The type 2C Ser/Thr phosphatase PP2Cgamma is a pre-mRNA splicing factor - PubMed (original) (raw)
The type 2C Ser/Thr phosphatase PP2Cgamma is a pre-mRNA splicing factor
M V Murray et al. Genes Dev. 1999.
Abstract
To identify activities involved in human pre-mRNA splicing, we have developed a procedure to separate HeLa cell nuclear extract into five complementing fractions. An activity called SCF1 was purified from one of these fractions by assaying for reconstitution of splicing in the presence of the remaining four fractions. A component of SCF1 is shown to be PP2Cgamma, a type 2C Ser/Thr phosphatase of previously unknown function. Previous work suggested that dephosphorylation of splicing factors may be important for catalysis after spliceosome assembly, although the identities of the specific phosphatases involved remain unclear. Here we show that human PP2Cgamma is physically associated with the spliceosome in vitro throughout the splicing reaction, but is first required during the early stages of spliceosome assembly for efficient formation of the A complex. The phosphatase activity is required for the splicing function of PP2Cgamma, as an active site mutant does not support spliceosome assembly. The requirement for PP2Cgamma is highly specific, as the closely related phosphatase PP2Calpha cannot substitute for PP2Cgamma. Consistent with a role in splicing, PP2Cgamma localizes to the nucleus in vivo. We conclude that at least one specific dephosphorylation event catalyzed by PP2Cgamma is required for formation of the spliceosome.
Figures
Figure 1
Fractionation of nuclear extract and reconstitution by ammonium sulfate precipitation. Nuclear extract was fractionated into high and low ammonium sulfate fractions, which were tested alone or in combination for pre-mRNA splicing. β-globin pre-mRNA was incubated with the indicated fractions for 2 hr. Three sets of fractionation experiments are shown, representing three different cuts, 20%–40%, 20%–45%, and 20%–50% saturation. Either purified HeLa SR proteins or the corresponding high ammonium sulfate cut was added to each of these fractions. The positions of the pre-mRNA, mRNA, lariat–exon 2, lariat intron, and debranched intron are shown.
Figure 2
Separation of the high ammonium sulfate fraction. (A) CsCl equilibrium density centrifugation of the high ammonium sulfate fraction. Fractions from the gradient were assayed for splicing activity with β-globin pre-mRNA in reactions that also contained the low ammonium sulfate fraction (20%–40%) and SR proteins. The sedimentation of bulk protein and nucleic acid are indicated. (B) Flow chart of SCF1 purification. (C) Fractions from the Poros 20 HQ step were assayed for splicing in combination with the 20%–40% fraction and SR proteins. In this assay, none of the HQ fractions had complementing activity in isolation (data not shown). (D) Coomassie-stained SDS gel of pooled fractions from the flow-through (FT) and the salt eluates of the Poros 20 HQ step.
Figure 3
Hydrophobic interaction chromatography. (A) Column profile. SCF1 from the Sephacryl S-300 chromatography step was loaded onto a Poros 20 PH column equilibrated in high salt, and the proteins were eluted by a reverse salt gradient. The _A_280 and conductivity tracing are shown. The splicing activity detected in B is indicated. (B) Splicing activity with β-globin pre-mRNA. Fractions from the gradient and flow-through were assayed in a reaction that contained 20%–40% fraction, SR proteins, HQ1M, and HQFT fractions. As the concentration of protein in the fractions is very low, activity is weaker than in previous steps. (C) Fractions at or flanking the peak of splicing activity were analyzed on a 10% SDS–polyacrylamide gel stained with a fluorescent protein stain, Sypro Orange (Molecular Probes). (*) Bands that comigrated with splicing activity.
Figure 3
Hydrophobic interaction chromatography. (A) Column profile. SCF1 from the Sephacryl S-300 chromatography step was loaded onto a Poros 20 PH column equilibrated in high salt, and the proteins were eluted by a reverse salt gradient. The _A_280 and conductivity tracing are shown. The splicing activity detected in B is indicated. (B) Splicing activity with β-globin pre-mRNA. Fractions from the gradient and flow-through were assayed in a reaction that contained 20%–40% fraction, SR proteins, HQ1M, and HQFT fractions. As the concentration of protein in the fractions is very low, activity is weaker than in previous steps. (C) Fractions at or flanking the peak of splicing activity were analyzed on a 10% SDS–polyacrylamide gel stained with a fluorescent protein stain, Sypro Orange (Molecular Probes). (*) Bands that comigrated with splicing activity.
Figure 3
Hydrophobic interaction chromatography. (A) Column profile. SCF1 from the Sephacryl S-300 chromatography step was loaded onto a Poros 20 PH column equilibrated in high salt, and the proteins were eluted by a reverse salt gradient. The _A_280 and conductivity tracing are shown. The splicing activity detected in B is indicated. (B) Splicing activity with β-globin pre-mRNA. Fractions from the gradient and flow-through were assayed in a reaction that contained 20%–40% fraction, SR proteins, HQ1M, and HQFT fractions. As the concentration of protein in the fractions is very low, activity is weaker than in previous steps. (C) Fractions at or flanking the peak of splicing activity were analyzed on a 10% SDS–polyacrylamide gel stained with a fluorescent protein stain, Sypro Orange (Molecular Probes). (*) Bands that comigrated with splicing activity.
Figure 4
Homology of PP2Cγ with other 2C Ser/Thr phosphatases. The amino acid sequence of human PP2Cγ (GenBank accession no. Y13936) was compared with those of human PP2Cα (S87759), S. cerevisiae PTC3 (U72346), and an ORF from C. elegans (U00051) by the PILEUP program (GCG) and manual adjustment. Identities are indicated by black shading and similarities by gray shading. PP2Cγ has an acidic domain from residues 117–319, and a similar domain is found in the C. elegans ORF. In this region, homology is not indicated, but the acidic residues are boxed. The crystal structure of PP2Cα showed that six highly conserved amino acids are involved in coordinating two active site metal ions. (•) Five of the six residues involved in metal ion coordination; (♦) the sixth residue, _Asp_496, which was mutated to Ala to make the active site mutant D496A.
Figure 5
Copurification of type 2C phosphatase activity with splicing activity. (A) Fractions from the upper half of a CsCl gradient were assayed for splicing activity with β-globin pre-mRNA in reactions that also contained the low ammonium sulfate fraction (20%–40%) and SR proteins. (B) The same fractions were assayed for type 2C phosphatase activity by incubating with 32P-labeled MBP. (C) Type 2C phosphatase activity in fractions from the HQ column step.
Figure 6
Phosphatase and splicing activity of recombinant PP2Cγ. (A) Coomassie-stained SDS gel of recombinant proteins. Mutant PP2Cγ has an alanine substitution for a highly conserved aspartate at position 496 (D496A). (B) Specific phosphatase activity of recombinant protein with 32P-labeled MBP as a substrate. The D496A mutation results in a nearly inactive form of the phosphatase. (C) rPP2Cγ was assayed for splicing with β-globin pre-mRNA in a reaction that contained the 20%–40% fraction, SR proteins, and HQFT and HQ1M fractions. Lanes 2 and 3 contain 1 and 2 μl, respectively, of the HQ2M fraction, which contains 0.1 U/μl of phosphatase activity (1 unit = 1 nmole/min). rPP2Cγ was added in lane 4 (50 ng, 0.02 unit), lane 5 (100 ng), lane 6 (200 ng), lane 7 (500 ng), lane 8 (1000 ng), and lane 9 (2000 ng). (D) Native gel analysis of spliceosomal complexes formed on β-globin pre-mRNA. The indicated combinations of fractions were incubated for 1 hr under splicing conditions and analyzed by native gel electrophoresis. Reactions contained either 0.4, 1, and 2 μl of the HQ2M fraction or 20, 50, and 100 ng of the indicated recombinant proteins. As a control, the complexes formed in a 30-min incubation with nuclear extract are also shown.
Figure 6
Phosphatase and splicing activity of recombinant PP2Cγ. (A) Coomassie-stained SDS gel of recombinant proteins. Mutant PP2Cγ has an alanine substitution for a highly conserved aspartate at position 496 (D496A). (B) Specific phosphatase activity of recombinant protein with 32P-labeled MBP as a substrate. The D496A mutation results in a nearly inactive form of the phosphatase. (C) rPP2Cγ was assayed for splicing with β-globin pre-mRNA in a reaction that contained the 20%–40% fraction, SR proteins, and HQFT and HQ1M fractions. Lanes 2 and 3 contain 1 and 2 μl, respectively, of the HQ2M fraction, which contains 0.1 U/μl of phosphatase activity (1 unit = 1 nmole/min). rPP2Cγ was added in lane 4 (50 ng, 0.02 unit), lane 5 (100 ng), lane 6 (200 ng), lane 7 (500 ng), lane 8 (1000 ng), and lane 9 (2000 ng). (D) Native gel analysis of spliceosomal complexes formed on β-globin pre-mRNA. The indicated combinations of fractions were incubated for 1 hr under splicing conditions and analyzed by native gel electrophoresis. Reactions contained either 0.4, 1, and 2 μl of the HQ2M fraction or 20, 50, and 100 ng of the indicated recombinant proteins. As a control, the complexes formed in a 30-min incubation with nuclear extract are also shown.
Figure 6
Phosphatase and splicing activity of recombinant PP2Cγ. (A) Coomassie-stained SDS gel of recombinant proteins. Mutant PP2Cγ has an alanine substitution for a highly conserved aspartate at position 496 (D496A). (B) Specific phosphatase activity of recombinant protein with 32P-labeled MBP as a substrate. The D496A mutation results in a nearly inactive form of the phosphatase. (C) rPP2Cγ was assayed for splicing with β-globin pre-mRNA in a reaction that contained the 20%–40% fraction, SR proteins, and HQFT and HQ1M fractions. Lanes 2 and 3 contain 1 and 2 μl, respectively, of the HQ2M fraction, which contains 0.1 U/μl of phosphatase activity (1 unit = 1 nmole/min). rPP2Cγ was added in lane 4 (50 ng, 0.02 unit), lane 5 (100 ng), lane 6 (200 ng), lane 7 (500 ng), lane 8 (1000 ng), and lane 9 (2000 ng). (D) Native gel analysis of spliceosomal complexes formed on β-globin pre-mRNA. The indicated combinations of fractions were incubated for 1 hr under splicing conditions and analyzed by native gel electrophoresis. Reactions contained either 0.4, 1, and 2 μl of the HQ2M fraction or 20, 50, and 100 ng of the indicated recombinant proteins. As a control, the complexes formed in a 30-min incubation with nuclear extract are also shown.
Figure 6
Phosphatase and splicing activity of recombinant PP2Cγ. (A) Coomassie-stained SDS gel of recombinant proteins. Mutant PP2Cγ has an alanine substitution for a highly conserved aspartate at position 496 (D496A). (B) Specific phosphatase activity of recombinant protein with 32P-labeled MBP as a substrate. The D496A mutation results in a nearly inactive form of the phosphatase. (C) rPP2Cγ was assayed for splicing with β-globin pre-mRNA in a reaction that contained the 20%–40% fraction, SR proteins, and HQFT and HQ1M fractions. Lanes 2 and 3 contain 1 and 2 μl, respectively, of the HQ2M fraction, which contains 0.1 U/μl of phosphatase activity (1 unit = 1 nmole/min). rPP2Cγ was added in lane 4 (50 ng, 0.02 unit), lane 5 (100 ng), lane 6 (200 ng), lane 7 (500 ng), lane 8 (1000 ng), and lane 9 (2000 ng). (D) Native gel analysis of spliceosomal complexes formed on β-globin pre-mRNA. The indicated combinations of fractions were incubated for 1 hr under splicing conditions and analyzed by native gel electrophoresis. Reactions contained either 0.4, 1, and 2 μl of the HQ2M fraction or 20, 50, and 100 ng of the indicated recombinant proteins. As a control, the complexes formed in a 30-min incubation with nuclear extract are also shown.
Figure 7
Association of PP2Cγ with the spliceosome and intracellular localization. (A) Western blot of HeLa nuclear extract probed with three different anti-PP2Cγ monoclonal antibodies. (B) Immunoprecipitations of β-globin splicing reactions after a 1-hr incubation. Radiolabeled RNA was recovered from the immunoprecipitates and analyzed by denaturing PAGE. The amount of input RNA shown is equivalent to 10% of the amount used for each immunoprecipitation. Anti-Sm (mAb Y12) and anti-trimethyl guanosine (mAb K121) immunoprecipitations were included as positive controls, and an anti-maltose-binding protein (α-MalE) antibody (mAb105) served as a negative control. (C) The intracellular localization of PP2Cγ in HeLa cells was determined by use of an anti-PP2Cγ monoclonal antibody (7-53) and FITC-conjugated secondary antibody. SC35 localization is included as a positive control and as a marker for the speckle region of the nucleus.
References
- Berglund JA, Chua K, Abovich N, Reed R, Rosbash M. The splicing factor BBP interacts specifically with the pre-mRNA branchpoint sequence UACUAAC. Cell. 1997;89:781–787. - PubMed
- Brosi R, Hauri HP, Krämer A. Separation of splicing factor SF3 into two components and purification of SF3a activity. J Biol Chem. 1993;268:17640–17646. - PubMed
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