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• W2037847135 abstract "Protein kinase CK2 regulates RNA polymerase III transcription of human U6 small nuclear RNA (snRNA) genes both negatively and positively depending upon whether the general transcription machinery or RNA polymerase III is preferentially phosphorylated. Human U1 snRNA genes share similar promoter architectures as that of U6 genes but are transcribed by RNA polymerase II. Herein, we report that CK2 inhibits U1 snRNA gene transcription by RNA polymerase II. Decreased levels of endogenous CK2 correlates with increased U1 expression, whereas CK2 associates with U1 gene promoters, indicating that it plays a direct role in U1 gene regulation. CK2 phosphorylates the general transcription factor small nuclear RNA-activating protein complex (SNAPC) that is required for both RNA polymerase II and III transcription, and SNAPC phosphorylation inhibits binding to snRNA gene promoters. However, restricted promoter access by phosphorylated SNAPC can be overcome by cooperative interactions with TATA-box-binding protein at a U6 promoter but not at a U1 promoter. Thus, CK2 may have the capacity to differentially regulate U1 and U6 transcription even though SNAPC is universally utilized for human snRNA gene transcription. Protein kinase CK2 regulates RNA polymerase III transcription of human U6 small nuclear RNA (snRNA) genes both negatively and positively depending upon whether the general transcription machinery or RNA polymerase III is preferentially phosphorylated. Human U1 snRNA genes share similar promoter architectures as that of U6 genes but are transcribed by RNA polymerase II. Herein, we report that CK2 inhibits U1 snRNA gene transcription by RNA polymerase II. Decreased levels of endogenous CK2 correlates with increased U1 expression, whereas CK2 associates with U1 gene promoters, indicating that it plays a direct role in U1 gene regulation. CK2 phosphorylates the general transcription factor small nuclear RNA-activating protein complex (SNAPC) that is required for both RNA polymerase II and III transcription, and SNAPC phosphorylation inhibits binding to snRNA gene promoters. However, restricted promoter access by phosphorylated SNAPC can be overcome by cooperative interactions with TATA-box-binding protein at a U6 promoter but not at a U1 promoter. Thus, CK2 may have the capacity to differentially regulate U1 and U6 transcription even though SNAPC is universally utilized for human snRNA gene transcription. Protein kinase CK2 is an important regulator of cellular growth (1Meggio F. Pinna L.A. FASEB J. 2003; 17: 349-368Crossref PubMed Scopus (1118) Google Scholar, 2Pinna L.A. J. Cell Sci. 2002; 115: 3873-3878Crossref PubMed Scopus (412) Google Scholar, 3Guerra B. Issinger O.G. Electrophoresis. 1999; 20: 391-408Crossref PubMed Scopus (367) Google Scholar, 4Pinna L.A. Meggio F. Prog. Cell Cycle Res. 1997; 3: 77-97Crossref PubMed Scopus (315) Google Scholar), and abnormal CK2 activity may contribute to tumor progression (5Tawfic S. Yu S. Wang H. Faust R. Davis A. Ahmed K. Histol. Histopathol. 2001; 16: 573-582PubMed Google Scholar). CK2 is a tetrameric enzyme composed of two catalytic subunits, α and α′, and two copies of the regulatory β subunit (6Niefind K. Guerra B. Ermakowa I. Issinger O.G. EMBO J. 2001; 20: 5320-5331Crossref PubMed Scopus (336) Google Scholar). One role for CK2 is to function as a regulatory protein that controls gene transcription. For example, general RNA synthesis in yeast is impaired when a temperature-sensitive mutant of the CK2 α′ subunit is shifted to a restrictive temperature (7Hanna D.E. Rethinaswamy A. Glover C.V. J. Biol. Chem. 1995; 270: 25905-25914Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). This decline in total RNA synthesis also suggests that expression of highly transcribed genes encoding ribosomal (r), transfer (t), and small nuclear RNA (snRNA) 1The abbreviations used are: snRNA, small nuclear RNA; TBP, TATA-box-binding protein; SNAPc, small nuclear RNA-activating protein complex; PTF, proximal sequence element transcription factor; PSE, proximal sequence element; RNAi, RNA interference; RT, reverse transcription; GST, glutathione S-transferase; DRB, d-ribofuranosylbenzimidazole; HA, hemagglutinin. is sensitive to levels of functional CK2. In yeast CK2 is important for active RNA polymerase III transcription (8Hockman D.J. Schultz M.C. Mol. Cell. Biol. 1996; 16: 892-898Crossref PubMed Scopus (44) Google Scholar) and yet, paradoxically, CK2 has been proposed to be the terminal effector in a DNA damage response pathway that represses RNA polymerase III transcription (9Ghavidel A. Schultz M.C. Cell. 2001; 106: 575-584Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). In humans, CK2 exhibits differential effects on gene transcription during the cell cycle. During mitosis, CK2 inhibits RNA polymerase III transcription, whereas at other stages CK2 can stimulate transcription (10Hu P. Samudre K. Wu S. Sun Y. Hernandez N. Mol. Cell. 2004; 16: 81-92Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The nature of the regulation is dictated by CK2 target selection. One key target for CK2 is the general transcription factor TFIIIB (11Ghavidel A. Schultz M.C. Genes Dev. 1997; 11: 2780-2789Crossref PubMed Scopus (49) Google Scholar, 12Johnston I.M. Allison S.J. Morton J.P. Schramm L. Scott P.H. White R.J. Mol. Cell. Biol. 2002; 22: 3757-3768Crossref PubMed Scopus (70) Google Scholar, 13Hu P. Wu S. Hernandez N. Mol. Cell. 2003; 12: 699-709Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). There are at least two versions of human TFIIIB that function for transcription of distinct classes of genes (14Schramm L. Hernandez N. Genes Dev. 2002; 16: 2593-2620Crossref PubMed Scopus (448) Google Scholar). The Brf1·TFIIIB complex functions for 5 S rRNA and tRNA transcription and is composed of the TATA-box-binding protein (TBP) and the TBP-associated factors, Bdp1 and Brf1. The Brf2·TFIIIB complex functions for U6 snRNA transcription and is composed of TBP plus Bdp1 but Brf2 instead of Brf1. Brf1·TFIIIB phosphorylation during M phase results in the selective release of Bdp1 from tRNA promoters (15Fairley J.A. Scott P.H. White R.J. EMBO J. 2003; 22: 5841-5850Crossref PubMed Scopus (56) Google Scholar). Hernandez and co-workers (10Hu P. Samudre K. Wu S. Sun Y. Hernandez N. Mol. Cell. 2004; 16: 81-92Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) further demonstrated that Bdp1 is the critical CK2 target within Brf2·TFIIIB for mitotic repression of U6 transcription. Because Bdp1 is a shared component of both TFIIIB complexes, CK2 may target this factor to repress global RNA polymerase III transcription. However, CK2 inhibitors also interfere with Brf1·TFIIIB binding to the TFIIIC complex (12Johnston I.M. Allison S.J. Morton J.P. Schramm L. Scott P.H. White R.J. Mol. Cell. Biol. 2002; 22: 3757-3768Crossref PubMed Scopus (70) Google Scholar), which itself recognizes intragenic promoter elements of 5 S rRNA and tRNA genes, suggesting that CK2 also has a stimulatory role in RNA polymerase III transcription through enhanced preinitiation complex assembly. Consistent with this positive role, CK2 can also activate RNA polymerase III transcription in human cells (12Johnston I.M. Allison S.J. Morton J.P. Schramm L. Scott P.H. White R.J. Mol. Cell. Biol. 2002; 22: 3757-3768Crossref PubMed Scopus (70) Google Scholar) and in this process may additionally phosphorylate RNA polymerase III itself (13Hu P. Wu S. Hernandez N. Mol. Cell. 2003; 12: 699-709Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Together, these data point to an important but complex role for CK2 control of RNA polymerase III transcription. Human U6 snRNA genes are interesting because they are transcribed by RNA polymerase III and yet their promoters are similar to other snRNA genes, such as U1 and U2, which are transcribed by RNA polymerase II (16Lobo S.M. Hernandez N.T. Conaway R.C. Conaway J.W. Transcription: Mechanisms and Regulation. Raven Press, Ltd., New York1994: 127-159Google Scholar, 17Hernandez N. J. Biol. Chem. 2001; 276: 26733-26736Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 18Henry R.W. Ford E. Mital R. Mittal V. Hernandez N. Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 111-120Crossref PubMed Scopus (32) Google Scholar). Consequently, the mechanisms regulating human snRNA gene transcription by RNA polymerases II and III may also be shared. Nonetheless, the RNA polymerase II-transcribed genes do not use TFIIIB and, thus, rely on other factors for regulatory intervention. Regardless of polymerase specificity, all human snRNA genes contain a distal sequence element encompassing an octamer element that is recognized by Oct-1. Additional sites for the Sp1 (19Ares Jr., M. Chung J.S. Giglio L. Weiner A.M. Genes Dev. 1987; 1: 808-817Crossref PubMed Scopus (63) Google Scholar) and STAF (20Schaub M. Myslinski E. Schuster C. Krol A. Carbon P. EMBO J. 1997; 16: 173-181Crossref PubMed Scopus (89) Google Scholar) transcriptional activator proteins are adjacently located to the distal sequence element at some snRNA genes (21Hernandez N. McKnight S. Yamamoto K. Transcriptional Regulation. Cold Spring Harbor Laboratory Press, Cold Spring Harbor1992: 281-313Google Scholar). Oct-1 activates snRNA transcription by direct protein contacts (22Mittal V. Ma B. Hernandez N. Genes Dev. 1999; 13: 1807-1821Crossref PubMed Scopus (56) Google Scholar, 23Ford E. Strubin M. Hernandez N. Genes Dev. 1998; 12: 3528-3540Crossref PubMed Scopus (50) Google Scholar, 24Hovde S. Hinkley C.S. Strong K. Brooks A. Gu L. Henry R.W. Geiger J. Genes Dev. 2002; 16: 2772-2777Crossref PubMed Scopus (18) Google Scholar) with the basal transcription factor called the snRNA-activating protein complex (SNAPC) (25Sadowski C.L. Henry R.W. Lobo S.M. Hernandez N. Genes Dev. 1993; 7: 1535-1548Crossref PubMed Scopus (142) Google Scholar), which is also referred to as the proximal sequence element transcription factor (PTF) (26Murphy S. Yoon J.B. Gerster T. Roeder R.G. Mol. Cell. Biol. 1992; 12: 3247-3261Crossref PubMed Scopus (150) Google Scholar). SNAPC binds to the proximal sequence element (PSE) common to the core promoters of human snRNA genes and functions for both RNA polymerase II and III transcription (18Henry R.W. Ford E. Mital R. Mittal V. Hernandez N. Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 111-120Crossref PubMed Scopus (32) Google Scholar, 25Sadowski C.L. Henry R.W. Lobo S.M. Hernandez N. Genes Dev. 1993; 7: 1535-1548Crossref PubMed Scopus (142) Google Scholar, 27Henry R.W. Sadowski C.L. Kobayashi R. Hernandez N. Nature. 1995; 374: 653-656Crossref PubMed Scopus (123) Google Scholar, 28Henry R.W. Ma B. Sadowski C.L. Kobayashi R. Hernandez N. EMBO J. 1996; 15: 7129-7136Crossref PubMed Scopus (50) Google Scholar, 29Henry R.W. Mittal V. Ma B. Kobayashi R. Hernandez N. Genes Dev. 1998; 12: 2664-2672Crossref PubMed Scopus (65) Google Scholar, 30Wong M.W. Henry R.W. Ma B. Kobayashi R. Klages N. Matthias P. Strubin M. Hernandez N. Mol. Cell. Biol. 1998; 18: 368-377Crossref PubMed Google Scholar, 31Sadowski C.L. Henry R.W. Kobayashi R. Hernandez N. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4289-4293Crossref PubMed Scopus (50) Google Scholar). SNAPC contains at least five proteins called SNAP190 (PTFα), SNAP50 (PTFβ), SNAP45 (PTFδ), SNAP43 (PTFγ), and SNAP19 (27Henry R.W. Sadowski C.L. Kobayashi R. Hernandez N. Nature. 1995; 374: 653-656Crossref PubMed Scopus (123) Google Scholar, 28Henry R.W. Ma B. Sadowski C.L. Kobayashi R. Hernandez N. EMBO J. 1996; 15: 7129-7136Crossref PubMed Scopus (50) Google Scholar, 29Henry R.W. Mittal V. Ma B. Kobayashi R. Hernandez N. Genes Dev. 1998; 12: 2664-2672Crossref PubMed Scopus (65) Google Scholar, 30Wong M.W. Henry R.W. Ma B. Kobayashi R. Klages N. Matthias P. Strubin M. Hernandez N. Mol. Cell. Biol. 1998; 18: 368-377Crossref PubMed Google Scholar, 31Sadowski C.L. Henry R.W. Kobayashi R. Hernandez N. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4289-4293Crossref PubMed Scopus (50) Google Scholar, 32Bai L. Wang Z. Yoon J.B. Roeder R.G. Mol. Cell. Biol. 1996; 16: 5419-5426Crossref PubMed Google Scholar, 33Yoon J.B. Roeder R.G. Mol. Cell. Biol. 1996; 16: 1-9Crossref PubMed Google Scholar). The largest subunit SNAP190 plays a centrally important role in human snRNA gene transcription first by serving as the scaffold for SNAPC assembly though interactions with most other members of SNAPC (34Ma B. Hernandez N. J. Biol. Chem. 2001; 276: 5027-5035Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 35Ma B. Hernandez N. Mol. Cell. Biol. 2002; 22: 8067-8078Crossref PubMed Scopus (33) Google Scholar). Once the complex is assembled, SNAP190 further recognizes the PSE through its Myb DNA binding domain (30Wong M.W. Henry R.W. Ma B. Kobayashi R. Klages N. Matthias P. Strubin M. Hernandez N. Mol. Cell. Biol. 1998; 18: 368-377Crossref PubMed Google Scholar) and is also the direct target for Oct-1 (23Ford E. Strubin M. Hernandez N. Genes Dev. 1998; 12: 3528-3540Crossref PubMed Scopus (50) Google Scholar, 36Mittal V. Cleary M.A. Herr W. Hernandez N. Mol. Cell. Biol. 1996; 16: 1955-1965Crossref PubMed Scopus (66) Google Scholar). In an unexpected twist, SNAP190 can make DNA contacts within the U1 distal sequence element and stimulate the binding of Oct-1 to this enhancer, suggesting that in some contexts coordinated binding of the activator and general transcription machinery is important for transcriptional activation (24Hovde S. Hinkley C.S. Strong K. Brooks A. Gu L. Henry R.W. Geiger J. Genes Dev. 2002; 16: 2772-2777Crossref PubMed Scopus (18) Google Scholar). Human U6 snRNA genes, but not U1 genes, also contain a TATA box that is located adjacently to the PSE, and this promoter arrangement dictates that transcription occurs by RNA polymerase III (37Lobo S.M. Hernandez N. Cell. 1989; 58: 55-67Abstract Full Text PDF PubMed Scopus (176) Google Scholar). The TATA box is recognized by the TBP component of the Brf2·TFIIIB complex (35Ma B. Hernandez N. Mol. Cell. Biol. 2002; 22: 8067-8078Crossref PubMed Scopus (33) Google Scholar, 38Hinkley C.S. Hirsch H.A. Gu L. LaMere B. Henry R.W. J. Biol. Chem. 2003; 278: 18649-18657Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 39Zhao X. Schramm L. Hernandez N. Herr W. Mol. Cell. 2003; 11: 151-161Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 40Cabart P. Murphy S. J. Biol. Chem. 2002; 277: 26831-26838Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 41Cabart P. Murphy S. J. Biol. Chem. 2001; 276: 43056-43064Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). SNAPC, through its SNAP190 subunit, stimulates TBP binding to the U6 TATA box as an early critical step in RNA polymerase III transcription (35Ma B. Hernandez N. Mol. Cell. Biol. 2002; 22: 8067-8078Crossref PubMed Scopus (33) Google Scholar, 38Hinkley C.S. Hirsch H.A. Gu L. LaMere B. Henry R.W. J. Biol. Chem. 2003; 278: 18649-18657Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). TBP is also required for human snRNA gene transcription by RNA polymerase II (25Sadowski C.L. Henry R.W. Lobo S.M. Hernandez N. Genes Dev. 1993; 7: 1535-1548Crossref PubMed Scopus (142) Google Scholar), but how TBP is recruited to these TATA-less promoters is unclear. Nonetheless, it is likely that SNAPC contributes to TBP activity at these genes. SNAPC and TBP co-purify extensively during the biochemical fractionation of SNAPC (27Henry R.W. Sadowski C.L. Kobayashi R. Hernandez N. Nature. 1995; 374: 653-656Crossref PubMed Scopus (123) Google Scholar), and those fractions enriched for SNAPC and TBP can reconstitute U1 snRNA transcription in vitro from extracts that have been depleted of endogenous TBP (25Sadowski C.L. Henry R.W. Lobo S.M. Hernandez N. Genes Dev. 1993; 7: 1535-1548Crossref PubMed Scopus (142) Google Scholar). Thus, SNAPC plays a pivotal role in snRNA gene transcription by providing core promoter recognition, serving as a target for transcription activation by Oct-1, and coordinating TBP activity and preinitiation complex assembly for both RNA polymerases II and III. Additional RNA polymerase II general transcription factors are also required for U1 transcription including TFIIA, TFIIB, TFIIE, and TFIIF (42Kuhlman T.C. Cho H. Reinberg D. Hernandez N. Mol. Cell. Biol. 1999; 19: 2130-2141Crossref PubMed Scopus (53) Google Scholar). As in RNA polymerase III transcription, CK2 also has a complex role in regulating RNA polymerase II transcription. CK2 phosphorylation of TFIIA and TFIIE stimulates preinitiation complex assembly at the adenovirus major late promoter, whereas TFIIF phosphorylation can stimulate RNA polymerase II elongation. In contrast, CK2 phosphorylation of RNA polymerase II inhibits transcription, potentially by impairing elongation (43Cabrejos M.E. Allende C.C. Maldonado E. J. Cell. Biochem. 2004; 93: 2-10Crossref PubMed Scopus (34) Google Scholar). The striking parallel between RNA polymerase II and III transcription of human snRNA genes prompted an investigation into the role of phosphorylation in U1 transcription. In this study we report that CK2 inhibits overall U1 snRNA gene transcription by RNA polymerase II and can phosphorylate SNAPC to inhibit its DNA binding. Interestingly, cooperative interactions of SNAPC with TBP at U6 but not at U1 promoter DNA can overcome the repressive effects of CK2. Together, these data suggest that CK2 may differentially affect preinitiation complex assembly for RNA polymerase II and III transcription of human snRNA genes depending upon the promoter architecture. Chromatin Immunoprecipitation Assays—Chromatin immunoprecipitation assays from HeLa cells were performed using the anti-CK2α(Ab245), anti-CK2β(Ab278) antibodies (44Yu I.J. Spector D.L. Bae Y.S. Marshak D.R. J. Cell Biol. 1991; 114: 1217-1232Crossref PubMed Scopus (88) Google Scholar) as well as anti-SNAP43 (CS48) and anti-TBP antibodies described previously (45Hirsch H.A. Jawdekar G.W. Lee K.A. Gu L. Henry R.W. Mol. Cell. Biol. 2004; 24: 5989-5999Crossref PubMed Scopus (37) Google Scholar). Enrichment of genomic sequences in the immunoprecipitation reactions was measured by PCR as previously described (45Hirsch H.A. Jawdekar G.W. Lee K.A. Gu L. Henry R.W. Mol. Cell. Biol. 2004; 24: 5989-5999Crossref PubMed Scopus (37) Google Scholar). RNA interference (RNAi)—CK2α and CK2α′ cDNA were generated with a T7 promoter at both ends by reverse transcription (RT)-PCR using total RNA from HeLa cells as a template. The primers for CK2α are CK2α forward, 5′-GCGTAATACGACTCACTATAGGAAATAATGAAAAAGTTGTTG-3′, and CK2α reverse, 5′-GCGTAATACGACTCACTATAGGCTCTTGCAGTAAGCCGTGAC-3′. The primers for CK2α′ are CK2α′ forward, 5′-GCGTAATACGACTCACTATAGGCAACAATGAGAGAGTGGTTG-3′, and CK2α′ reverse, 5′-GCGTAATACGACTCACTATAGGTCTGTTGATGGTC GTATCGC-3′. LacZ cDNA with a T7 promoter at both ends was generated by PCR using pPelican-lacZ as a template. The primers used are lacZ forward, 5′-TTAATACGACTCACTATAGGGAGACGATAACCACCACGCTCATCG-3′, and lacZ reverse, 5′-TTAATACGACTCACTATAGGGAGAGCGTTACCCAACTTAATCGCC-3′. Resultant cDNAs were subjected to in vitro transcription with T7 polymerase to produce double-stranded RNA. After DNase I treatment, double-stranded RNA was incubated with recombinant Dicer, and resultant Dicer-generated small interfering RNA were purified according to the manufacturer's instructions (Invitrogen). Approximately 250 ng of Dicer-generated small interfering RNA for lacZ, CK2α, or CK2α plus 250 ng of CK2α′ Dicer-generated small interfering RNA were transfected into HeLa cells using Lipofectamine 2000 (Invitrogen). Cells were harvested 30 h later, and total RNA was extracted using TRIzol (Invitrogen). RT-PCR was carried out using Titan One Tube RT-PCR System (Roche Applied Science). The primers used to amplify U1 primary transcript are U1prim forward, 5′-ACTTGCTGCTTCACCACGAA-3′, and U1prim reverse, 5′-ACAGCCTCATACGCCTCACT-3′. The primers used to amplify the total U1 snRNA population are U1 forward, 5′-ATACTTACCTGGCAGGGGAG-3′, and U1 reverse, 5′-CAGGGGAAAGCGCGAACGCA-3′. RT-PCR products were separated by 3% Tris borate EDTA-agarose electrophoresis, stained with ethidium bromide, and visualized with Kodak imaging software. In Vitro Transcription Assays—In vitro transcription of human U1 and U6 snRNA genes were performed as described previously (37Lobo S.M. Hernandez N. Cell. 1989; 58: 55-67Abstract Full Text PDF PubMed Scopus (176) Google Scholar) with the following modifications. The HeLa cell nuclear extracts were preincubated with Dignam buffer D either with or without recombinant CK2 and kinase inhibitors for 60 min at 30 °C before initiating transcription by the addition of transcription buffers, nucleotide triphosphates, and DNA templates. The amounts of recombinant CK2 and kinase inhibitors used are indicated in the legend to Fig. 2. Transcripts were separated by denaturing PAGE and visualized by PhosphorImager analysis (Amersham Biosciences). Expression and Purification of Recombinant Proteins—GST-SNAP190-(1–719) was expressed in Escherichia coli BL21 (DE3) using the vector pSBet-GST-SNAP190-(1–719) and was purified for in vitro kinase assays by affinity chromatography using glutathione-Sepharose beads (Amersham Biosciences). Recombinant mini-SNAPC containing SNAP190-(1–719), SNAP43, and SNAP50 was co-expressed in E. coli using the vector combination pSBet-GST-SNAP190-(1–719) and pET21-His-SNAP43/HA-SNAP50. Recombinant mini-SNAPC was affinity-purified using glutathione-agarose beads followed by digestion with thrombin to release the complex from the GST tag and dialysis against Dignam buffer D containing 80 mm KCl. Immunoprecipitation and in Vitro Kinase Assays—For the experiment presented in Fig. 3A, 180 μl of HeLa cell nuclear extract (∼10 mg/ml) was incubated with 20 μl of rabbit anti-SNAP43 (CS48 (27Henry R.W. Sadowski C.L. Kobayashi R. Hernandez N. Nature. 1995; 374: 653-656Crossref PubMed Scopus (123) Google Scholar)), anti-SNAP190 (CS398, CS402 (30Wong M.W. Henry R.W. Ma B. Kobayashi R. Klages N. Matthias P. Strubin M. Hernandez N. Mol. Cell. Biol. 1998; 18: 368-377Crossref PubMed Google Scholar)), anti-CK2α (Ab245 (44Yu I.J. Spector D.L. Bae Y.S. Marshak D.R. J. Cell Biol. 1991; 114: 1217-1232Crossref PubMed Scopus (88) Google Scholar)), or preimmune antibodies covalently coupled to protein-G agarose beads. Recovered proteins were analyzed by Western blot using a mouse monoclonal antibody against CK2α (Transduction Laboratories). For Fig. 3B, 40 μl of HeLa cell nuclear extract was used for each immunoprecipitation. After extensive washing with HEMGT-150 buffer (20 mm Hepes, pH 7.9, 0.1 mm EDTA, 5 mm MgCl2, 10% glycerol, 0.5% Tween 20, 150 mm KCl), the beads were suspended in 40 μl of HEMGT-150 buffer containing 2 μl of [γ-32P]ATP (6000 Ci/mmol, 150 mCi/ml), and the samples were incubated at 30 °C for 15 min. The beads were then washed extensively in HEMGT-150 buffer, and proteins were separated by 12.5% SDS-PAGE. Radiolabeled proteins were visualized by autoradiography. For Fig. 3C, 100 μl of HeLa cell nuclear extracts were used for immunoprecipitation. After kinase reactions, proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Radiolabeled proteins were detected first by autoradiography. Subsequently, Western blot analyses were performed using anti-SNAP190 (CS402) antibodies. For Fig. 5, B and C, ∼5 μg of GST-SNAP190-(1–719) was bound to glutathione-agarose beads (10 μl). Immobilized GST-SNAP190-(1–719) was incubated with 10 μl of HeLa cell nuclear extract for 30 min at 30 °C. The beads were washed extensively with HEMGT-150. In vitro kinase assays were then performed directly on the beads. Kinase reactions were also performed using untreated GST-SNAP190-(1–719) plus 10 units of recombinant CK2 (New England Biolabs). Where indicated, kinase reactions were performed in the presence of 20 nm [γ-32P]ATP or [γ-32P]GTP with or without d-ribofuranosylbenzimidazole (DRB; Sigma) or 3,3′,4′,5,7-pentahyroxyflavone (quercetin; Sigma). Tryptic Phosphopeptide Mapping—To obtain material for thin layer chromatography (TLC) analysis, ∼1 μg of GST-SNAP190-(1–719) was labeled with [γ-32P]ATP by using HeLa cell nuclear extracts or recombinant CK2. Phosphorylated GST-SNAP190-(1–719) was gel purified before digestion with sequencing grade modified trypsin (Promega). The tryptic fragments from each of these reactions were spotted individually or were combined at a 1:1 ratio and spotted onto a cellulose TLC plate. Peptides were separated in the first dimension by electrophoresis in pH 1.9 buffer (formic acid (88% w/v)/glacial acetic acid/distilled H2O, 25:78: 897, v/v/v) and in the second dimension by chromatography in chromatography buffer (n-butanol/pyridine/glacial acetic acid/distilled H2O, 15:10:13:12, v/v/v/v) before detection by PhosphorImager analysis (46van der Geer P. Hunter T. Electrophoresis. 1994; 15: 544-554Crossref PubMed Scopus (125) Google Scholar). Phosphoamino Acid Analysis—Endogenous SNAP190 was immunoprecipitated, phosphorylated in vitro in the presence of [γ-32P]ATP, separated by 7.5% SDS-PAGE, and transferred to nitrocellulose membrane. The ∼190-kDa radioactive protein corresponding to SNAP190 was excised and hydrolyzed in 5.7 n HCl for 1 h at 100 °C. Recovered phosphoamino acids were vacuum-dried and dissolved in 10 μl of pH 1.9 buffer containing cold phosphoserine, phosphothreonine, and phosphotyrosine mixture. The mixture was separated by one-dimensional electrophoresis (500 V) on cellulose TLC plates (Eastman Kodak Co.) for 1 h at 0 °C in pH 2.5 buffer (67% pH 3.5 buffer (glacial acetic acid, pyridine, water, 50:5:945, v/v/v, containing 0.5 mm EDTA) and 33% pH 1.9 buffer (glacial acetic acid, 88% formic acid, water, 78:25:897, v/v/v)). Unlabeled amino acid standards were visualized by spraying the cellulose plates with ninhydrin. The 32P-labeled amino acid residues were visualized by autoradiography with a PhosphorImager. Electrophoretic Mobility Shift Assays—Approximately 100 ng of purified mini-SNAPc and/or 30 ng of TBP were preincubated alone or with 15 or 150 units CK2 and/or 7 mm ATP for 30 min at 30 °C. Electrophoretic mobility shift assays were then performed using DNA probes containing a wild-type mouse U6 PSE with a wild-type or mutant human U6 TATA box as described previously (38Hinkley C.S. Hirsch H.A. Gu L. LaMere B. Henry R.W. J. Biol. Chem. 2003; 278: 18649-18657Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 47Mittal V. Hernandez N. Science. 1997; 275: 1136-1140Crossref PubMed Scopus (77) Google Scholar). CK2 Inhibits U1 snRNA Gene Transcription—The conservation of similar promoter architectures among the human snRNA gene family suggests that these genes could be coordinately regulated. That CK2 regulates human U6 snRNA gene transcription by RNA polymerase III (13Hu P. Wu S. Hernandez N. Mol. Cell. 2003; 12: 699-709Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) prompted us to examine whether CK2 similarly regulates human snRNA gene transcription by RNA polymerase II. First, chromatin immunoprecipitation experiments were performed to determine whether endogenous CK2 could associate with the promoter regions of both U6 and U1 snRNA genes. As shown in Fig. 1A, both U6 and U1 promoter regions were enriched in immunoprecipitation reactions using anti-CK2α antibodies (lane 7), whereas U1 but not U6 promoter regions were enriched in the anti-CK2β immunoprecipitation reactions (lane 8). Similar results were obtained in experiments performed with different antibodies directed against CK2α and CK2β (data not shown). Possibly the epitopes recognized by the CK2β antibodies may be occluded by other transcription factors at the U6 promoter. However, this result stands in contrast with that noted previously (13Hu P. Wu S. Hernandez N. Mol. Cell. 2003; 12: 699-709Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) wherein a more robust CK2β association with this U6 promoter was noted and only weak U1 promoter association by any CK2 subunit was detected. The reason for this discrepancy is unclear, but differences in chromatin immunoprecipitation or cell growth conditions could potentially affect promoter recovery by CK2 antibodies. The levels of U1 and U6 promoter recovery in this reaction were less than that those observed in either anti-SNAP43 (lane 6) or anti-TBP (lane 9) reactions but markedly greater than that seen in reactions using IgG (lane 5). No significant enrichment of the glyceraldehyde-3-phosphate dehydrogenase exon 2 or U1 upstream regions was observed in any reactions. Therefore, endogenous CK2 associates with the promoter regions of both U1 and U6 genes and suggests the possibility that CK2 could additionally affect human snRNA gene expression by RNA polymerase II. To test whether endogenous CK2 influences human U1 gene expression in living cells, CK2 levels were reduced by RNAi, and the effect on U1 snRNA production was monitored by RT-PCR (Fig. 1B). As a negative control, RNAi was also performed using lacZ-specific RNA. Because it was demonstrated that phosphorylation of the carboxyl-terminal domain of RNA polymerase II contributes to 3′ processing of human U2 snRNA (48Jacobs E.Y. Ogiwara I. Weiner A.M. Mol. Cell. Biol. 2004; 24: 846-855Crossref PubMed Scopus (34) Google Scholar, 49Medlin J.E. Uguen P. Taylor A. Bentley D.L. Murphy S. EMBO J. 2003; 22: 925-934Crossref PubMed Scopus (69) Google Scholar), it was possible that CK2 could also play a role in U1 3′ processing. Therefore, in this experiment different primer combinations were used to detect either the primary U1 snRNA transcript that is normally processed rapidly or the total U1 snRNA steady state population. Lanes 1–5 (top panel) show that amplification of the U1 p" @default.
• W2037847135 created "2016-06-24" @default.
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• W2037847135 date "2005-07-01" @default.
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• W2037847135 title "Cooperation between Small Nuclear RNA-activating Protein Complex (SNAPC) and TATA-box-binding Protein Antagonizes Protein Kinase CK2 Inhibition of DNA Binding by SNAPC" @default.
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• W2037847135 doi "https://doi.org/10.1074/jbc.m503206200" @default.
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