Volume 10 Issue 4
Apr.  2019
Turn off MathJax
Article Contents
Jinbo Huang, Xu Dong, Zhou Gong, Ling-Yun Qin, Shuai Yang, Yue-Ling Zhu, Xiang Wang, Delin Zhang, Tingting Zou, Ping Yin, Chun Tang. Solution structure of the RNA recognition domain of METTL3-METTL14 N6-methyladenosine methyltransferase[J]. Protein&Cell, 2019, 10(4): 272-284. doi: 10.1007/s13238-018-0518-7
Citation: Jinbo Huang, Xu Dong, Zhou Gong, Ling-Yun Qin, Shuai Yang, Yue-Ling Zhu, Xiang Wang, Delin Zhang, Tingting Zou, Ping Yin, Chun Tang. Solution structure of the RNA recognition domain of METTL3-METTL14 N6-methyladenosine methyltransferase[J]. Protein&Cell, 2019, 10(4): 272-284. doi: 10.1007/s13238-018-0518-7

Solution structure of the RNA recognition domain of METTL3-METTL14 N6-methyladenosine methyltransferase

doi: 10.1007/s13238-018-0518-7

The work was supported by National Key R&D Program of China (2016YFA0501200 to C. Tang), the Chinese Ministry of Science and Technology (2015CB910900 to P. Yin), the National Natural Science Foundation of China (91753132 and 31770799 to C. Tang, 31722017 to P. Yin, 31400735 to Z. Gong, 31400644 to X. Dong), the Fok Ying-Tong Education Foundation (151021 to P. Yin), and the Fundamental Research Funds for the Central Universities (2017PY031 to P. Yin).

  • Received Date: 2018-01-14
  • N6-methyladenosine (m6A), a ubiquitous RNA modification, is installed by METTL3-METTL14 complex. The structure of the heterodimeric complex between the methyltransferase domains (MTDs) of METTL3 and METTL14 has been previously determined. However, the MTDs alone possess no enzymatic activity. Here we present the solution structure for the zinc finger domain (ZFD) of METTL3, the inclusion of which fulfills the methyltransferase activity of METTL3-METTL14. We show that the ZFD specifically binds to an RNA containing 5'-GGACU-3' consensus sequence, but does not to one without. The ZFD thus serves as the target recognition domain, a structural feature previously shown for DNA methyltransferases, and cooperates with the MTDs of METTL3-METTL14 for catalysis. However, the interaction between the ZFD and the specific RNA is extremely weak, with the binding affinity at several hundred micromolar under physiological conditions. The ZFD contains two CCCH-type zinc fingers connected by an anti-parallel β-sheet. Mutational analysis and NMR titrations have mapped the functional interface to a contiguous surface. As a division of labor, the RNAbinding interface comprises basic residues from zinc finger 1 and hydrophobic residues from β-sheet and zinc finger 2. Further we show that the linker between the ZFD and MTD of METTL3 is flexible but partially folded, which may permit the cooperation between the two domains during catalysis. Together, the structural characterization of METTL3 ZFD paves the way to elucidate the atomic details of the entire process of RNA m6A modification.
  • loading
  • [1]
    Amann BT, Worthington MT, Berg JM (2003) A Cys3His zinc-binding domain from Nup475/tristetraprolin:a novel fold with a disklike structure. Biochemistry 42:217-221
    Antos JM, Truttmann MC, Ploegh HL (2016) Recent advances in sortase-catalyzed ligation methodology. Curr Opin Struct Biol 38:111-118
    Bheemanaik S, Reddy YV, Rao DN (2006) Structure, function and mechanism of exocyclic DNA methyltransferases. Biochem J 399:177-190
    Case D, Babin V, Berryman J, Betz R, Cai Q, Cerutti D, Cheatham Iii T, Darden T, Duke R, Gohlke H (2014) Amber 14. University of Calforina, San Francisco Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe:a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277-293
    Dominguez C, Schubert M, Duss O, Ravindranathan S, Allain FH (2011) Structure determination and dynamics of protein-RNA complexes by NMR spectroscopy. Prog Nucl Magn Reson Spectrosc 58:1-61
    Dominissini D et al (2012) Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 485:201-206
    Franke D et al (2017) ATSAS 2.8:a comprehensive data analysis suite for small-angle scattering from macromolecular solutions. J Appl Crystallogr 50:1212-1225
    Freiburger L, Sonntag M, Hennig J, Li J, Zou P, Sattler M (2015) Efficient segmental isotope labeling of multi-domain proteins using Sortase A. J Biomol NMR 63:1-8
    Fu M, Blackshear PJ (2017) RNA-binding proteins in immune regulation:a focus on CCCH zinc finger proteins. Nat. Rev. Immunol. 17:130-143
    Fu Y, Dominissini D, Rechavi G, He C (2014) Gene expression regulation mediated through reversible m(6)A RNA methylation. Nat Rev Genet 15:293-306
    Gokhale NS et al (2016) N6-methyladenosine in flaviviridae viral RNA genomes regulates infection. Cell Host Microbe 20:654-665
    Iwahara J, Tang C, Clore GM (2007) Practical aspects of 1H transverse paramagnetic relaxation enhancement measurements on macromolecules. J Magn Reson 184:185-195
    Iyer LM, Zhang D, Aravind L (2016) Adenine methylation in eukaryotes:Apprehending the complex evolutionary history and functional potential of an epigenetic modification. Bioessays 38:27-40
    Kay LE, Torchia DA, Bax A (1989) Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy:application to staphylococcal nuclease. Biochemistry 28:8972-8979
    Kleckner IR, Foster MP (2011) An introduction to NMR-based approaches for measuring protein dynamics. Biochim Biophys Acta 1814:942-968
    Laskowski RA, Rullmannn JA, MacArthur MW, Kaptein R, Thornton JM (1996) AQUA and PROCHECK-NMR:programs for checking the quality of protein structures solved by NMR. J Biomol NMR 8:477-486
    Lence T et al (2016) m6A modulates neuronal functions and sex determination in Drosophila. Nature 540:242-247
    Liu N, Pan T (2016) N6-methyladenosine-encoded epitranscriptomics. Nat Struct Mol Biol 23:98-102
    Liu Z, Zhang WP, Xing Q, Ren X, Liu M, Tang C (2012) Noncovalent dimerization of ubiquitin. Angew Chem Int Ed Engl 51:469-472
    Liu J et al (2014) A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation. Nat Chem Biol 10:93-95
    Liu Z, Gong Z, Dong X, Tang C (2015) Transient protein-protein interactions visualized by solution NMR. BBA Proteins Proteomics 1864:115-122
    Murn J, Teplova M, Zarnack K, Shi Y, Patel DJ (2016) Recognition of distinct RNA motifs by the clustered CCCH zinc fingers of neuronal protein Unkempt. Nat Struct Mol Biol 23:16-23
    Park S, Phukan PD, Zeeb M, Martinez-Yamout MA, Dyson HJ, Wright PE (2017) Structural basis for interaction of the tandem zinc finger domains of human muscleblind with cognate RNA from human cardiac troponin T. Biochemistry 56:4154-4168
    Patil DP, Chen CK, Pickering BF, Chow A, Jackson C, Guttman M, Jaffrey SR (2016) m(6)A RNA methylation promotes XISTmediated transcriptional repression. Nature 537:369-373
    Pelton JG, Torchia DA, Meadow ND, Roseman S (1993) Tautomeric states of the active-site histidines of phosphorylated and unphosphorylated ⅢGlc, a signal-transducing protein from Escherichia coli, using two-dimensional heteronuclear NMR techniques. Protein Sci 2:543-558
    Peters MB, Yang Y, Wang B, Fusti-Molnar L, Weaver MN, Merz KM Jr (2010) Structural survey of zinc containing proteins and the development of the zinc AMBER force field (ZAFF). J Chem Theory Comput 6:2935-2947
    Ping XL et al (2014) Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase. Cell Res 24:177-189
    Rieping W, Habeck M, Bardiaux B, Bernard A, Malliavin TE, Nilges M (2007) ARIA2:automated NOE assignment and data integration in NMR structure calculation. Bioinformatics 23:381-382
    Roundtree IA, Evans ME, Pan T, He C (2017) Dynamic RNA modifications in gene expression regulation. Cell 169:1187-1200
    Schibler U, Kelley DE, Perry RP (1977) Comparison of methylated sequences in messenger RNA and heterogeneous nuclear RNA from mouse L cells. J Mol Biol 115:695-714
    Schlundt A, Tants JN, Sattler M (2017) Integrated structural biology to unravel molecular mechanisms of protein-RNA recognition. Methods 118-119:119-136
    Schwartz S et al (2014) Perturbation of m6A writers reveals two distinct classes of mRNA methylation at internal and 5' sites. Cell Rep. 8:284-296
    Schwieters CD, Clore GM (2014) Using small angle solution scattering data in Xplor-NIH structure calculations. Prog Nucl Magn Reson Spectrosc 80:1-11
    Schwieters CD, Kuszewski JJ, Clore GM (2006) Using Xplor-NIH for NMR molecular structure determination. Prog Nucl Magn Reson Spectrosc 48:47-62
    Shen Y, Delaglio F, Cornilescu G, Bax A (2009) TALOS+:a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts. J Biomol NMR 44:213-223
    Sledz P, Jinek M (2016) Structural insights into the molecular mechanism of the m(6)A writer complex. eLife 5:e18434
    Song J, Yi C (2017) Chemical modifications to RNA:a new layer of gene expression regulation. ACS Chem Biol 12:316-325
    Song J, Rechkoblit O, Bestor TH, Patel DJ (2011) Structure of DNMT1-DNA complex reveals a role for autoinhibition in maintenance DNA methylation. Science 331:1036-1040
    Sun WJ, Li JH, Liu S, Wu J, Zhou H, Qu LH, Yang JH (2016a) RMBase:a resource for decoding the landscape of RNA modifications from high-throughput sequencing data. Nucleic Acids Res 44:D259-265
    Sun ZJ, Bhanu MK, Allan MG, Arthanari H, Wagner G, Hanna J (2016b) Solution structure of the Cuz1 AN1 zinc finger domain:an exposed LDFLP motif defines a subfamily of AN1 proteins. PloS ONE 11:e0163660
    Tomczak K, Czerwinska P, Wiznerowicz M (2015) The cancer genome atlas (TCGA):an immeasurable source of knowledge. Contemp Oncol 19:A68-77
    Wang Y, Li Y, Toth JI, Petroski MD, Zhang Z, Zhao JC (2014) N6-methyladenosine modification destabilizes developmental regulators in embryonic stem cells. Nat Cell Biol 16:191-198
    Wang P, Doxtader KA, Nam Y (2016a) Structural basis for cooperative function of Mettl3 and Mettl14 methyltransferases. Mol Cell 63:306-317
    Wang X et al (2016b) Structural basis of N(6)-adenosine methylation by the METTL3-METTL14 complex. Nature 534:575-578
    Wang X, Huang J, Zou T, Yin P (2017) Human m(6)A writers:two subunits, 2 roles. RNA Biol 14:300-304
    Xiang Y et al (2017) RNA m6A methylation regulates the ultravioletinduced DNA damage response. Nature 543:573-576
    Xing Q et al (2014) Visualizing an ultra-weak protein-protein interaction in phosphorylation signaling. Angew Chem Int Ed Engl 53:11501-11505
    Yamazaki T, Formankay JD, Kay LE (1993) 2-Dimensional Nmr experiments for correlating C-13-beta and H-1-delta/epsilon chemical-shifts of aromatic residues in C-13-labeled proteins via scalar couplings. J Am Chem Soc 115:11054-11055
    Yoon KJ et al (2017) Temporal control of mammalian cortical neurogenesis by m(6)A methylation. Cell 171(877-889):e817
    Zheng Q, Hou J, Zhou Y, Li Z, Cao X (2017) The RNA helicase DDX46 inhibits innate immunity by entrapping m(6)A-demethylated antiviral transcripts in the nucleus. Nat Immunol 18:1094-1103
    Zhong S, Li H, Bodi Z, Button J, Vespa L, Herzog M, Fray RG (2008) MTA is an Arabidopsis messenger RNA adenosine methylase and interacts with a homolog of a sex-specific splicing factor. Plant Cell 20:1278-1288
    Zhou H, Di Palma S, Preisinger C, Peng M, Polat AN, Heck AJ, Mohammed S (2013) Toward a comprehensive characterization of a human cancer cell phosphoproteome. J Proteome Res 12:260-271
  • 加载中


    通讯作者: 陈斌, bchen63@163.com
    • 1. 

      沈阳化工大学材料科学与工程学院 沈阳 110142

    1. 本站搜索
    2. 百度学术搜索
    3. 万方数据库搜索
    4. CNKI搜索


    Article Metrics

    Article views (876) PDF downloads(1480) Cited by()
    Proportional views


    DownLoad:  Full-Size Img  PowerPoint