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• W2092321887 abstract "The 5′ end of kinetoplastid mRNA possesses a hypermethylated cap 4 structure, which is derived from standard m7GpppN (cap 0) with additional methylations at seven sites within the first four nucleosides on the spliced leader RNA. In addition to TbCe1 guanylyltransferase and TbCmt1 (guanine N-7) methyltransferase, Trypanosoma brucei encodes a second cap 0 forming enzyme. TbCgm1 (T. brucei cap guanylyltransferase-methyltransferase) is a novel bifunctional capping enzyme consisting of an amino-terminal guanylyltransferase domain and a carboxyl-terminal methyltransferase domain. Recombinant TbCgm1 transfers the GMP to spliced leader RNA (SL RNA) via a covalent enzyme-GMP intermediate, and methylates the guanine N-7 position of the GpppN-terminated RNA to form cap 0 structure. The two domains can function autonomously in vitro. TbCGM1 is essential for parasite growth. Silencing of TbCGM1 by RNA interference increased the abundance of uncapped SL RNA and lead to accumulation of hypomethylated SL RNA. In contrast, silencing of TbCE1 and TbCMT1 did not affect parasite growth or SL RNA capping. We conclude that TbCgm1 specifically cap SL RNA, and cap 0 is a prerequisite for subsequent methylation events leading to the formation of mature SL RNA. The 5′ end of kinetoplastid mRNA possesses a hypermethylated cap 4 structure, which is derived from standard m7GpppN (cap 0) with additional methylations at seven sites within the first four nucleosides on the spliced leader RNA. In addition to TbCe1 guanylyltransferase and TbCmt1 (guanine N-7) methyltransferase, Trypanosoma brucei encodes a second cap 0 forming enzyme. TbCgm1 (T. brucei cap guanylyltransferase-methyltransferase) is a novel bifunctional capping enzyme consisting of an amino-terminal guanylyltransferase domain and a carboxyl-terminal methyltransferase domain. Recombinant TbCgm1 transfers the GMP to spliced leader RNA (SL RNA) via a covalent enzyme-GMP intermediate, and methylates the guanine N-7 position of the GpppN-terminated RNA to form cap 0 structure. The two domains can function autonomously in vitro. TbCGM1 is essential for parasite growth. Silencing of TbCGM1 by RNA interference increased the abundance of uncapped SL RNA and lead to accumulation of hypomethylated SL RNA. In contrast, silencing of TbCE1 and TbCMT1 did not affect parasite growth or SL RNA capping. We conclude that TbCgm1 specifically cap SL RNA, and cap 0 is a prerequisite for subsequent methylation events leading to the formation of mature SL RNA. The 5′ cap is an essential feature of eukaryotic mRNAs and snRNAs, 3The abbreviations used are: snRNA, small nuclear RNA; AdoMet, S-adenosylmethionine; AdoHcy, S-adenosylhomocysteine; DTT, dithiothreitol; pol II, polymerase II; SL RNA, spliced leader RNA; RNAi, RNA interference; nt, nucleotide(s). and is required for RNA stability and efficient translation (1.Furuichi Y. Shatkin A.J. Adv. Virus Res. 2000; 55: 135-184Crossref PubMed Google Scholar). The m7GpppN (cap 0) is formed by sequential action of three enzymatic activities. The 5′ triphosphate of the nacent RNA is hydrolyzed to a diphosphate by RNA triphosphatase, the diphosphate end is capped with GMP by guanylyltransferase, and the GpppN cap is methylated at the N-7 position by (guanine N-7) methyltransferase (2.Shuman S. Prog. Nucleic Acids Res. Mol. Biol. 2001; 66: 1-40Crossref PubMed Google Scholar). Whereas the three-step capping reaction is universal to all eukaryotes, organization of individual activities differs between multicellular and unicellular eukaryotes (3.Shuman S. Nat. Rev. Mol. Cell. Biol. 2002; 3: 619-625Crossref PubMed Scopus (114) Google Scholar). Metazoans and plants have a two-component capping system consisting of a bifunctional triphosphatase-guanylyltransferase protein and a separate (guanine N-7) methyltransferase protein (4.Ho C.K. Sriskanda V. McCracken S. Bentley D. Schwer B. Shuman S. J. Biol. Chem. 1998; 273: 9577-9585Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 5.Takagi T. Walker A.K. Sawa C. Diehn F. Takase Y. Blackwell T.K. Buratowski S. J. Biol. Chem. 2003; 278: 14174-14184Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 6.Tsukamoto T. Shibagaki Y. Murakoshi T. Suzuki M. Nakamura A. Gotoh H. Mizumoto K. Biochem. Biophys. Res. Commun. 1998; 243: 101-108Crossref PubMed Scopus (30) Google Scholar, 7.Yue Z. Maldonado E. Pillutla R. Cho H. Reinberg D. Shatkin A.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12898-12903Crossref PubMed Scopus (196) Google Scholar, 8.Wang S.P. Deng L. Ho C.K. Shuman S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9573-9578Crossref PubMed Scopus (100) Google Scholar, 9.Yamada-Okabe T. Doi R. Shimmi O. Arisawa M. Yamada-Okabe H. Nucleic Acids Res. 1998; 26: 1700-1706Crossref PubMed Scopus (41) Google Scholar). In contrast, fungi and other unicellular eukaryotes, including Encephalitozoon cuniculi, Giardia lamblia, and Plasmodium falciparum have a three-component system consisting of separate RNA triphosphatase, guanylyltransferase, and (guanine N-7) methyltransferase gene products (10.Hausmann S. Vivares C.P. Shuman S. J. Biol. Chem. 2002; 277: 96-103Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 11.Tsukamoto T. Shibagaki Y. Imajoh-Ohmi S. Murakoshi T. Suzuki M. Nakamura A. Gotoh H. Mizumoto K. Biochem. Biophys. Res. Commun. 1997; 239: 116-122Crossref PubMed Scopus (79) Google Scholar, 12.Hausmann S. Altura M.A. Witmer M. Singer S.M. Elmendorf H.G. Shuman S. J. Biol. Chem. 2005; 280: 12077-12086Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 13.Ho C.K. Shuman S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3050-3055Crossref PubMed Scopus (38) Google Scholar, 14.Ho C.K. Schwer B. Shuman S. Mol. Cell. Biol. 1998; 18: 5189-5198Crossref PubMed Google Scholar, 15.Pei Y. Schwer B. Hausmann S. Shuman S. Nucleic Acids Res. 2001; 29: 387-396Crossref PubMed Google Scholar, 16.Yamada-Okabe T. Mio T. Matsui M. Kashima Y. Arisawa M. Yamada-Okabe H. FEBS Lett. 1998; 435: 49-54Crossref PubMed Scopus (30) Google Scholar). Cap is the earliest modification to the nascent transcripts synthesized by pol II. This specificity is achieved through the direct interaction of one or more components of the capping apparatus with the phosphorylated carboxyl-terminal domain of the large subunit of RNA pol II (7.Yue Z. Maldonado E. Pillutla R. Cho H. Reinberg D. Shatkin A.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12898-12903Crossref PubMed Scopus (196) Google Scholar, 17.McCracken S. Fong N. Rosonina E. Yankulov K. Brothers G. Siderovski D. Hessel A. Foster S. Shuman S. Bentley D.L. Genes Dev. 1997; 11: 3306-3318Crossref PubMed Scopus (433) Google Scholar, 18.Cho E.J. Takagi T. Moore C.R. Buratowski S. Genes Dev. 1997; 11: 3319-3326Crossref PubMed Scopus (373) Google Scholar, 19.Pillutla R.C. Yue Z. Maldonado E. Shatkin A.J. J. Biol. Chem. 1998; 273: 21443-21446Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 20.Ho C.K. Shuman S. Mol. Cell. 1999; 3: 405-411Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). In kinetoplastid protozoa, such as Trypanosoma brucei, capping is not restricted to pol II transcripts. Kinetoplastid mRNAs possess a unique hypermethylated cap structure called cap 4, which consists of cap 0 with 2′-O-methylations on the first four ribose sugars (AmAmCmUm), and additional base methylations on the first adenine (m6,6A) and the fourth uracil (m3U) (21.Bangs J.D. Crain P.F. Hashizume T. McCloskey J.A. Boothroyd J.C. J. Biol. Chem. 1992; 267: 9805-9815Abstract Full Text PDF PubMed Google Scholar). The cap 4 structure is formed exclusively on the SL RNA synthesized by pol II, and is transferred via trans-splicing to the 5′ end of individual pre-mRNAs derived from a polycistronic transcript to form mature mRNAs (reviewed in Refs. 22.Adams M.D. Rudner D.Z. Rio D.C. Curr. Opin. Cell Biol. 1996; 8: 331-339Crossref PubMed Scopus (117) Google Scholar, 23.Agabian N. Cell. 1990; 61: 1157-1160Abstract Full Text PDF PubMed Scopus (291) Google Scholar, 24.Liang X.-h. Haritan A. Uliel S. Michaeli S. Eukaryot. Cell. 2003; 2: 830-840Crossref PubMed Scopus (268) Google Scholar). Trypanosome U2, U3, and U4 snRNAs have a 2,2,7-trimethylguanosine cap (m2,2,7G) derived from cap 0 (25.Mottram J. Perry K.L. Lizardi P.M. Luhrmann R. Agabian N. Nelson R.G. Mol. Cell. Biol. 1989; 9: 1212-1223Crossref PubMed Scopus (98) Google Scholar, 26.Marchetti M.A. Tschudi C. Silva E. Ullu E. Nucleic Acids Res. 1998; 26: 3591-3598Crossref PubMed Scopus (25) Google Scholar). However, these snRNAs appear to be synthesized by pol III (27.Fantoni A. Dare A.O. Tschudi C. Mol. Cell. Biol. 1994; 14: 2021-2028Crossref PubMed Scopus (63) Google Scholar, 28.Gunzl A. Tschudi C. Nakaar V. Ullu E. J. Biol. Chem. 1995; 270: 17287-17291Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), whereas other kinetoplastid pol III transcripts, such as tRNA, 5S RNA, and 7SL RNA, lack a cap structure. These findings suggest that the kinetoplastid capping enzyme is capable of capping pol II as well as selected pol III transcripts, and that the recruitment of the capping enzyme to the site of transcription is likely to be different from other eukaryotes. Earlier studies indicated that the T. brucei capping apparatus resembles that of yeast, with separate triphosphatase (TbCet1), guanylyltransferase (TbCe1), and methyltransferase (TbCmt1) components. TbCet1 is a metal-dependent phosphohydrolase that catalyzes the removal of the terminal phosphate from triphosphate-terminated RNA (29.Ho C.K. Shuman S. J. Biol. Chem. 2001; 276: 46182-46186Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). TbCe1 is mechanistically related to other cellular guanylyltransferases except that it contains an amino-terminal extension of 250 amino acids of unknown function (30.Silva E. Ullu E. Kobayashi R. Tschudi C. Mol. Cell. Biol. 1998; 18: 4612-4619Crossref PubMed Scopus (30) Google Scholar). TbCmt1 catalyzes the guanine N-7 methylation on a GpppN-terminated RNA (31.Hall M.P. Ho C.K. RNA (Cold Spring Harbor). 2006; 12: 488-497Google Scholar). In addition, two separate 2′-O-nucleoside methyltransferases implicated in SL RNA cap 4 methylation have been identified and characterized (32.Arhin G.K. Li H. Ullu E. Tschudi C. RNA (Cold Spring Harbor). 2006; 12: 53-62Google Scholar, 33.Arhin G.K. Ullu E. Tschudi C. Mol. Biochem. Parasitol. 2006; 147: 137-139Crossref PubMed Scopus (24) Google Scholar, 34.Hall M.P. Ho C.K. Nucleic Acids Res. 2006; 34: 5594-5602Crossref PubMed Scopus (17) Google Scholar, 35.Zamudio J.R. Mittra B. Zeiner G.M. Feder M. Bujnicki J.M. Sturm N.R. Campbell D.A. Eukaryot. Cell. 2006; 5: 905-915Crossref PubMed Scopus (26) Google Scholar). We recently reported the identification of a second candidate T. brucei capping enzyme, which we named TbCgm1 (31.Hall M.P. Ho C.K. RNA (Cold Spring Harbor). 2006; 12: 488-497Google Scholar). The primary structure of TbCgm1 suggests that the enzyme consists of a guanylyltransferase domain and a methyltransferase domain (Fig. 1A). The amino-terminal portion of TbCgm1 contains the defining sequence motifs of the covalent nucleotidyltransferase superfamily (I, III, IIIa, IV, V, and VI) involved in GTP binding and catalysis, except that the 139-amino acid interval between motifs I and III of TbCgm1 is slightly longer than that of other cellular guanylyltransferases. Motif I (127KADGTR132) contains the presumptive active site lysine to which GMP becomes covalently linked via a phosphoamide bond (8.Wang S.P. Deng L. Ho C.K. Shuman S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9573-9578Crossref PubMed Scopus (100) Google Scholar, 36.Schwer B. Shuman S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4328-4332Crossref PubMed Scopus (61) Google Scholar). Residues that are essential in Saccharomyces cerevisiae guanylyltransferase Ceg1 are conserved in TbCgm1, as well as those residues that make direct contacts with the GTP substrate, as deduced from the Chlorella virus guanylyltransferase-GTP cocrystal structure (8.Wang S.P. Deng L. Ho C.K. Shuman S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9573-9578Crossref PubMed Scopus (100) Google Scholar, 37.Hakansson K. Doherty A.J. Shuman S. Wigley D.B. Cell. 1997; 89: 545-553Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 38.Hakansson K. Wigley D.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1505-1510Crossref PubMed Scopus (58) Google Scholar). The carboxyl-terminal portion of TbCgm1 contains an AdoMet binding motif (785VADLCSGRGG794), along with a number of key residues that make direct contacts with the GpppA cap as shown in the crystal structure of E. cuniculi methyltransferase (39.Fabrega C. Hausmann S. Shen V. Shuman S. Lima C.D. Mol. Cell. 2004; 13: 77-89Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Here, we show that purified recombinant TbCgm1 is a bifunctional capping enzyme with an amino-terminal guanylyltransferase (amino acids: 1–567) domain and a carboxyl-terminal (guanine N-7) methyltransferase domain (amino acids 717–1050). Each domain can function autonomously in vitro. TbCGM1 is essential for parasite growth. RNAi-mediated down-regulation of TbCGM1 shows reduced levels of cap 4 methylation on SL RNA. In contrast, down-regulation of TbCE1 and TbCMT1 were not essential for viability and did not affect SL RNA capping. Together, these results demonstrate that the bifunctional TbCgm1 is responsible for m7GpppN formation on the SL RNA. TbCGM1 Expression Plasmids−The TbCGM1 gene (accession number XP_840738.1) was PCR amplified from total T. brucei brucei genomic DNA (a gift of Laurie Read, SUNY at Buffalo) and cloned into BamHI and XhoI sites of pET28-His-Smt3 vector (a gift of Chris Lima, Sloan-Kettering Institute) to fuse the 1050-amino acid TbCgm1 polypeptide in-frame to the amino-terminal His-Smt3 tag to obtain pET-HisSmt3-TbCGM1. The carboxyl-terminal truncation mutant, TbCGM1-(1–567), was constructed by PCR amplification using sense primer that introduced an NdeI site at a start codon and an antisense primer that introduced an XhoI site immediately downstream of the new stop codon at Glu568. TbCGM1-(717–1050) was constructed by PCR amplification using a sense primer that introduced a translation start codon at Leu716 with an NdeI site at the new start codon. The PCR products were digested with NdeI and XhoI, and then inserted into pET16b to obtain pET-TbCGM1-(1–567) and pET-TbCGM1-(717–1050), respectively. Expression and Purification of Recombinant TbCgm1−pET-HisSmt3-TbCGM1 was transformed into Escherichia coli BL21(ROS2). A 1-liter culture amplified from a single transformant colony was grown at 37 °C in LB medium containing 60 μg/ml kanamycin and 100 μg/ml chloramphenicol until the A600 reached 0.4. The culture was adjusted to 2% ethanol and incubated at 17 °C for 18 h. Cells were harvested by centrifugation and stored at –80 °C. Thawed bacteria were resuspended in 50 ml of Buffer A (50 mm Tris-HCl, pH 7.5, 0.25 m NaCl, 10% sucrose). Lysozyme and Triton X-100 were added to final concentrations of 50 μg/ml and 0.1%, respectively. The lysates were sonicated to reduce viscosity, and insoluble material was removed by centrifugation for 45 min at 14,000 × g in a Beckman T14-50 rotor. The soluble lysate was applied to 1.5-ml columns of nickel-nitrilotriacetic acid-agarose (Qiagen) equilibrated with Buffer A containing 0.1% Triton X-100. The column was washed with 15 ml of the same buffer and eluted stepwise with 3 ml of Buffer B (50 mm Tris-HCl, pH 8, 0.25 m NaCl, 10% glycerol, 0.05% Triton X-100) containing 0, 0.005, 0.05, 0.1, 0.2, 0.5, and 1 m imidazole. The recombinant His-Smt3-TbCgm1 was recovered in the 0.2 m imidazole eluate (0.5 mg of proteins per 1-liter culture). Two hundred micrograms of His-Smt3-TbCgm1 polypeptide was incubated with 60 μg of His-tagged ULP1 protease on ice for 1 h to cleave the NH2-terminal His-Smt3 tag. Sample was then diluted to 0.05 m imidazole in Buffer B and applied to 0.5 ml of nickel-agarose equilibrated with Buffer B. The native TbCgm1 protein was recovered in a flow-though fraction and concentrated to 0.17 mg/ml. All enzyme fractions were stored at –80 °C and thawed on ice just prior to use. Protein concentrations were determined using the Bio-Rad dye-binding assay with bovine serum albumin as a standard. TbCgm1-(1–567) and TbCgm1-(717–1050) proteins were expressed in 500-ml cultures of E. coli BL21(DE3) in LB medium containing 100 μg/ml ampicillin at 37 °C until the A600 reached 0.4. The cultures were adjusted to 0.4 mm isopropyl β-d-thiogalactoside and 2% ethanol, and incubated for 18 h at 17 °C. Cells were harvested by centrifugation and stored at –80 °C. Thawed bacteria were resuspended in 20 ml of Buffer A. Soluble lysates were prepared as described for full-length TbCgm1 and then applied to 1-ml columns of nickel-nitrilotriacetic acid-agarose equilibrated with Buffer A containing 0.1% Triton X-100. Columns were washed with 10 ml of Buffer A containing 0.1% Triton X-100 and eluted stepwise with Buffer B containing 0, 0.05, 0.1, 0.2, 0.5, and 1 m imidazole. Recombinant proteins retained on the column and were recovered predominantly in the 0.2 m imidazole eluate (1.6 mg of TbCgm1-(1–567) and 3.3 mg of TbCgm1-(717–1050) per 500 ml of culture). Guanylyltransferase Assay−Standard reaction mixtures (10 μl) containing 50 mm Tris-HCl (pH 7.5), 5 mm DTT, 2 mm MgCl2, 20 μm [α-32P]GTP, and enzyme were incubated at 30 °C for 15 min. The reactions were quenched with SDS loading buffer and the products were resolved by 10% SDS-PAGE. Enzyme-[32P]GMP adduct was visualized by autoradiography of the dried gel and quantified by scanning the gel with a Storm 860 PhosphorImager. Cap Methyltransferase Assay−Triphosphate-terminated poly(A) was synthesized (40.Shuman S. Surks M. Furneaux H. Hurwitz J. J. Biol. Chem. 1980; 255: 11588-11598Abstract Full Text PDF PubMed Google Scholar) and then converted to 32P cap-labeled poly(A) (m7GpppA terminated poly(A): boldface indicates radiolabeled phosphate) as described previously (41.Ho C.K. Van Etten J.L. Shuman S. J. Virol. 1996; 70: 6658-6664Crossref PubMed Google Scholar). The length of cap-labeled poly(A) was between 150 and 250 nt. Standard reaction mixtures (10 μl) containing 50 mm Tris acetate (pH 6), 2 mm DTT, 20 μm AdoMet, 67 fmol of 32P cap-labeled poly(A), and either TbCgm1 or TbCgm1-(717–1050) were incubated for 30 min at 30 °C. The reaction mixtures were adjusted to 100 mm sodium acetate (pH 5.5) and incubated with 100 ng of nuclease P1 for 60 min at 37 °C. Aliquots (3 μl) were spotted onto a PEI cellulose thin-layer chromatography (TLC) plate, which was developed with 0.45 m ammonium sulfate. The extent of methylation (m7GpppA/[m7GpppA + GpppA]) was quantified by scanning the TLC plate with a phosphorimager. RNA Interference, Cell Lines, and Growth Analysis−A SalI-HindIII fragment of TbCGM1 (500 bp, 2089–2587 nt) was cloned into XhoI-HindIII sites of the pZJMβ vector (42.Wang Z. Morris J.C. Drew M.E. Englund P.T. J. Biol. Chem. 2000; 275: 40174-40179Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar) to generate pZJM-TbCGM1. DNA fragments specific for TbCE1 (347 bp, 106–453 nt) and for TbCMT1 (410 bp, 565–975 nt) were PCR amplified and each fragment was inserted into XhoI-BamHI sites of the p2T7-177 vector (43.Wickstead B. Ersfeld K. Gull K. Mol. Biochem. Parasitol. 2002; 125: 211-216Crossref PubMed Scopus (205) Google Scholar), to generate p2T7-TbCE1 and p2T7-TbCMT1, respectively. In all three RNAi constructs, expression of double-stranded RNA is under control of opposing tetracycline-inducible T7 promoters. RNAi constructs were linearized with NotI, phenol-chloroform extracted, ethanol precipitated, and electroporated into procyclic T. brucei 29.13 cells, which constitutively express T7 RNA polymerase and Tet repressor proteins (44.Wirtz E. Leal S. Ochatt C. Cross G.A. Mol. Biochem. Parasitol. 1999; 99: 89-101Crossref PubMed Scopus (1126) Google Scholar). This procyclic cell line was cultivated in SDM 79 medium supplemented with 15% fetal bovine serum (Hyclone) containing 50 μg/ml hygromycin B and 15 μg/ml G418. The transfectants were selected with 2.5 μg/ml phleomycin. For induction of RNAi, transfectants were cultured in medium supplemented with 1.0 μg/ml tetracycline. Cells were counted at different time intervals under a microscope using a hemocytometer. Growth curves represent the log of the direct cell count multiplied by the dilution factor. Parasite density was maintained between 1 × 106 and 1 × 107 cells/ml. Control parasites were from clonal population of cells not treated with tetracycline but grown in parallel with tetracycline-induced cells. For preparation of whole cell extract, 2 × 108 cells were resuspended in 1 ml of buffer C (50 mm Tris-HCl, pH 7.5, 20 mm NaCl, 10% sucrose, 0.1% Triton X-100 and 1× protease inhibitor mixture (Research Product International)). For guanylyltransferase assay, aliquots (12.5 μg) of whole cell extract were incubated in reaction mixtures (16 μl) containing 12.5 mm MgCl2 and 4 μm [α-32P]GTP. The enzyme-GMP complex was resolved on a 10% SDS-PAGE and visualized by phosphorimager. For Western blot analysis, 25 μg of uninduced and induced TbCMT1-RNAi whole cell extracts were resuspended in SDS loading buffer. Samples were separated by electrophoresis on 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. TbCmt1 was detected using rabbit polyclonal serum against recombinant TbCmt1 protein (1:500 dilution, prepared by Pocono Rabbit Farm & Laboratory, PA) and horseradish peroxidase-conjugated secondary antibody (1:5,000 dilution), and developed using an ECL kit from Pierce. RNA Analysis−Total RNA was purified from 2 × 107 cells by TRIzol reagent (Invitrogen). 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Total RNA (20 μg) extracted from both tetracycline-induced and -uninduced RNAi cells at the indicated time points were incubated with anti-m2,2,7G beads (20 μl) in 75 μl of buffer D (10 mm Tris-HCl, pH 7.5, 0.1 m NaCl and 1 mm EDTA) for 1 h at 16°C with gentle rocking. Immune complexes were recovered by centrifugation at 10,000 × g for 5 min and washed three times with 0.5 ml of buffer D containing 0.2 m NaCl. Bound RNA was resuspended in 75 μl of buffer D. Five microliters of bound and unbound fractions were used for primer extension analysis as described above. For RNase H digestion, SL-(29–50), 5′-GCTTCTCATACCAATATAGTAC, and SL-(18–39), 5′-CAATATAGTACAGAAACTGTTC, oligonucleotides were used in addition to the SL-(35–56), U2, and 5S oligonucleotides listed above. TbCgm1 Is a Bifunctional Capping Enzyme with Guanylyltransferase and Methyltransferase Activities−To determine whether TbCgm1 has an intrinsic capping activity, we expressed the recombinant protein in E. coli as an NH2-terminal His-Smt3-tagged fusion protein to facilitate solubility and purification. The fusion protein was purified from soluble bacterial lysate by adsorption to nickel-agarose and elution with 0.2 m imidazole (Fig. 1B, lanes 1 and 2). The native TbCgm1 protein was obtained by cleaving the fusion protein with His-tagged ULP1 protease, followed by removal of the His-smt3 tag by a second round of nickel-agarose chromatography (Fig. 1B, lane 3). TbCgm1 was further purified away from a ∼65-kDa bacteria contaminant (Fig. 1B, asterisk) by glycerol gradient sedimentation (see below). The guanylyltransferase reaction entails two sequential nucleotidyl transfer steps. In the first step, nucleophilic attack on the α-phosphate of GTP results in formation of a covalent EpG intermediate and liberation of PPi. The guanylyltransferase activity of TbCgm1 was evinced by label transfer from [α-32P]GTP to the TbCgm1 polypeptide to form a SDS-stable nucleotidyl-protein adduct that migrated as a 116-kDa species (Fig. 1C, lane 3). Note that three other polypeptides in the range of 60–100 kDa were labeled with GMP to a lesser extent, which suggested that these were proteolytic fragments of TbCgm1. We conclude that TbCgm1 is a covalent nucleotidyltransferase. The methyltransferase activity of TbCgm1 was assayed by conversion of 32P cap-labeled poly(A) to methylated cap-labeled poly(A) in the presence of AdoMet. Digestion by nuclease P1 liberated a labeled species that co-migrated with m7GpppA, generated in a parallel reaction mixture containing purified yeast Abd1 (Fig. 1D, lanes 2 and 5, respectively). The activity was dependent on AdoMet and the inclusion of AdoHcy was inhibitory to the reaction (Fig. 1D, lane 4). We further verified that methylation occurs at the terminal guanosine nucleoside by digesting the reaction products with nucleotide pyrophosphatase, a nuclease that cleaves between the γ and β phosphates in the capped structure. Both the TbCgm1 and Abd1 reaction products liberated m7Gp (data not shown). We conclude that TbCgm1 catalyzes methylation at the N-7 position of the terminal cap guanosine to form cap 0. The native size of TbCgm1 was gauged by sedimentation through a 15–30% glycerol gradient and the fractions were assayed for enzyme-GMP formation and methyltransferase activities. The guanylyltransferase and methyltransferase activities co-sedimented as a 6.7 S peak (Fig. 1E). Based on the predicted molecular weight, we conclude that TbCgm1 is a monomeric protein in solution. The Amino-terminal Domain of TbCgm1 Can Function Autonomously as Guanylyltransferase−To evaluate whether the amino-terminal portion of TbCgm1 constituted an autonomous functional guanylyltransferase domain, we expressed the TbCgm1 segment from residues 1–567 as a His-tagged fusion protein (Fig. 2A). The choice of residue 567 as a domain breakpoint was based on the location of motif VI in TbCgm1, which is situated at residues 491–500 in TbCgm1 (Fig. 1A). In most cellular guanylyltransferases, motif VI is positioned ∼50 amino acids upstream of the carboxyl-terminal end (20.Ho C.K. Shuman S. Mol. Cell. 1999; 3: 405-411Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 21.Bangs J.D. Crain P.F. Hashizume T. McCloskey J.A. Boothroyd J.C. J. Biol. Chem. 1992; 267: 9805-9815Abstract Full Text PDF PubMed Google Scholar). The isopropyl β-d-thiogalactoside-induced bacteria accumulated substantial amounts of a soluble 65-kDa polypeptid" @default.
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• W2092321887 title "Trypanosoma brucei Encodes a Bifunctional Capping Enzyme Essential for Cap 4 Formation on the Spliced Leader RNA" @default.
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