NIPP1 fragments on the accumulation of these complexes. In Fig. 3 it is shown that the C-complex was no longer formed in the presence of 10 µM of the FHA domain. Moreover, in the latter condition there appeared to be an accumulation of the B-complex as expected from a block at the B  C transition. Similar data were obtained with NIPP1-(1-224) (not shown).

View larger version (82K): [in this window] [in a new window]   Fig. 3.   NIPP1-(1-142) inhibits the assembly of the spliceosomal C-complex. -globin pre-mRNA was incubated under standard splicing conditions in the absence (control) or presence of 10 µM NIPP1-(1-142). The splicing reactions were arrested at the indicated time points by the addition of heparin, and the spliceosomal complexes were separated by native gel electrophoresis. Radioactively labeled RNA was detected by autoradiography. The positions of the A-, B-, and C-complexes are indicated. H refers to unspecific pre-RNP complexes.

PP1 Is Not Required for in Vitro Splicing-- In view of a report that hinted at an essential role for PP1 in constitutive splicing in vitro (7), it was surprising that NIPP1, a potent inhibitor of PP1, did not affect splicing of -globin pre-mRNA (Figs. 2 and 4A). It was therefore important to examine whether NIPP1 was inhibitory to PP1 in nuclear extracts under conditions that were identical to those that were used for pre-mRNA splicing (see "Experimental Procedures"). As a protein phosphatase substrate we used glycogen phosphorylase a, a well known substrate for PP1 and PP2A. The phosphorylase phosphatase activity in splicing extracts was inhibited for about 90% by saturating concentrations of NIPP1 (Fig. 4B), in accordance with the previous demonstration that showed that nearly all the phosphorylase phosphatase activity in nuclear extracts stems from PP1 (9). The remainder of the activity was most likely derived from PP2A because this activity was blocked by low concentrations of okadaic acid (50 nM in Fig. 4B), which were only marginally inhibitory for PP1 in splicing extracts (Fig. 5B). Similar data on splicing and phosphatase activities were obtained with inhibitor-2, a structurally unrelated inhibitor of PP1 (Fig. 4). These data demonstrated that PP1 activity was not required for in vitro splicing of the -globin transcript.

View larger version (34K): [in this window] [in a new window]   Fig. 4.   Inhibition of PP1 does not affect splicing. A, the in vitro splicing of labeled -globin pre-mRNA was carried out for 2 h at 30 °C in the absence (control) or presence of the indicated concentrations of NIPP1 and inhibitor 2 (I2). The splice products were separated by denaturing PAGE and visualized by autoradiography. B, in the same splicing extracts the phosphorylase phosphatase activity was measured. In one assay, the phosphatase activity was assayed in the presence of 10 µM NIPP1 plus 50 nM okadaic acid. The results are shown as means ± S.E. (n = 4).

View larger version (41K): [in this window] [in a new window]   Fig. 5.   Splicing inhibition by okadaic acid is not mediated by PP1. A, splicing of -globin pre-mRNA was carried out for 30 or 120 min at 30 °C in the presence of the indicated concentrations of okadaic acid. The progression of splicing was followed by denaturing PAGE of the phenol-extracted RNA. B shows the phosphorylase phosphatase activity in the same extracts represented as means ± S.E. (n = 4).

The evidence that implicated PP1 in constitutive in vitro splicing was largely based on the use of toxins that differentially inhibit various types of protein serine/threonine phosphatases (7). For example, low concentrations of okadaic acid were reported to inhibit the second step of splicing catalysis while higher concentrations of this toxin also blocked the first step. Because PP1 is relatively insensitive to inhibition by okadaic acid, these data were taken as evidence for a role of PP1 in the first step of splicing. However, in these studies the phosphatase and splicing assays were done at hugely different dilutions of the nuclear extracts and with very different assay mixtures. We have therefore reevaluated the effects of okadaic acid on pre-mRNA splicing and on PP1 activity in identical circumstances except for the use of a different substrate (pre-mRNA versus phosphorylase a). Under the adopted assay conditions okadaic acid slowed down pre-mRNA splicing but did not block the process entirely (Fig. 5A). Indeed, while okadaic acid prevented the accumulation of mRNA at an earlier time point (30 min), this was no longer apparent at a later time point (120 min). On the other hand, okadaic acid clearly caused an accumulation of splicing intermediates at 120 min. These findings suggested that one or more okadaic acid-sensitive protein phosphatases determined the kinetics of the second catalytic step of splicing but did not affect the first step of splicing. At variance with the data of Mermoud et al. (7), we found that the effects of okadaic acid on splicing were maximal at concentrations between 40 and 200 nM (Fig. 5A), and these concentrations were only partially inhibitory for PP1 in the extracts (Fig. 5B). Conversely, the nearly complete inhibition of PP1 by high concentrations of okadaic acid (1-5 µM) did not reproducibly cause an additional splicing deficiency. Thus, the splicing effects of okadaic acid were not correlated with an inhibition of PP1.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

NIPP1 Is a Novel Component of the Spliceosomes-- Spliceosomes are highly complex and dynamic molecular machines. They consist of up to five snRNAs and 100 different polypeptides (16). The present data show that NIPP1 is one of the spliceosomal components in nuclear extracts. NIPP1 was recruited during or shortly after the ATP-dependent formation of the A-complex (Fig. 1A) and remained associated with the spliceosomes throughout the splicing process, as indicated by its association with complexes that contained pre-mRNA, splicing intermediates, and splicing products (Fig. 1B). The recruitment of NIPP1 to the spliceosomes was antagonized by the addition of the FHA domain (Fig. 1A), but this effect was not seen when the FHA domain was mutated in its phosphate-binding loop. This indicates that the association of NIPP1 with the spliceosomes is mediated by the binding of the FHA domain to a phosphoprotein(s). It remains to be investigated whether NIPP1 is targeted to the spliceosomes by the same proteins (CDC5L and SAP155) that also mediate its association with the speckles (6).² The likely phosphorylation-dependence of the association of NIPP1 with the spliceosomes also explains why NIPP1 has hitherto not been recognized as a splicing factor. Indeed, the described purification procedures of spliceosomal complexes do not include the use of inhibitors of protein phosphatases (17, 18) and are therefore expected to result in the loss of proteins that interact through phosphopeptide modules.

The Role of NIPP1 in Spliceosome Assembly-- NIPP1-(1-142) (Fig. 1A) and NIPP1-(1-224) (not shown) displaced endogenous NIPP1 from the spliceosomes and blocked the transition between the spliceosomal B- and C-complexes (Fig. 3). Because NIPP1-(1-329) did not display such effects, these data suggest that residues 225-329 are somehow involved in the rearrangements between spliceosomal components that accompany the conversion of the B-complex into the C-complex. We speculate that residues 225-329 of NIPP1 constitute an interaction site for a spliceosomal component and that this interaction is essential for the B  C transition. It is possible that this C-terminal interactor of NIPP1 is SAP155, which also interacts with the FHA domain of NIPP1.² An interaction between SAP155 and the C-terminal domain of NIPP1 would explain why the dominant negative mutants of NIPP1 hampered the tight association of SAP155 with the spliceosomes (Fig. 1A).

Although the amount of RNA that co-precipitated with NIPP1 was doubled by the addition of recombinant NIPP1 (Fig. 1), this did not measurably stimulate splicing catalysis (Fig. 2). This indicates that not all complexes that contained NIPP1 and RNA were converted into mature spliceosomes, probably because other splicing factors besides NIPP1 were also present at limiting concentrations.

An unexpected finding was that the function of NIPP1 in spliceosome assembly appeared unrelated to its ability to interact with PP1 (Fig. 2). Our results are at variance with those of Trinkle-Mulcahy et al. (4), who reported that NIPP1 became inhibitory to in vitro splicing following mutation of the PP1-binding RVXF-motif in the central domain of NIPP1. We did not observe any effect of this mutation on in vitro splicing (Fig. 2) in accordance with our failure to detect a role for PP1 in splicing (Fig. 5). It should also be pointed out that the mutation of the RVXF-motif is not sufficient to abolish the interaction between NIPP1 and PP1 because NIPP1 also harbors a PP1 interaction domain in its C-terminal residues (3).

The Role of Protein Phosphatases in Pre-mRNA Splicing-- Mammalian genomes contain 100-200 genes that encode protein phosphatases (19). These enzymes have been classified into four families based on their substrate specificity and structural conservation. All protein phosphatase families are represented in the nucleus. However, members of the family of protein tyrosine phosphatases do not appear to be essential for in vitro splicing because their inhibition by 1 mM vanadate did not affect the splicing of -globin (not shown). Neither was splicing affected by 120 mM KCl (not shown), which inhibits the protein serine/threonine phosphatase FCP1, the only known member of the FCP family (20). On the other hand, the Mg²⁺-dependent protein serine/threonine phosphatase PP2C, a member of the PPM family, has been identified as a splicing factor that is involved in the assembly of the A-complex (12) (Fig. 6). The large PPP family of protein serine/threonine protein phosphatases comprises the structurally related subfamilies PP1, PP2A (including PP4 and PP6), PP2B (calcineurin), and PP5, and these phosphatases are inhibited by toxins such as okadaic acid and microcystin that also inhibit in vitro splicing (7). We found that the specific inhibition of PP2B by 1 mM EGTA (not shown) and of PP1 by NIPP1 or inhibitor-2 (Fig. 4) did not affect in vitro splicing. This suggests that the inhibition of splicing by the toxins most likely stems from their effects on PP5 and/or PP2A (Fig. 6).

View larger version (36K): [in this window] [in a new window]   Fig. 6.   The role of protein phosphatases in pre-mRNA splicing. The figure shows where protein phosphatases and NIPP1 contribute to spliceosomal assembly, splicing catalysis, spliceosome disassembly, and the shuttling of splicing factors between the splicing factor compartments (SFCs) and the spliceosomes. Potential substrates of protein phosphatases are boxed.

In accordance with previous studies (7), we noted that microcystin and okadaic acid inhibited splicing catalysis but did not affect the accumulation of the spliceosomal A-, B-, and C-complexes (not shown). A remarkable difference in the effects of these toxins was that microcystin blocked both step of catalysis (Ref. 7, not shown), while okadaic acid only inhibited the second step (Fig. 5). Because PP1 is clearly not implicated in pre-mRNA splicing in vitro (Figs. 4 and 5), these data are difficult to explain by different sensitivities of known protein phosphatases to these toxins. Perhaps, microcystin can inhibit a protein phosphatase that is buried inside the spliceosomal complex and that can not be accessed by okadaic acid due to its hydrophobic nature. Alternatively, the first step of splicing is controlled by an hitherto unknown protein phosphatase that is inhibited by microcystin but not by okadaic acid.

There is currently only limited information available on the spliceosomal substrates of protein phosphatases (Fig. 6). An obvious candidate is the splicing factor SF1, which inhibits the assembly of the A-complex in its phosphorylated form (21). Phosphorylation of the splicing factor SF2/ASF stimulates its interaction with the U1-snRNP-associated U1-70K phosphoprotein and thereby facilitates the initial association of the U1 snRNP with the 5'-splice site (22). Both the SF2/ASF and the U1-70K protein have to be dephosphorylated at a later step, before the first step of catalysis. SAP155 is known to be phosphorylated shortly before or during the first step of splicing, but the function of this phosphorylation and the timing of dephosphorylation has not yet been explored (23).

Although protein tyrosine phosphatases, FCP1, PP2B, and PP1 do not appear to be rate-limiting for in vitro splicing, we do not rule out their involvement in spliceosome disassembly, in the shuttling of splicing factors to the speckles, or in the control of alternative splicing. As a matter of fact, PP1 has been suggested to modulate alternative 5'-splice site selection in nuclear extracts (24). PP1 has also been implicated in the dephosphorylation of splicing factors of the SR family (25), necessary for their shuttling to the speckles after splicing catalysis (Fig. 6). The dephosphorylation of other splicing factors (e.g. SAP155 or CDC5L) by PP1 may also be required for spliceosome disassembly or for the subsequent relocalization of these splicing factors (6).² The regulatory subunit that targets these splicing factors for dephosphorylation by PP1 has not yet been identified, but NIPP1 seems an obvious candidate. Thus, NIPP1 could turn out to be a bifunctional protein, i.e. it has a PP1-independent function in spliceosome assembly, and after splicing catalysis, NIPP1 is possibly involved in the regeneration of splicing-competent splicing factors through their dephosphorylation by NIPP1-associated PP1 (Fig. 6).

	ACKNOWLEDGEMENTS

We thank Prof. W. Stalmans for his continuous support and for commenting critically on the manuscript. An Boudrez affinity-purified the SAP155 and CDC5L antibodies and Nicole Sente provided expert technical assistance.

	FOOTNOTES

^* This work was supported by the Fund for Scientific Research-Flanders Grant G.0374.01 and by a Flemish Concerted Research Action.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Postdoctoral fellow of the National Fund for Scientific Research-Flanders.

^§ To whom correspondence should be addressed: Afdeling Biochemie, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. Tel.: 32-16-34-57-01; Fax: 32-16-34-59-95; E-mail: Mathieu.Bollen@med.kuleuven.ac.be.

Published, JBC Papers in Press, March 21, 2002, DOI 10.1074/jbc.M200847200

² A. Boudrez, M. Beullens, S. Keppens, E. Waelkens, W. Stalmans, and M. Bollen, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: NIPP1, nuclear inhibitor of protein phosphatase-1; PP1, protein phosphatase-1; FHA, forkhead-associated; snRNP, small nuclear ribonucleoprotein.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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