PP1/PP2A Phosphatases Are Required for the Second Step of Pre-mRNA Splicing and Target Specific snRNP Proteins  Yongsheng Shi, Bharat Reddy, James L. Manley  Molecular Cell  Volume 23, Issue 6, Pages 819-829 (September 2006) DOI: 10.1016/j.molcel.2006.07.022 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Depletion of PP1/PP2A Phosphatases Blocks the Second Step of Splicing (A) HeLa nuclear extract (NE) was untreated, mock depleted (mock), PP2A depleted (Δ2A), or PP1/PP2A depleted (ΔPPP). Levels of PP1, PP2A, PP4, PP5, PP5, and SR proteins were monitored by western blotting. (B) Splicing activities of these extracts were examined in vitro by using 32P-labeled β-globin pre-mRNA. RNAs were analyzed by denaturing 6% PAGE and autoradiography. Splicing intermediates and products are indicated schematically. The radiointensities of pre-mRNAs, first-step (5′ exon and lariat intermediate), and second-step (mRNA and the lariat) products were quantified by using the Scion Image program. The levels of RNAs in NE were arbitrarily set to 1, and the relative levels of RNAs in other samples were calculated. (C) Splicing was carried out with mock or ΔPPP NE with the AdMLΔAG substrate for 40 or 60 min. RNAs were analyzed as described. The first-step RNAs were quantified by using a phosphorimager. The levels of these RNAs in mock NE were arbitrarily set to 1, and relative RNA levels in ΔPPP were calculated. (D) Spliceosome assembly assays were carried out by using mock or ΔPPP NE. Splicing complexes were resolved on native agarose gel and visualized by autoradiography. Molecular Cell 2006 23, 819-829DOI: (10.1016/j.molcel.2006.07.022) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 PP1 and Certain PP2A Phosphatases Restore Splicing in the Phosphatase-Depleted Extract (A) Increasing amounts of recombinant PP1α catalytic subunit were added to mock or ΔPPP NE, and splicing activity was assayed by using β-globin substrate. (B) Increasing amounts of recombinant PP2A holoenzyme (ABαC) were added to mock or ΔPPP NE, and splicing activity was determined. (C) Mock IP (MK), immunopurified PP1 wild-type (WT), or H125A mutant (MT) was added to mock or ΔPPP NE, and splicing activity was determined. (D) Mock IP (MK), immunopurified PP1, PP4, PP5, or PP6 was added to mock or ΔPPP NE, and splicing activity was determined. Molecular Cell 2006 23, 819-829DOI: (10.1016/j.molcel.2006.07.022) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 PP1/PP2A Phosphatases Are Associated with the Spliceosome and snRNPs (A) Pull-down assays and IPs were carried out with β-globin splicing reaction using control antibody (control Ab), anti-SC35 antibody, anti-PP2A A antibody, or microcystin beads (MC). Recovered RNAs were extracted, resolved by denaturing PAGE, and visualized by autoradiography. (B) Pull-down assays were carried out with NE using control or MC beads. Recovered RNAs were extracted, labeled with pCp, and resolved by denaturing PAGE. Recovered proteins were resolved by SDS-PAGE and subjected to western blotting. RNA and proteins from NE were treated the same way and used for comparison. Molecular Cell 2006 23, 819-829DOI: (10.1016/j.molcel.2006.07.022) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Spliceosomal Substrates for the PP1/PP2A Phosphatases (A) Splicing reactions were carried out with mock or ΔPPP NE in the presence of ATP or ATPγS using β-globin substrate. (B and C) Splicing reactions were carried out in the presence of γ32P-ATP by using AdMLΔAG substrate, and IPs were performed by using control or SC35 antibody (αSC35). RNAs were extracted from the input splicing reaction, IPs (IP), and the supernatants (SN), resolved by denaturing PAGE, and visualized by autoradiography (B). The same samples were treated with RNase A, and released proteins were resolved on SDS-PAGE and visualized by autoradiography (C). Molecular Cell 2006 23, 819-829DOI: (10.1016/j.molcel.2006.07.022) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 Spliceosomal Substrates for the PP1/PP2A Phosphatases (A) NE was incubated with Mg2+ and γ32P-ATP, and snRNPs were IPed with anti-m3G cap antibodies. Proteins were resolved by SDS-PAGE and visualized by silver staining (left) or autoradiography (right). (B) MC pull-down assay or IP with anti-Sm antibody (Y12) was carried out with NE incubated with Mg2+ and γ32P-ATP. Proteins were resolved by SDS-PAGE along with IPed spliceosomes as in Figures 4B and 4C and visualized by autoradiography (left) and western blotting (right). A nonspecific band recognized by SAP155 antibody is marked with an asterisk. (C) Spliceosomes assembled on AdMLΔAG in the presence of γ32P-ATP were IPed as in (B). Spliceosomal proteins were released by RNase A treatment and denatured before being IPed with preimmune serum (control) or antibodies against U5-116 kDa and SAP155. Proteins were resolved by SDS-PAGE and visualized by autoradiography. Molecular Cell 2006 23, 819-829DOI: (10.1016/j.molcel.2006.07.022) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 Protein Dephosphorylation during the Second Step of Splicing (A) Spliceosomes assembled on AdMLΔAG or AdML substrates in the presence of γ32P-ATP were IPed as in Figures 4 and 5, incubated with buffer D (Bfr D) or micrococcal nuclease-treated NE (MNE) under splicing condition for 90 min. RNAs from input reaction (input), supernatant (SN), and IPed spliceosomes before (0) or after incubation with Bfr D (Bfr D 90) or MNE (MNE 90) were extracted and resolved by denaturing PAGE. (B) Proteins were extracted from samples in (A) and resolved by SDS-PAGE and visualized by using a phosphorimager. (C) Protein dephosphorylation observed in (B) was quantitated, by using a phosphorimager, from intensities of individual proteins or all spliceosomal proteins at time 0 (I0) and 90 min (I90) by the following formula: percentage dephosphorylation = (I0 − I90) / I0. Molecular Cell 2006 23, 819-829DOI: (10.1016/j.molcel.2006.07.022) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 7 PP1/PP2A Phosphatases Dephosphorylate U5-116 kDa and SAP155 during the Second Step of Splicing (A) Spliceosomes assembled on a β-globin substrate in the presence of γ32P-ATP were IPed and incubated with buffer D (Bfr D), MNE, micrococcal nuclease-treated ΔPPP extract (ΔPPP∗), or ΔPPP∗ plus recombinant PP1 (ΔPPP∗+PP1) for 90 min. RNAs were extracted from these samples and input reaction, and resolved by denaturing PAGE. A shorter exposure was used for the upper half of the gel to emphasize differences among samples, and the two exposures are delineated by a line. (B) Quantitation of results in (A). To monitor progression of the second step, the ratio between first-step RNAs and the final splicing products was calculated. Results from three independent experiments were represented as mean ± standard deviation. ΔPPP∗+PP1 is labeled as PP1. (C) Proteins from the same samples shown in (A) were extracted, resolved by SDS-PAGE, and visualized by using a phosphorimager. (D) Quantitation of results in (C). Intensities of U5-116 kDa and SAP155 bands in BfrD were arbitrarily set to 1, and the relative phosphorylation levels of these proteins were calculated and shown in the graph. Molecular Cell 2006 23, 819-829DOI: (10.1016/j.molcel.2006.07.022) Copyright © 2006 Elsevier Inc. Terms and Conditions