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Enzymatic characterization of three human RNA adenosine methyltransferases reveals diverse substrate affinities and reaction optima

2021; Elsevier BV; Volume: 296; Linguagem: Inglês

10.1016/j.jbc.2021.100270

ISSN

1083-351X

Autores

Dan Yu, Gundeep Kaur, Robert Blumenthal, Xing Zhang, Xiaodong Cheng,

Tópico(s)

RNA regulation and disease

Resumo

RNA methylations of varied RNA species (mRNA, tRNA, rRNA, non-coding RNA) generate a range of modified nucleotides, including N6-methyladenosine. Here we study the enzymology of three human RNA methyltransferases that methylate the adenosine amino group in diverse contexts, when it is: the first transcribed nucleotide after the mRNA cap (PCIF1), at position 1832 of 18S rRNA (MettL5-Trm112 complex), and within a hairpin in the 3′ UTR of the S-adenosyl-l-methionine synthetase (MettL16). Among these three enzymes, the catalytic efficiency ranges from PCIF1, with the fastest turnover rate of >230 h−1 μM−1 on mRNA cap analog, down to MettL16, which has the lowest rate of ∼3 h−1 μM−1 acting on an RNA hairpin. Both PCIF1 and MettL5 have a binding affinity (Km) of ∼1 μM or less for both substrates of SAM and RNA, whereas MettL16 has significantly lower binding affinities for both (Km >0.4 mM for SAM and ∼10 μM for RNA). The three enzymes are active over a wide pH range (∼5.4–9.4) and have different preferences for ionic strength. Sodium chloride at 200 mM markedly diminished methylation activity of MettL5-Trm112 complex, whereas MettL16 had higher activity in the range of 200 to 500 mM NaCl. Zinc ion inhibited activities of all three enzymes. Together, these results illustrate the diversity of RNA adenosine methyltransferases in their enzymatic mechanisms and substrate specificities and underline the need for assay optimization in their study. RNA methylations of varied RNA species (mRNA, tRNA, rRNA, non-coding RNA) generate a range of modified nucleotides, including N6-methyladenosine. Here we study the enzymology of three human RNA methyltransferases that methylate the adenosine amino group in diverse contexts, when it is: the first transcribed nucleotide after the mRNA cap (PCIF1), at position 1832 of 18S rRNA (MettL5-Trm112 complex), and within a hairpin in the 3′ UTR of the S-adenosyl-l-methionine synthetase (MettL16). Among these three enzymes, the catalytic efficiency ranges from PCIF1, with the fastest turnover rate of >230 h−1 μM−1 on mRNA cap analog, down to MettL16, which has the lowest rate of ∼3 h−1 μM−1 acting on an RNA hairpin. Both PCIF1 and MettL5 have a binding affinity (Km) of ∼1 μM or less for both substrates of SAM and RNA, whereas MettL16 has significantly lower binding affinities for both (Km >0.4 mM for SAM and ∼10 μM for RNA). The three enzymes are active over a wide pH range (∼5.4–9.4) and have different preferences for ionic strength. Sodium chloride at 200 mM markedly diminished methylation activity of MettL5-Trm112 complex, whereas MettL16 had higher activity in the range of 200 to 500 mM NaCl. Zinc ion inhibited activities of all three enzymes. Together, these results illustrate the diversity of RNA adenosine methyltransferases in their enzymatic mechanisms and substrate specificities and underline the need for assay optimization in their study. Postsynthetic methylations of DNA and RNA are common and are well known to play significant roles in a wide range of cellular functions in bacterial and archaea (1Nye T.M. Fernandez N.L. Simmons L.A. A positive perspective on DNA methylation: Regulatory functions of DNA methylation outside of host defense in Gram-positive bacteria.Crit. Rev. Biochem. Mol. Biol. 2020; 55: 576-591Crossref PubMed Scopus (4) Google Scholar, 2Sanchez-Romero M.A. Casadesus J. The bacterial epigenome.Nat. Rev. Microbiol. 2020; 18: 7-20Crossref PubMed Scopus (49) Google Scholar, 3Deng X. Chen K. Luo G.Z. Weng X. Ji Q. Zhou T. He C. Widespread occurrence of N6-methyladenosine in bacterial mRNA.Nucleic Acids Res. 2015; 43: 6557-6567Crossref PubMed Scopus (103) Google Scholar), such as adenine methylation-directed mismatch repair in Escherichia coli (4Wyrzykowski J. Volkert M.R. The Escherichia coli methyl-directed mismatch repair system repairs base pairs containing oxidative lesions.J. Bacteriol. 2003; 185: 1701-1704Crossref PubMed Scopus (61) Google Scholar). These enzyme-driven chemical reactions use S-adenosyl-l-methionine (SAM) as the methyl donor and transfer the methyl group onto DNA or RNA at the ring carbon C5 of cytosine (yielding 5-methylcytosine, 5mC) or at the exocyclic amino groups of either cytosine at N4 (yielding N4-methylcytosine, N4mC) or adenine at N6 (yielding N6-methyladenine, N6mA) (5Cheng X. Blumenthal R.M. S-Adenosylmethionine-Dependent Methyltransferases: Structures and Functions. World Scientifc Publishing Co, River Edge, NJ1999Crossref Google Scholar). [There are also methyltransferases (MTases) that modify other parts of the nucleic acid (e.g., (6Ayadi L. Galvanin A. Pichot F. Marchand V. Motorin Y. RNA ribose methylation (2'-O-methylation): Occurrence, biosynthesis and biological functions.Biochim. Biophys. Acta Gene Regul. Mech. 2019; 1862: 253-269Crossref PubMed Scopus (54) Google Scholar) and references therein), but here we are considering only modification of the bases.] Mammalian DNA 5mC is a major epigenetic regulator in development and disease (7Greenberg M.V.C. Bourc'his D. The diverse roles of DNA methylation in mammalian development and disease.Nat. Rev. Mol. Cell. Biol. 2019; 20: 590-607Crossref PubMed Scopus (436) Google Scholar), while in mammals the amino modification of cytosine in DNA (N4mC) has not been established and that of adenine in DNA (N6mA) is controversial. While noted immunochemically as early as 1983 (8Achwal C.W. Iyer C.A. Chandra H.S. Immunochemical evidence for the presence of 5mC, 6mA and 7mG in human, Drosophila and mealybug DNA.FEBS Lett. 1983; 158: 353-358Crossref PubMed Scopus (61) Google Scholar), N6mA was reported in mammalian DNA using very sensitive approaches only in 2016 (9Wu T.P. Wang T. Seetin M.G. Lai Y. Zhu S. Lin K. Liu Y. Byrum S.D. Mackintosh S.G. Zhong M. Tackett A. Wang G. Hon L.S. Fang G. Swenberg J.A. et al.DNA methylation on N(6)-adenine in mammalian embryonic stem cells.Nature. 2016; 532: 329-333Crossref PubMed Scopus (338) Google Scholar), and its existence in mammals is still debatable (10Schiffers S. Ebert C. Rahimoff R. Kosmatchev O. Steinbacher J. Bohne A.V. Spada F. Michalakis S. Nickelsen J. Muller M. Carell T. Quantitative LC-MS provides no evidence for m(6) dA or m(4) dC in the genome of mouse embryonic stem cells and tissues.Angew. Chem. Int. Ed. Engl. 2017; 56: 11268-11271Crossref PubMed Scopus (60) Google Scholar, 11Douvlataniotis K. Bensberg M. Lentini A. Gylemo B. Nestor C.E. No evidence for DNA N (6)-methyladenine in mammals.Sci. Adv. 2020; 6eaay3335Crossref PubMed Scopus (31) Google Scholar). Remaining unsettled questions include how N6mA is generated in mammalian DNA (12Musheev M.U. Baumgartner A. Krebs L. Niehrs C. The origin of genomic N(6)-methyl-deoxyadenosine in mammalian cells.Nat. Chem. Biol. 2020; 16: 630-634Crossref PubMed Scopus (22) Google Scholar, 13Liu X. Lai W. Li Y. Chen S. Liu B. Zhang N. Mo J. Lyu C. Zheng J. Du Y.R. Jiang G. Xu G.L. Wang H. N(6)-methyladenine is incorporated into mammalian genome by DNA polymerase.Cell Res. 2020; 31: 94-97Crossref PubMed Scopus (15) Google Scholar) and identification of the potential adenine DNA MTase(s) (14Xie Q. Wu T.P. Gimple R.C. Li Z. Prager B.C. Wu Q. Yu Y. Wang P. Wang Y. Gorkin D.U. Zhang C. Dowiak A.V. Lin K. Zeng C. Sui Y. et al.N(6)-methyladenine DNA modification in glioblastoma.Cell. 2018; 175: 1228-1243Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 15Woodcock C.B. Yu D. Zhang X. Cheng X. Human HemK2/KMT9/N6AMT1 is an active protein methyltransferase, but does not act on DNA in vitro, in the presence of Trm112.Cell Discov. 2019; 5: 50Crossref PubMed Scopus (13) Google Scholar, 16Li W. Shi Y. Zhang T. Ye J. Ding J. Structural insight into human N6amt1-Trm112 complex functioning as a protein methyltransferase.Cell Discov. 2019; 5: 51Crossref PubMed Scopus (10) Google Scholar). In contrast, the RNA methylation generating N6mA has been found in most eukaryotic RNA molecules including mRNA, tRNA, rRNA, noncoding RNA, and chromosome-associated regulatory RNA (17Liu S. Zhu A. He C. Chen M. REPIC: A database for exploring the N(6)-methyladenosine methylome.Genome Biol. 2020; 21: 100Crossref PubMed Scopus (18) Google Scholar, 18Liu J. Dou X. Chen C. Chen C. Liu C. Xu M.M. Zhao S. Shen B. Gao Y. Han D. He C. N (6)-methyladenosine of chromosome-associated regulatory RNA regulates chromatin state and transcription.Science. 2020; 367: 580-586Crossref PubMed Scopus (136) Google Scholar, 19Ru W. Zhang X. Yue B. Qi A. Shen X. Huang Y. Lan X. Lei C. Chen H. Insight into m(6)A methylation from occurrence to functions.Open Biol. 2020; 10: 200091Crossref PubMed Scopus (7) Google Scholar, 20Boo S.H. Kim Y.K. The emerging role of RNA modifications in the regulation of mRNA stability.Exp. Mol. Med. 2020; 52: 400-408Crossref PubMed Scopus (55) Google Scholar, 21Frye M. Jaffrey S.R. Pan T. Rechavi G. Suzuki T. RNA modifications: What have we learned and where are we headed?.Nat. Rev. Genet. 2016; 17: 365-372Crossref PubMed Scopus (132) Google Scholar). Here, we chose three recently identified human RNA adenine MTases: PCIF1, acting on mRNA, MettL5 on rRNA, and MettL16 on snRNA. PCIF1—named as phosphorylated RNA polymerase II CTD interacting factor 1 (22Fan H. Sakuraba K. Komuro A. Kato S. Harada F. Hirose Y. PCIF1, a novel human WW domain-containing protein, interacts with the phosphorylated RNA polymerase II.Biochem. Biophys. Res. Commun. 2003; 301: 378-385Crossref PubMed Scopus (31) Google Scholar)—methylates adenosine when it is the first transcribed nucleotide after the mRNA cap (23Akichika S. Hirano S. Shichino Y. Suzuki T. Nishimasu H. Ishitani R. Sugita A. Hirose Y. Iwasaki S. Nureki O. Suzuki T. Cap-specific terminal N (6)-methylation of RNA by an RNA polymerase II-associated methyltransferase.Science. 2019; 363eaav0080Crossref PubMed Scopus (138) Google Scholar, 24Sun H. Zhang M. Li K. Bai D. Yi C. Cap-specific, terminal N(6)-methylation by a mammalian m(6)Am methyltransferase.Cell Res. 2019; 29: 80-82Crossref PubMed Scopus (76) Google Scholar, 25Boulias K. Toczydlowska-Socha D. Hawley B.R. Liberman N. Takashima K. Zaccara S. Guez T. Vasseur J.J. Debart F. Aravind L. Jaffrey S.R. Greer E.L. Identification of the m(6)Am methyltransferase PCIF1 reveals the location and functions of m(6)Am in the transcriptome.Mol. Cell. 2019; 75: 631-643Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 26Sendinc E. Valle-Garcia D. Dhall A. Chen H. Henriques T. Navarrete-Perea J. Sheng W. Gygi S.P. Adelman K. Shi Y. PCIF1 catalyzes m6Am mRNA methylation to regulate gene expression.Mol. Cell. 2019; 75: 620-630Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). PCIF1 affects mRNA levels in mouse (27Pandey R.R. Delfino E. Homolka D. Roithova A. Chen K.M. Li L. Franco G. Vagbo C.B. Taillebourg E. Fauvarque M.O. Pillai R.S. The mammalian cap-specific m(6)Am RNA methyltransferase PCIF1 regulates transcript levels in mouse tissues.Cell Rep. 2020; 32: 108038Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar), though this effect might be mediated via its direct interaction with the phosphorylated C-terminal domain of RNA polymerase II, as has been demonstrated for the mouse, human, and Drosophila orthologs (27Pandey R.R. Delfino E. Homolka D. Roithova A. Chen K.M. Li L. Franco G. Vagbo C.B. Taillebourg E. Fauvarque M.O. Pillai R.S. The mammalian cap-specific m(6)Am RNA methyltransferase PCIF1 regulates transcript levels in mouse tissues.Cell Rep. 2020; 32: 108038Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 28Hirose Y. Iwamoto Y. Sakuraba K. Yunokuchi I. Harada F. Ohkuma Y. Human phosphorylated CTD-interacting protein, PCIF1, negatively modulates gene expression by RNA polymerase II.Biochem. Biophys. Res. Commun. 2008; 369: 449-455Crossref PubMed Scopus (18) Google Scholar). MettL5 forms a heterodimer with Trm112, a conserved protein that binds to and stabilizes various MTase proteins and was named initially for its role in tRNA methylation (29Bourgeois G. Letoquart J. van Tran N. Graille M. Trm112, a protein activator of methyltransferases modifying actors of the eukaryotic translational apparatus.Biomolecules. 2017; 7: 7Crossref Scopus (30) Google Scholar, 30van Tran N. Muller L. Ross R.L. Lestini R. Letoquart J. Ulryck N. Limbach P.A. de Crecy-Lagard V. Cianferani S. Graille M. Evolutionary insights into Trm112-methyltransferase holoenzymes involved in translation between archaea and eukaryotes.Nucleic Acids Res. 2018; 46: 8483-8499Crossref PubMed Scopus (20) Google Scholar). The MettL5-Trm112 complex is responsible for 18S rRNA adenine methylation at position 1832 (31van Tran N. Ernst F.G.M. Hawley B.R. Zorbas C. Ulryck N. Hackert P. Bohnsack K.E. Bohnsack M.T. Jaffrey S.R. Graille M. Lafontaine D.L.J. The human 18S rRNA m6A methyltransferase METTL5 is stabilized by TRMT112.Nucleic Acids Res. 2019; 47: 7719-7733Crossref PubMed Scopus (107) Google Scholar, 32Ignatova V.V. Stolz P. Kaiser S. Gustafsson T.H. Lastres P.R. Sanz-Moreno A. Cho Y.L. Amarie O.V. Aguilar-Pimentel A. Klein-Rodewald T. Calzada-Wack J. Becker L. Marschall S. Kraiger M. Garrett L. et al.The rRNA m(6)A methyltransferase METTL5 is involved in pluripotency and developmental programs.Genes Dev. 2020; 34: 715-729Crossref PubMed Scopus (29) Google Scholar, 33Leismann J. Spagnuolo M. Pradhan M. Wacheul L. Vu M.A. Musheev M. Mier P. Andrade-Navarro M.A. Graille M. Niehrs C. Lafontaine D.L. Roignant J.Y. The 18S ribosomal RNA m(6) A methyltransferase Mettl5 is required for normal walking behavior in Drosophila.EMBO Rep. 2020; 21e49443Crossref PubMed Scopus (22) Google Scholar, 34Rong B. Zhang Q. Wan J. Xing S. Dai R. Li Y. Cai J. Xie J. Song Y. Chen J. Zhang L. Yan G. Zhang W. Gao H. Han J.J. et al.Ribosome 18S m(6)A methyltransferase METTL5 promotes translation initiation and breast cancer cell growth.Cell Rep. 2020; 33: 108544Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 35Xing M. Liu Q. Mao C. Zeng H. Zhang X. Zhao S. Chen L. Liu M. Shen B. Guo X. Ma H. Chen H. Zhang J. The 18S rRNA m(6)A methyltransferase METTL5 promotes mouse embryonic stem cell differentiation.EMBO Rep. 2020; 21e49863Crossref PubMed Scopus (12) Google Scholar). Finally, MettL16 catalyzes adenine methylation in the conserved sequence UACAGAGAA, within hairpins in the 3′ UTR of the SAM synthetase (MAT2A) mRNA and in U6 snRNA (36Shima H. Matsumoto M. Ishigami Y. Ebina M. Muto A. Sato Y. Kumagai S. Ochiai K. Suzuki T. Igarashi K. S-adenosylmethionine synthesis is regulated by selective N(6)-adenosine methylation and mRNA degradation involving METTL16 and YTHDC1.Cell Rep. 2017; 21: 3354-3363Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 37Pendleton K.E. Chen B. Liu K. Hunter O.V. Xie Y. Tu B.P. Conrad N.K. The U6 snRNA m(6)A methyltransferase METTL16 regulates SAM synthetase intron retention.Cell. 2017; 169: 824-835Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar, 38Warda A.S. Kretschmer J. Hackert P. Lenz C. Urlaub H. Hobartner C. Sloan K.E. Bohnsack M.T. Human METTL16 is a N(6)-methyladenosine (m(6)A) methyltransferase that targets pre-mRNAs and various non-coding RNAs.EMBO Rep. 2017; 18: 2004-2014Crossref PubMed Scopus (227) Google Scholar). The discovery of adenine methylation is often aided by the sensitive mass spectrometry approach (23Akichika S. Hirano S. Shichino Y. Suzuki T. Nishimasu H. Ishitani R. Sugita A. Hirose Y. Iwasaki S. Nureki O. Suzuki T. Cap-specific terminal N (6)-methylation of RNA by an RNA polymerase II-associated methyltransferase.Science. 2019; 363eaav0080Crossref PubMed Scopus (138) Google Scholar, 26Sendinc E. Valle-Garcia D. Dhall A. Chen H. Henriques T. Navarrete-Perea J. Sheng W. Gygi S.P. Adelman K. Shi Y. PCIF1 catalyzes m6Am mRNA methylation to regulate gene expression.Mol. Cell. 2019; 75: 620-630Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 32Ignatova V.V. Stolz P. Kaiser S. Gustafsson T.H. Lastres P.R. Sanz-Moreno A. Cho Y.L. Amarie O.V. Aguilar-Pimentel A. Klein-Rodewald T. Calzada-Wack J. Becker L. Marschall S. Kraiger M. Garrett L. et al.The rRNA m(6)A methyltransferase METTL5 is involved in pluripotency and developmental programs.Genes Dev. 2020; 34: 715-729Crossref PubMed Scopus (29) Google Scholar, 34Rong B. Zhang Q. Wan J. Xing S. Dai R. Li Y. Cai J. Xie J. Song Y. Chen J. Zhang L. Yan G. Zhang W. Gao H. Han J.J. et al.Ribosome 18S m(6)A methyltransferase METTL5 promotes translation initiation and breast cancer cell growth.Cell Rep. 2020; 33: 108544Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 36Shima H. Matsumoto M. Ishigami Y. Ebina M. Muto A. Sato Y. Kumagai S. Ochiai K. Suzuki T. Igarashi K. S-adenosylmethionine synthesis is regulated by selective N(6)-adenosine methylation and mRNA degradation involving METTL16 and YTHDC1.Cell Rep. 2017; 21: 3354-3363Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). We note that these three MTases all possess conserved sequence motifs for binding cofactor SAM (motif I) and for catalysis of amino (NH2)-methylation (motif IV) in a particular order in their amino acid sequence (39Malone T. Blumenthal R.M. Cheng X. Structure-guided analysis reveals nine sequence motifs conserved among DNA amino-methyltransferases, and suggests a catalytic mechanism for these enzymes.J. Mol. Biol. 1995; 253: 618-632Crossref PubMed Scopus (418) Google Scholar) and retain the characteristic overall fold of seven-stranded class-I MTases (40Schubert H.L. Blumenthal R.M. Cheng X. Many paths to methyltransfer: A chronicle of convergence.Trends Biochem. Sci. 2003; 28: 329-335Abstract Full Text Full Text PDF PubMed Scopus (617) Google Scholar) (Fig. S1). Here, we found a surprisingly wide range of optimal conditions for the in vitro enzymatic activity of each enzyme acting on its known RNA substrates. We first optimized the enzymatic activity of purified recombinant full-length human PCIF1 (Fig. S2, A–C), using as substrate the O-methylated cap analog, which has a 2'-O-methyladenosine at the +1 site [m7G(5')ppp(5')(2'OmeA)pG]. PCIF1 is active over a wide pH range of ∼5 to 10 (Fig. 1A), but is sensitive to increased ionic strength of sodium chloride beyond 200 mM (Fig. 1B). Interestingly, we observed greater activity at both ends of the tested pH range, at either pH 5.4 or pH 9.4 (Fig. 1A), which was independent of the buffering agent used (Fig. 1C). We next measured the PCIF1 kinetic parameters at three different pH values (5.4, 8.0, and 9.4) by varying, respectively, concentrations of the RNA substrate (Fig. 1D) and methyl donor SAM (Fig. 1E). Like the pH activity curve, the kcat values for the RNA and SAM substrates are each ∼2 to 3× higher at pH 5.4 and 9.4 than at pH 8.0 (summarized in Fig. 1F). However, binding affinities for the RNA substrate (as reflected by Km values) are approximately the same (∼0.3 μM) at the two higher pH conditions (8.0 and 9.6) and fourfold lower (i.e., increased binding affinity) than at pH 5.4 (Km value ∼1.2 μM). For comparison, Akichika et al. (23Akichika S. Hirano S. Shichino Y. Suzuki T. Nishimasu H. Ishitani R. Sugita A. Hirose Y. Iwasaki S. Nureki O. Suzuki T. Cap-specific terminal N (6)-methylation of RNA by an RNA polymerase II-associated methyltransferase.Science. 2019; 363eaav0080Crossref PubMed Scopus (138) Google Scholar) reported Km = 3.5 μM for m7Gppp(2'OmeA). That Km for the cap substrate is about an order of magnitude higher than the one we determined (0.3 μM at pH 8.0 versus 3.5 μM at pH 7.5). Their study was performed in a reaction mixture containing 50 mM HEPES-KOH [pH 7.5, so near where we saw lowest activity (Fig. 1A)]. On the other hand, the binding affinities for SAM (again, as reflected by Km) remain relatively constant at ∼0.7 to 0.9 μM over a range of pH values. Taken together, the catalytic efficiency of PCIF1 on the mRNA cap analog (comparing kcat/Km values) is the highest at pH 9.4 (3.9 min−1 μM−1; Fig. 1F), more than 1.5× higher than that at pH 8.0 (2.2 min−1 μM−1) and >2.5× higher than at pH 5.4 (1.5 min−1 μM−1). The higher kcat value at lower pH (5.4) is probably unique to the cap analog, as there are two titratable groups on the ligand (41Dlugosz M. Blachut-Okrasinska E. Bojarska E. Darzynkiewicz E. Antosiewicz J.M. Effects of pH on kinetics of binding of mRNA-cap analogs by translation initiation factor eIF4E.Eur. Biophys. J. 2003; 31: 608-616Crossref PubMed Scopus (11) Google Scholar) that might be directly involved in the binding. Direct interactions with the cap have not been characterized structurally with human PCIF1, but the zebrafish ortholog uses both positively and negatively charged residues (Arg269 and Glu563) in its interactions with the ribose and guanine moieties of m7G, as well as Arg239 interaction with one of the phosphate groups in the cap (23Akichika S. Hirano S. Shichino Y. Suzuki T. Nishimasu H. Ishitani R. Sugita A. Hirose Y. Iwasaki S. Nureki O. Suzuki T. Cap-specific terminal N (6)-methylation of RNA by an RNA polymerase II-associated methyltransferase.Science. 2019; 363eaav0080Crossref PubMed Scopus (138) Google Scholar). These charged residues are all conserved in human PCIF1. We did not observe a similar kcat versus pH phenomenon on a short RNA oligonucleotide—its methylation increased monotonically with increasing pH (Fig. 1G). Compared with the mRNA cap analog, we observed greatly reduced activity (by a factor of ∼100) on the conventional RNA oligo (without any pre-modifications) (Fig. 1G). Human PCIF1 has been structurally characterized as two separate fragments (23Akichika S. Hirano S. Shichino Y. Suzuki T. Nishimasu H. Ishitani R. Sugita A. Hirose Y. Iwasaki S. Nureki O. Suzuki T. Cap-specific terminal N (6)-methylation of RNA by an RNA polymerase II-associated methyltransferase.Science. 2019; 363eaav0080Crossref PubMed Scopus (138) Google Scholar): an NMR structure of the RNA polymerase-binding WW domain (residues 40–86) and a crystal structure of a large C-terminal fragment (residues 165–668) containing the MTase domain in complex with S-adenosyl-l-homocysteine (SAH) (Fig. 2A). To exclude the possibility of protein aggregation influencing activity at different pH values, we used three biophysical methods to measure the molecular mass of the full-length PCIF1 at pH 7.5 or 8.0, where we observed the lowest kcat value on cap analog (Fig. 1A). The sequence-predicted monomeric MW of PCIF1 (704 residues) is 80.7 kDa. First, the samples were subjected to size-exclusion chromatography (SEC), which gave the apparent molecular weight of ∼80 kDa (Fig. S2A). Second, SEC coupled with synchrotron-based multiangle light scattering (MALS) (42Some D. Amartely H. Tsadok A. Lebendiker M. Characterization of proteins by size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS).J. Vis. Exp. 2019; 148e59615Google Scholar) gave the absolute mass of 86 kDa with an averaged hydrodynamic radius of 4.8 ± 0.4 nm (Fig. 2B and Fig. S2, E and F). The same SEC fractions were simultaneously examined by synchrotron-based small-angle X-ray scattering (SAXS) (43Kikhney A.G. Svergun D.I. A practical guide to small angle X-ray scattering (SAXS) of flexible and intrinsically disordered proteins.FEBS Lett. 2015; 589: 2570-2577Crossref PubMed Scopus (262) Google Scholar), which gave the molecular weight of 86 kDa (Fig. 2B and Fig. S3). In sum, the observed molecular mass from all three methods agrees with the calculated mass of a monomeric form of PCIF1 (Fig. 2B). The overall shape of the SAXS envelope agrees with a negative stain electron microscopy (EM) model, with the maximum dimension of the molecule to be 115 to 120 Å (Fig. 2, C and D and Fig. S4). The overall contour of the low-resolution models can be fitted with the X-ray structure of the C-terminal MTase domain (Fig. 2C). The additional unaccounted-for density near one corner of the SAXS envelope might be where the N-terminal WW domain is located (red circle in Fig. 2, C and D). In addition, we observed extra density in the middle of the SAXS envelope, where the nucleic acid substrate might be bound. We generated a homology model for the full-length PCIF1 using I-TASSER (44Yang J. Yan R. Roy A. Xu D. Poisson J. Zhang Y. The I-TASSER suite: Protein structure and function prediction.Nat. Methods. 2015; 12: 7-8Crossref PubMed Scopus (3118) Google Scholar), including the missing N-terminal residues, the linker region between the WW and MTase domains, and the C-terminal residues. Interestingly, the extreme C-terminal 15-residues form an alpha helix that extends to the central bulged density (Fig. 2D). Helical wheel analysis (Fig. 2E) suggests that this helix has a basic face that incorporates the three arginine residues in the sequence KDRDSGREQGPSREPHPTCOOH, consistent with possible nucleic acid binding. The C-terminal 25 residues, specifically including these three arginines, are highly conserved among the Mammalia (other than monotremes, Fig. 2F). These residues follow a conserved polyserine tract that, in other proteins, can be phosphorylated and affect subnuclear localization (45Wolf A. Mantri M. Heim A. Muller U. Fichter E. Mackeen M.M. Schermelleh L. Dadie G. Leonhardt H. Venien-Bryan C. Kessler B.M. Schofield C.J. Bottger A. The polyserine domain of the lysyl-5 hydroxylase Jmjd6 mediates subnuclear localization.Biochem. J. 2013; 453: 357-370Crossref PubMed Scopus (27) Google Scholar, 46Cozza G. Moro E. Black M. Marin O. Salvi M. Venerando A. Tagliabracci V.S. Pinna L.A. The Golgi 'casein kinase' Fam20C is a genuine 'phosvitin kinase' and phosphorylates polyserine stretches devoid of the canonical consensus.FEBS J. 2018; 285: 4674-4683Crossref PubMed Scopus (9) Google Scholar). Finally, we used dynamic light scattering to determine the size distribution of PCIF1 at five different pH values (Fig. 2G). We observed protein aggregation only at pH < 5, whereas PCIF1 is stable over the pH range of 5.4 to 11, with a hydrodynamic radius of 4.5 to 5 nm (or diameter of ∼9–10 nm) in agreement with SAXS and the EM model (Fig. 2, C and D). In addition, the protein has the least dynamic nonuniformity, as indicated by smallest polydispersity of ∼5%, at pH 8.0. This suggests that the pH-dependent lower kcat value on cap analog is substrate specific. During our study, two publications reported the identification of MettL5, an RNA adenine MTase that methylates A1832 of 18S rRNA (31van Tran N. Ernst F.G.M. Hawley B.R. Zorbas C. Ulryck N. Hackert P. Bohnsack K.E. Bohnsack M.T. Jaffrey S.R. Graille M. Lafontaine D.L.J. The human 18S rRNA m6A methyltransferase METTL5 is stabilized by TRMT112.Nucleic Acids Res. 2019; 47: 7719-7733Crossref PubMed Scopus (107) Google Scholar, 32Ignatova V.V. Stolz P. Kaiser S. Gustafsson T.H. Lastres P.R. Sanz-Moreno A. Cho Y.L. Amarie O.V. Aguilar-Pimentel A. Klein-Rodewald T. Calzada-Wack J. Becker L. Marschall S. Kraiger M. Garrett L. et al.The rRNA m(6)A methyltransferase METTL5 is involved in pluripotency and developmental programs.Genes Dev. 2020; 34: 715-729Crossref PubMed Scopus (29) Google Scholar). This 18S rRNA N6mA, first identified about 35 years ago (47Maden B.E. Identification of the locations of the methyl groups in 18 S ribosomal RNA from Xenopus laevis and man.J. Mol. Biol. 1986; 189: 681-699Crossref PubMed Scopus (66) Google Scholar), is located in the 3′ minor domain of 18S, at the very base of helix h44, and only a few nucleotides away from the decoding center. Van Tran et al. (31van Tran N. Ernst F.G.M. Hawley B.R. Zorbas C. Ulryck N. Hackert P. Bohnsack K.E. Bohnsack M.T. Jaffrey S.R. Graille M. Lafontaine D.L.J. The human 18S rRNA m6A methyltransferase METTL5 is stabilized by TRMT112.Nucleic Acids Res. 2019; 47: 7719-7733Crossref PubMed Scopus (107) Google Scholar) showed that MettL5 must form a heterodimeric complex with Trm112 for its stability in cells and determined an X-ray structure of the MettL5-Trm112 complex with bound SAM (PDB 6H2U). Unexpectedly, despite extensive efforts, the authors "were unable to recapitulate METTL5–TRMT112 enzymatic activity in vitro with short single- or double-stranded RNAs corresponding to the sequences surrounding m6A1832 in the mature ribosome" (31van Tran N. Ernst F.G.M. Hawley B.R. Zorbas C. Ulryck N. Hackert P. Bohnsack K.E. Bohnsack M.T. Jaffrey S.R. Graille M. Lafontaine D.L.J. The human 18S rRNA m6A methyltransferase METTL5 is stabilized by TRMT112.Nucleic Acids Res. 2019; 47: 7719-7733Crossref PubMed Scopus (107) Google Scholar). On the other hand, a GST-MettL5 fusion purified from E. coli (in the absence of Trm112) was active in vitro on total RNA, on RNAs >200 nt long, and on dsRNAs (32Ignatova V.V. Stolz P. Kaiser S. Gustafsson T.H. Lastres P.R. Sanz-Moreno A. Cho Y.L. Amarie O.V. Aguilar-Pimentel A. Klein-Rodewald T. Calzada-Wack J. Becker L. Marschall S. Kraiger M. Garrett L. et al.The rRNA m(6)A methyltransferase METTL5 is involved in pluripotency and developmental programs.Genes Dev. 2020; 34: 715-729Crossref PubMed Scopus (29) Google Scholar). To resolve this discrepancy, regarding MettL5 activity in vitro, we purified recombinant MettL5-Trm112 complex (Fig. 3A). Because we do not know the conditions under which the unsuccessful experiments were conducted, we designed a short linear 14-mer RNA oligo corresponding to the sequence surrounding A1832 of 18S rRNA and tested for in vitro MettL5-Trim112 activity under minimal buffer conditions (20 mM Tris-HCl pH 8.0 and 1 mM DTT). We observed activity at no or low NaCl concentration (0–50 mM), but the activity was undetectable at 200 mM NaCl (Fig. 3B). Unlike PCIF1, MettL5-Trm112 methylase complex exhibited the highest activity at pH 8.0, followed by pH 9.4 and 5.4 (Fig. 3C). Under the linear reaction conditions of pH 8.0 and 50 mM NaCl for 20 min (Fig. 3D), the enzyme complex has similar Km values (∼1 μM) for the RNA oligo substrate and for SAM, while showing kcat values of ∼13 h−1

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