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• W2157746712 abstract "Human Tudor staphylococcal nuclease (Tudor-SN) is composed of four tandem repeats of staphylococcal nuclease (SN)-like domains, followed by a tudor and SN-like domain (TSN) consisting of a central tudor flanked by two partial SN-like sequences. The crystal structure of the tudor domain displays a conserved aromatic cage, which is predicted to hook methyl groups. Here, we demonstrated that the TSN domain of Tudor-SN binds to symmetrically dimethylarginine (sDMA)-modified SmB/B′ and SmD1/D3 core proteins of the spliceosome. We demonstrated that this interaction ability is reduced by the methyltransferase inhibitor 5-deoxy-5-(methylthio)adenosine. Mutagenesis experiments indicated that the conserved amino acids (Phe-715, Tyr-721, Tyr-738, and Tyr-741) in the methyl-binding cage of the TSN domain are required for Tudor-SN-SmB interaction. Furthermore, depletion of Tudor-SN affects the association of Sm protein with snRNAs and, as a result, inhibits the assembly of uridine-rich small ribonucleoprotein mediated by the Sm core complex in vivo. Our results reveal the molecular basis for the involvement of Tudor-SN in regulating small nuclear ribonucleoprotein biogenesis, which provides novel insight related to the biological activity of Tudor-SN. Human Tudor staphylococcal nuclease (Tudor-SN) is composed of four tandem repeats of staphylococcal nuclease (SN)-like domains, followed by a tudor and SN-like domain (TSN) consisting of a central tudor flanked by two partial SN-like sequences. The crystal structure of the tudor domain displays a conserved aromatic cage, which is predicted to hook methyl groups. Here, we demonstrated that the TSN domain of Tudor-SN binds to symmetrically dimethylarginine (sDMA)-modified SmB/B′ and SmD1/D3 core proteins of the spliceosome. We demonstrated that this interaction ability is reduced by the methyltransferase inhibitor 5-deoxy-5-(methylthio)adenosine. Mutagenesis experiments indicated that the conserved amino acids (Phe-715, Tyr-721, Tyr-738, and Tyr-741) in the methyl-binding cage of the TSN domain are required for Tudor-SN-SmB interaction. Furthermore, depletion of Tudor-SN affects the association of Sm protein with snRNAs and, as a result, inhibits the assembly of uridine-rich small ribonucleoprotein mediated by the Sm core complex in vivo. Our results reveal the molecular basis for the involvement of Tudor-SN in regulating small nuclear ribonucleoprotein biogenesis, which provides novel insight related to the biological activity of Tudor-SN. Tudor staphylococcal nuclease (Tudor-SN), 4The abbreviations used are:SNstaphylococcal nuclease-like domainTSNtudor and SN-like domainU snRNPsuridine-rich small ribonucleoproteinsDMAsymmetrical dimethylarginineTCLtotal cell lysateQ-PCRquantitative real time PCRTMGtrimethylguanosinesnRNPsmall nuclear ribonucleoproteinRIPRNA-binding protein immunoprecipitationMTA5-deoxy-5-(methylthio)adenosineSMNsurvival of motor neuron. also known as SND1 (staphylococcal nuclease domain containing 1) or p100, has been identified as a ubiquitous protein present in humans, cattle (1.Broadhurst M.K. Lee R.S. Hawkins S. Wheeler T.T. The p100 EBNA-2 co-activator. A highly conserved protein found in a range of exocrine and endocrine cells and tissues in cattle.Biochim. Biophys. Acta. 2005; 1681: 126-133Crossref PubMed Scopus (29) Google Scholar), rats (2.Rodríguez L. Ochoa B. Martínez M.J. NF-Y and Sp1 are involved in transcriptional regulation of rat SND p102 gene.Biochem. Biophys. Res. Commun. 2007; 356: 226-232Crossref PubMed Scopus (12) Google Scholar), zebrafish (3.Zhao C.T. Shi K.H. Su Y. Liang L.Y. Yan Y. Postlethwait J. Meng A.M. Two variants of zebrafish p100 are expressed during embryogenesis and regulated by Nodal signaling.FEBS Lett. 2003; 543: 190-195Crossref PubMed Scopus (15) Google Scholar), Tetrahymena thermophila (4.Howard-Till R.A. Yao M.C. Tudor nuclease genes and programmed DNA rearrangements in Tetrahymena thermophila.Eukaryot. Cell. 2007; 6: 1795-1804Crossref PubMed Scopus (19) Google Scholar), rice (5.Sami-Subbu R. Choi S.B. Wu Y. Wang C. Okita T.W. Identification of a cytoskeleton-associated 120-kDa RNA-binding protein in developing rice seeds.Plant Mol. Biol. 2001; 46: 79-88Crossref PubMed Scopus (39) Google Scholar), peas (6.Abe S. Sakai M. Yagi K. Hagino T. Ochi K. Shibata K. Davies E. A Tudor protein with multiple SNc domains from pea seedlings. Cellular localization, partial characterization, sequence analysis, and phylogenetic relationships.J. Exp. Bot. 2003; 54: 971-983Crossref PubMed Scopus (29) Google Scholar), and many other species. Tudor-SN is a multifunctional protein implicated in a variety of cellular processes, such as gene transcription, pre-mRNA splicing, formation of stress granules, as well as the RNA-induced silencing complex in which small RNAs are complexed with ribonucleoproteins to ensure an RNAi-mediated gene (7.Li C.L. Yang W.Z. Chen Y.P. Yuan H.S. Structural and functional insights into human Tudor-SN, a key component linking RNA interference and editing.Nucleic Acids Res. 2008; 36: 3579-3589Crossref PubMed Scopus (78) Google Scholar, 8.Paukku K. Kalkkinen N. Silvennoinen O. Kontula K.K. Lehtonen J.Y. p100 increases AT1R expression through interaction with AT1R 3′-UTR.Nucleic Acids Res. 2008; 36: 4474-4487Crossref PubMed Scopus (52) Google Scholar, 9.Caudy A.A. Ketting R.F. Hammond S.M. Denli A.M. Bathoorn A.M. Tops B.B. Silva J.M. Myers M.M. Hannon G.J. Plasterk R.H. A micrococcal nuclease homologue in RNAi effector complexes.Nature. 2003; 425: 411-414Crossref PubMed Scopus (350) Google Scholar, 10.Gao X. Ge L. Shao J. Su C. Zhao H. Saarikettu J. Yao X. Yao Z. Silvennoinen O. Yang J. Tudor-SN interacts with and co-localizes with G3BP in stress granules under stress conditions.FEBS Lett. 2010; 584: 3525-3532Crossref PubMed Scopus (55) Google Scholar, 11.Shaw N. Zhao M. Cheng C. Xu H. Saarikettu J. Li Y. Da Y. Yao Z. Silvennoinen O. Yang J. Liu Z.J. Wang B.C. Rao Z. The multifunctional human p100 protein “hooks” methylated ligands.Nat. Struct. Mol. Biol. 2007; 14: 779-784Crossref PubMed Scopus (64) Google Scholar, 12.Yang J. Aittomäki S. Pesu M. Carter K. Saarinen J. Kalkkinen N. Kieff E. Silvennoinen O. Identification of p100 as a co-activator for STAT6 that bridges STAT6 with RNA polymerase II.EMBO J. 2002; 21: 4950-4958Crossref PubMed Scopus (143) Google Scholar, 13.Yang J. Välineva T. Hong J. Bu T. Yao Z. Jensen O.N. Frilander M.J. Silvennoinen O. Transcriptional co-activator protein p100 interacts with snRNP proteins and facilitates the assembly of the spliceosome.Nucleic Acids Res. 2007; 35: 4485-4494Crossref PubMed Scopus (98) Google Scholar, 14.Välineva T. Yang J. Palovuori R. Silvennoinen O. The transcriptional co-activator protein p100 recruits histone acetyltransferase activity to STAT6 and mediates interaction between the CREB-binding protein and STAT6.J. Biol. Chem. 2005; 280: 14989-14996Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Combination of the modeled three-dimensional structures and x-ray crystallography indicates that the full-length structure of Tudor-SN resembles a stick with a hook, where SN-like domains form the stick and the tudor domain makes up the hook (11.Shaw N. Zhao M. Cheng C. Xu H. Saarikettu J. Li Y. Da Y. Yao Z. Silvennoinen O. Yang J. Liu Z.J. Wang B.C. Rao Z. The multifunctional human p100 protein “hooks” methylated ligands.Nat. Struct. Mol. Biol. 2007; 14: 779-784Crossref PubMed Scopus (64) Google Scholar). It indicates that different domains of Tudor-SN protein may recruit different protein complexes to play different roles. In line with this concept, we have demonstrated that Tudor-SN functions as a transcriptional co-activator of STAT6 via interaction with the SN-like domains (12.Yang J. Aittomäki S. Pesu M. Carter K. Saarinen J. Kalkkinen N. Kieff E. Silvennoinen O. Identification of p100 as a co-activator for STAT6 that bridges STAT6 with RNA polymerase II.EMBO J. 2002; 21: 4950-4958Crossref PubMed Scopus (143) Google Scholar, 15.Dong L. Zhang X. Fu X. Zhang X. Gao X. Zhu M. Wang X. Yang Z. Jensen O.N. Saarikettu J. Yao Z. Silvennoinen O. Yang J. PTB-associated splicing factor (PSF) functions as a repressor of STAT6-mediated Ig ϵ gene transcription by recruitment of HDAC1.J. Biol. Chem. 2011; 286: 3451-3459Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), whereas the TSN domain is involved in the spliceosome assembly and accelerates the kinetics of precursor-messenger RNA (pre-mRNA) splicing (13.Yang J. Välineva T. Hong J. Bu T. Yao Z. Jensen O.N. Frilander M.J. Silvennoinen O. Transcriptional co-activator protein p100 interacts with snRNP proteins and facilitates the assembly of the spliceosome.Nucleic Acids Res. 2007; 35: 4485-4494Crossref PubMed Scopus (98) Google Scholar). However, the precise molecular mechanism underlying the involvement of Tudor-SN in pre-mRNA processing has not been fully elucidated. staphylococcal nuclease-like domain tudor and SN-like domain uridine-rich small ribonucleoprotein symmetrical dimethylarginine total cell lysate quantitative real time PCR trimethylguanosine small nuclear ribonucleoprotein RNA-binding protein immunoprecipitation 5-deoxy-5-(methylthio)adenosine survival of motor neuron. The pre-mRNA splicing process is essential for the successful execution of eukaryotic gene expression and mediates the production of mature mRNA through excision of introns and ligation of exons in pre-mRNAs by the spliceosome machinery. The spliceosome consists of five conserved snRNPs formed by an ordered binding of specific protein complexes onto metabolically stable U snRNAs, including U1, U2, U5, and base-paired U4/U6, which are highly abundant in eukaryotic cells (16.Will C.L. Lührmann R. Spliceosomal UsnRNP biogenesis, structure, and function.Curr. Opin. Cell Biol. 2001; 13: 290-301Crossref PubMed Scopus (550) Google Scholar, 17.Kambach C. Walke S. Nagai K. Structure and assembly of the spliceosomal small nuclear ribonucleoprotein particles.Curr. Opin. Struct Biol. 1999; 9: 222-230Crossref PubMed Scopus (110) Google Scholar, 18.Jurica M.S. Moore M.J. Pre-mRNA splicing. Awash in a sea of proteins.Mol. Cell. 2003; 12: 5-14Abstract Full Text Full Text PDF PubMed Scopus (792) Google Scholar, 19.Nagai K. Muto Y. Pomeranz Krummel D.A. Kambach C. Ignjatovic T. Walke S. Kuglstatter A. Structure and assembly of the spliceosomal snRNPs. Novartis Medal Lecture.Biochem. Soc. Trans. 2001; 29: 15-26Crossref PubMed Google Scholar). The biogenesis of U snRNPs is a stepwise process, and the hallmark of which is the formation of an Sm protein ring consisting of seven polypeptides (B/B′, D1, D2, D3, E, F, and G) around the U snRNAs (20.Achsel T. Stark H. Lührmann R. The Sm domain is an ancient RNA-binding motif with oligo(U) specificity.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 3685-3689Crossref PubMed Scopus (107) Google Scholar, 21.Kambach C. Walke S. Young R. Avis J.M. de la Fortelle E. Raker V.A. Lührmann R. Li J. Nagai K. Crystal structures of two Sm protein complexes and their implications for the assembly of the spliceosomal snRNPs.Cell. 1999; 96: 375-387Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar, 22.Stark H. Dube P. Lührmann R. Kastner B. Arrangement of RNA and proteins in the spliceosomal U1 small nuclear ribonucleoprotein particle.Nature. 2001; 409: 539-542Crossref PubMed Scopus (192) Google Scholar). Notably, arginine residues in SmB/B′, SmD1, and SmD3 proteins can be methylated by protein-arginine methyltransferases (23.Boisvert F.M. Chénard C.A. Richard S. Protein interfaces in signaling regulated by arginine methylation.Sci. STKE 2005. 2005; : re2Google Scholar, 24.Lee J.H. Cook J.R. Yang Z.H. Mirochnitchenko O. Gunderson S.I. Felix A.M. Herth N. Hoffmann R. Pestka S. PRMT7, a new protein arginine methyltransferase that synthesizes symmetric dimethylarginine.J. Biol. Chem. 2005; 280: 3656-3664Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 25.Meister G. Eggert C. Bühler D. Brahms H. Kambach C. Fischer U. Methylation of Sm proteins by a complex containing PRMT5 and the putative U snRNP assembly factor pICln.Curr. Biol. 2001; 11: 1990-1994Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 26.Cheng D. Côté J. Shaaban S. Bedford M.T. The arginine methyltransferase CARM1 regulates the coupling of transcription and mRNA processing.Mol. Cell. 2007; 25: 71-83Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 27.Gonsalvez G.B. Tian L. Ospina J.K. Boisvert F.M. Lamond A.I. Matera A.G. Two distinct arginine methyltransferases are required for biogenesis of Sm-class ribonucleoproteins.J. Cell Biol. 2007; 178: 733-740Crossref PubMed Scopus (117) Google Scholar). There are two general types (i.e. type I and type II) of protein-arginine methyltransferase responsible for protein arginine methylation. Type I methyltransferases (PRMT1, -3, -4, and -6) mainly generate monomethylarginine and asymmetrical dimethylarginine, whereas type II methyltransferases (PRMT5, -7, and -9) predominantly generate monomethylarginine as well as symmetrical dimethylarginine (sDMA) (24.Lee J.H. Cook J.R. Yang Z.H. Mirochnitchenko O. Gunderson S.I. Felix A.M. Herth N. Hoffmann R. Pestka S. PRMT7, a new protein arginine methyltransferase that synthesizes symmetric dimethylarginine.J. Biol. Chem. 2005; 280: 3656-3664Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 25.Meister G. Eggert C. Bühler D. Brahms H. Kambach C. Fischer U. Methylation of Sm proteins by a complex containing PRMT5 and the putative U snRNP assembly factor pICln.Curr. Biol. 2001; 11: 1990-1994Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 26.Cheng D. Côté J. Shaaban S. Bedford M.T. The arginine methyltransferase CARM1 regulates the coupling of transcription and mRNA processing.Mol. Cell. 2007; 25: 71-83Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 27.Gonsalvez G.B. Tian L. Ospina J.K. Boisvert F.M. Lamond A.I. Matera A.G. Two distinct arginine methyltransferases are required for biogenesis of Sm-class ribonucleoproteins.J. Cell Biol. 2007; 178: 733-740Crossref PubMed Scopus (117) Google Scholar). sDMA is detected on both nuclear and cytoplasmic Sm proteins, whereas asymmetrical dimethylarginine is only identified on nuclear Sm proteins (28.Miranda T.B. Khusial P. Cook J.R. Lee J.H. Gunderson S.I. Pestka S. Zieve G.W. Clarke S. Spliceosome Sm proteins D1, D3, and B/B′ are asymmetrically dimethylated at arginine residues in the nucleus.Biochem. Biophys. Res. Commun. 2004; 323: 382-387Crossref PubMed Scopus (33) Google Scholar). The tudor domain has previously been shown to be able to bind methylated proteins. For example, sDMA-modified Sm proteins are recognized by the tudor domains of SMN and SPF30 (29.Sprangers R. Groves M.R. Sinning I. Sattler M. High resolution x-ray and NMR structures of the SMN Tudor domain. Conformational variation in the binding site for symmetrically dimethylated arginine residues.J. Mol. Biol. 2003; 327: 507-520Crossref PubMed Scopus (142) Google Scholar, 30.Côté J. Richard S. Tudor domains bind symmetrical dimethylated arginines.J. Biol. Chem. 2005; 280: 28476-28483Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 31.Tripsianes K. Madl T. Machyna M. Fessas D. Englbrecht C. Fischer U. Neugebauer K.M. Sattler M. Structural basis for dimethylarginine recognition by the Tudor domains of human SMN and SPF30 proteins.Nat. Struct. Mol. Biol. 2011; 18: 1414-1420Crossref PubMed Scopus (134) Google Scholar). The formation of the spliceosome complex is a dynamic process of snRNP particles occurring on pre-mRNA. The U5 snRNP binds the 5′ and 3′ splice sites of exons, allowing the spliceosome to “tether” the exons at the 5′ splice site, and it intermediately carries out the first catalytic step and then aligns with the 3′ splice site of exon for the second step. Prp8, U5–116, and hBrr2 are three key components of U5 snRNP that interact extensively with each other and play essential roles in the formation, activation, and remodeling of the spliceosome. We reported earlier that purified Tudor-SN could accelerate the first catalytic step of splicing, but it did not affect the overall level of splicing (13.Yang J. Välineva T. Hong J. Bu T. Yao Z. Jensen O.N. Frilander M.J. Silvennoinen O. Transcriptional co-activator protein p100 interacts with snRNP proteins and facilitates the assembly of the spliceosome.Nucleic Acids Res. 2007; 35: 4485-4494Crossref PubMed Scopus (98) Google Scholar). The association of Tudor-SN- and U5 snRNP-specific proteins may explain the phenomenon. However, it cannot explain how Tudor-SN could associate with different U snRNAs, such as U1, U2, U4, U5, and U6 snRNAs. The tudor domain in Tudor-SN shows a high level of homology to the tudor domain of the SMN protein (13.Yang J. Välineva T. Hong J. Bu T. Yao Z. Jensen O.N. Frilander M.J. Silvennoinen O. Transcriptional co-activator protein p100 interacts with snRNP proteins and facilitates the assembly of the spliceosome.Nucleic Acids Res. 2007; 35: 4485-4494Crossref PubMed Scopus (98) Google Scholar). SMN protein associates with the spliceosomal Sm proteins via its tudor domain and plays essential roles in the assembly of U snRNPs (32.Kolb S.J. Battle D.J. Dreyfuss G. Molecular functions of the SMN complex.J. Child Neurol. 2007; 22: 990-994Crossref PubMed Scopus (120) Google Scholar, 33.Bühler D. Raker V. Lührmann R. Fischer U. Essential role for the Tudor domain of SMN in spliceosomal U snRNP assembly. Implications for spinal muscular atrophy.Hum. Mol. Genet. 1999; 8: 2351-2357Crossref PubMed Scopus (219) Google Scholar). Thus, it is possible that Tudor-SN protein participates in snRNP assembly through similar interaction mechanisms, which led us to investigate the relationship between the TSN domain and Sm proteins. In addition, surface electrostatic potential plots of tudor domain in Tudor-SN protein also reveal negatively charged surfaces, which are involved in recognition and binding of methylation marks (11.Shaw N. Zhao M. Cheng C. Xu H. Saarikettu J. Li Y. Da Y. Yao Z. Silvennoinen O. Yang J. Liu Z.J. Wang B.C. Rao Z. The multifunctional human p100 protein “hooks” methylated ligands.Nat. Struct. Mol. Biol. 2007; 14: 779-784Crossref PubMed Scopus (64) Google Scholar). The aim of this study was thus to investigate the molecular mechanisms of the Tudor-SN protein in the pre-mRNA splicing process. In this study, we demonstrate that the interaction of Tudor-SN and snRNPs involves the efficient association of Sm protein, apart from the interaction with U5 snRNPs. HeLa cells and COS-7 cells were cultured as reported previously (12.Yang J. Aittomäki S. Pesu M. Carter K. Saarinen J. Kalkkinen N. Kieff E. Silvennoinen O. Identification of p100 as a co-activator for STAT6 that bridges STAT6 with RNA polymerase II.EMBO J. 2002; 21: 4950-4958Crossref PubMed Scopus (143) Google Scholar). COS-7 cells were transiently transfected with expression plasmids by electroporation with a Bio-Rad gene pulser at 220 V/950 microfarads. siRNA was transfected into HeLa cells using Lipofectamine RNAi MAX (Invitrogen), according to the manufacturer's instructions. Tudor-SN siRNA was generated as reported previously (14.Välineva T. Yang J. Palovuori R. Silvennoinen O. The transcriptional co-activator protein p100 recruits histone acetyltransferase activity to STAT6 and mediates interaction between the CREB-binding protein and STAT6.J. Biol. Chem. 2005; 280: 14989-14996Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), and SMN siRNA was purchased from Santa Cruz Biotechnology. pCMV6-AC-GFP-SMN plasmid was purchased from OriGene Technologies. The pSG5-Tudor-SN expression plasmid containing the full-length cDNA of human Tudor-SN and the GST-TSN plasmid containing the TSN domain (640–885 amino acids) of Tudor-SN protein were constructed as described previously (12.Yang J. Aittomäki S. Pesu M. Carter K. Saarinen J. Kalkkinen N. Kieff E. Silvennoinen O. Identification of p100 as a co-activator for STAT6 that bridges STAT6 with RNA polymerase II.EMBO J. 2002; 21: 4950-4958Crossref PubMed Scopus (143) Google Scholar). GST-Tudor was constructed by cloning PCR products corresponding to amino acids 678–769 of human TSN into pGEXT-4T-1 vector with EcoRI and NotI. GST-SN1 (26–184 amino acids), SN2 (186–341 amino acids), SN3 (342–504 amino acids), or SN4 (509–673 amino acids) were constructed by cloning PCR products corresponding to amino acids into pGEX-4T-1 vector with EcoRI and NotI. GST-TSN mutant constructs (F715A, Y721A, F715A/Y721A, Y741A, Y738A, and Y738A/Y741A) were generated as described previously (11.Shaw N. Zhao M. Cheng C. Xu H. Saarikettu J. Li Y. Da Y. Yao Z. Silvennoinen O. Yang J. Liu Z.J. Wang B.C. Rao Z. The multifunctional human p100 protein “hooks” methylated ligands.Nat. Struct. Mol. Biol. 2007; 14: 779-784Crossref PubMed Scopus (64) Google Scholar). All PCR products were sequenced. The total cell lysates (TCLs) of HeLa cells were harvested with Nonidet P-40 lysis buffer (50 mm Tris-HCl (pH 7.6), 300 mm NaCl, 0.1 mm EDTA, 0.5% Nonidet P-40, 20% glycerol, 0.1 mm sodium orthovanadate, 1 mm sodium butyrate). The TCLs were layered on a 10-ml gradient of 10–30% glycerol and centrifuged at 24,000 rpm (Beckman SW-41Ti rotor) for 20 h at 4 °C. All glycerol solutions were prepared with 25 mm Tris-HCl buffer (pH 7.5), 1.5 mm EGTA, 15 mm MgCl2, 50 mm NaCl, 0.1 mm NaF (pH 7.2), 1 mm DTT, 0.04% Nonidet P-40. and 10–30% glycerol. Eighteen fractions of 500 μl each were collected from top to bottom. Each fraction was run on SDS-PAGE and analyzed by Western blotting with corresponding antibodies. The extracted snRNAs from each fraction were also analyzed by Northern blotting with U1, U2, U4, U5, and U6 snRNA probes as described previously (34.Pessa H.K. Ruokolainen A. Frilander M.J. The abundance of the spliceosomal snRNPs is not limiting the splicing of U12-type introns.RNA. 2006; 12: 1883-1892Crossref PubMed Scopus (27) Google Scholar). Radiolabeled probes of U1, U2, U4, U5, and U6 snRNA were made by in vitro transcription of the linearized snRNA plasmids (34.Pessa H.K. Ruokolainen A. Frilander M.J. The abundance of the spliceosomal snRNPs is not limiting the splicing of U12-type introns.RNA. 2006; 12: 1883-1892Crossref PubMed Scopus (27) Google Scholar). HeLa cells (2.0 × 107) were incubated with 1% formaldehyde for 10 min at 37 °C to cross-link RNA protein. Then the glycine (125 mm, pH 7.0) was added to quench the cross-linking. Total cell lysates were harvested with SDS-lysis buffer (Upstate Biotechnology), supplemented with protease inhibitor mixture (Roche Applied Science) and RiboLock Ribonuclease inhibitor (MBI E00381) for 10 min on ice. Then the lysates were incubated with corresponding antibodies conjugated with protein G Dynabeads (Invitrogen) or protein A-agarose (Upstate Biotechnology) in the binding buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mm EDTA, 167 mm NaCl, 16.7 mm Tris (pH 8.1), 400 units/ml MBL RiboLock Ribonuclease inhibitor and Roche Applied Science protease inhibitors mixture) at 4 °C overnight with head-over-tail rotation. The precipitated complexes were sequentially washed with buffer containing 500 mm NaCl, 150 mm NaCl, TE buffer (pH 8.0) and then incubated with the elution buffer (1% SDS, 0.1 m NaHCO3, 400 units/ml MBL RiboLock Ribonuclease inhibitor) at 4 °C for 15 min and at 65 °C for 2 h with addition of 5 m NaCl. The bound RNAs were isolated using TRIzol reagent (Invitrogen) and used for the first-strand cDNA synthesis with reverse transcriptase M-MLV(RNase H−) and random hexamer primers (Takara, Japan), according to the manufacturer's protocol. The quantitative real time PCR (Q-PCR) assay was performed to detect the presence of precipitated U1, U2, U4, U5, and U6 snRNA with the specific primers (35.Zhang Z. Lotti F. Dittmar K. Younis I. Wan L. Kasim M. Dreyfuss G. SMN deficiency causes tissue-specific perturbations in the repertoire of snRNAs and widespread defects in splicing.Cell. 2008; 133: 585-600Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar). Amplification specificity was detected by gel electrophoresis and dissociation curve analysis. For the Q-PCR, the RIP fold changes were calculated validly using the ΔΔCt (cycle time) method (36.Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real time quantitative PCR and the 2−ΔΔC(T)) Method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (123298) Google Scholar, 37.Chakrabarti S.K. James J.C. Mirmira R.G. Quantitative assessment of gene targeting in vitroin vivo by the pancreatic transcription factor Pdx1. Importance of chromatin structure in directing promoter binding.J. Biol. Chem. 2002; 277: 13286-13293Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). The ΔCt value of bound snRNAs with either experimental (anti-Y12 or anti-TMG) or control (anti-IgG) beads was normalized to the Input fraction Ct value in the same assay. Then the ΔΔCt value was calculated by subtracting the ΔCt(normalized anti-IgG control RIP) from the ΔCt(normalized Y12 or anti-TMG RIP). Fold change value (experimental RIP versus control RIP) was determined by raising 2 to the power of the negative ΔΔCt. GST pulldown assays were performed as described previously (12.Yang J. Aittomäki S. Pesu M. Carter K. Saarinen J. Kalkkinen N. Kieff E. Silvennoinen O. Identification of p100 as a co-activator for STAT6 that bridges STAT6 with RNA polymerase II.EMBO J. 2002; 21: 4950-4958Crossref PubMed Scopus (143) Google Scholar). GST alone or GST fusion proteins were produced in Escherichia coli BL21 bacteria and bound to glutathione-Sepharose 4B beads (Amersham Biosciences) according to the manufacturer's instructions. The presence of GST fusion protein in the lysate was separated by SDS-PAGE, followed by Coomassie Blue staining. TCLs of COS-7 cells or HeLa cells were collected with Nonidet P-40 lysis buffer (20 mm Tris-HCl (pH 7.6), 100 mm NaCl, 10% glycerol, 1% Nonidet P-40, 2 mm EDTA, and 50 mm NaF) supplemented with protease inhibitor mixture (P8340, Sigma). Protein concentrations of TCLs were measured using the protein assay system from Bio-Rad. TCLs of HeLa cells were treated with or without 100 μl of RNase mixture (MBI Fermentas) at 37 °C for 30 min. The information on the RNase mixture system used in protein binding assay is shown in supplemental Table S1. The bead-bound fusion proteins were incubated with TCLs overnight at 4 °C with head-over-tail rotation and then washed five times with binding buffer containing 75 mm NaCl. The bound proteins were separated by SDS-PAGE and blotted with monoclonal anti-SmB (Clone 12F5, Sigma) or anti-Sm (Y12, ab3138, Abcam). HeLa or COS-7 cells were incubated with or without the methyltransferase inhibitor, 5-deoxy-5-(methylthio)adenosine (250 μm, MTA, Sigma) for 20 h. The total cell lysates of HeLa or COS-7 cells were collected and then incubated with corresponding antibodies conjugated with either protein G Dynabeads (Invitrogen) or protein A-agarose (Upstate Biotechnology) at 4 °C for 12 h with head-over-tail rotation. Bound proteins were subjected to SDS-PAGE and detected by blotting with corresponding antibodies. The following antibodies were used: anti-trimethylguanosine (TMG)-agarose beads and anti-TMG-cap antibody (K121, Calbiochem); mouse monoclonal anti-Tudor-SN antibody and rabbit polyclonal anti-IgG antibody (Santa Cruz Biotechnology); monoclonal anti-SmB antibody (Clone 12F5, Sigma); anti-Sm (Y12, Abcam); mouse monoclonal anti-FLAGM2 coupled with agarose and anti-His-agarose (H-0767, Sigma); goat anti-Tudor-SN (C-17, Santa Cruz Biotechnology); rabbit polyclonal anti-sDMA antibody (anti-dimethylarginine, symmetric, SYM10, Upstate Biotechnology); and rabbit anti-SMN (H-195, Santa Cruz Biotechnology). The rabbit polyclonal anti-Tudor-SN antibody was generated against TSN domain (amino acids 640–885) of Tudor-SN. [32P]UTP-labeled adenovirus-splicing substrates was produced by in vitro transcription with T7 RNA polymerase (Promega) using the Adenovirus Major Late (AdML) plasmid linearized with HindIII as template and purified as described previously (13.Yang J. Välineva T. Hong J. Bu T. Yao Z. Jensen O.N. Frilander M.J. Silvennoinen O. Transcriptional co-activator protein p100 interacts with snRNP proteins and facilitates the assembly of the spliceosome.Nucleic Acids Res. 2007; 35: 4485-4494Crossref PubMed Scopus (98) Google Scholar, 38.Zhou Z. Licklider L.J. Gygi S.P. Reed R. Comprehensive proteomic analysis of the human spliceosome.Nature. 2002; 419: 182-185Crossref PubMed Scopus (720) Google Scholar). The wild- type TSN proteins or TSN with single or double amino acid substitutions were purified as described previously (11.Shaw N. Zhao M. Cheng C. Xu H. Saarikettu J. Li Y. Da Y. Yao Z. Silvennoinen O. Yang J. Liu Z.J. Wang B.C. Rao Z. The multifunctional human p100 protein “hooks” methylated ligands.Nat. Struct. Mol. Biol. 2007; 14: 779-784Crossref PubMed Scopus (64) Google Scholar). The in vitro splicing reactions, containing 40% (v/v) HeLa extracts, 2 mm MgCl2, 10 mm DTT, 20 mm creatine phosphate, and 2 mm ATP, were supplemented with different purified proteins and preincubated at 30 °C for 10 min on ice, followed by addition of 10,000 cpm of the AdML pre-mRNA. The splicing reactions were incubated at 30 °C for different time points and stopped by placing the reaction on ice. Heparin (1 mg/ml final concentration) was added to the splicing reactions prior to loading. Native gel electrophoresis on 4% acrylamide was performed as described previously (39.Frilander M.J. Meng X. Proximity of the U12 snRNA with both the 5′ splice site and the branch point during early stages of spliceosome assembly.Mol. Cell. Biol. 2005; 25: 4813-4825Crossref PubMed Scopus (9) Google Scholar) and visualized by autoradiograph" @default.
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• W2157746712 date "2012-05-01" @default.
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• W2157746712 title "Tudor Staphylococcal Nuclease (Tudor-SN) Participates in Small Ribonucleoprotein (snRNP) Assembly via Interacting with Symmetrically Dimethylated Sm Proteins" @default.
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• W2157746712 doi "https://doi.org/10.1074/jbc.m111.311852" @default.
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