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

ABSTRACTN6-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 RNA-binding 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.

[1]  J. Berg,et al.  A Cys3His zinc-binding domain from Nup475/tristetraprolin: a novel fold with a disklike structure. , 2003, Biochemistry.

[2]  S. Grzesiek,et al.  NMRPipe: A multidimensional spectral processing system based on UNIX pipes , 1995, Journal of biomolecular NMR.

[3]  Ping Wang,et al.  Structural Basis for Cooperative Function of Mettl3 and Mettl14 Methyltransferases. , 2016, Molecular cell.

[4]  U. Schibler,et al.  Comparison of methylated sequences in messenger RNA and heterogeneous nuclear RNA from mouse L cells. , 1977, Journal of molecular biology.

[5]  Nian Liu,et al.  N6-methyladenosine–encoded epitranscriptomics , 2016, Nature Structural &Molecular Biology.

[6]  L. Kay,et al.  Two-dimensional NMR experiments for correlating carbon-13.beta. and proton.delta./.epsilon. chemical shifts of aromatic residues in 13C-labeled proteins via scalar couplings , 1993 .

[7]  A. Bax,et al.  TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts , 2009, Journal of biomolecular NMR.

[8]  Miguel A. Andrade-Navarro,et al.  m6A modulates neuronal functions and sex determination in Drosophila , 2016, Nature.

[9]  Shivakumara Bheemanaik,et al.  Structure, function and mechanism of exocyclic DNA methyltransferases. , 2006, The Biochemical journal.

[10]  M. Jinek,et al.  Structural insights into the molecular mechanism of the m(6)A writer complex , 2016 .

[11]  Michael Nilges,et al.  ARIA2: Automated NOE assignment and data integration in NMR structure calculation , 2007, Bioinform..

[12]  G. Wagner,et al.  Solution Structure of the Cuz1 AN1 Zinc Finger Domain: An Exposed LDFLP Motif Defines a Subfamily of AN1 Proteins , 2016, PloS one.

[13]  Schraga Schwartz,et al.  Perturbation of m6A writers reveals two distinct classes of mRNA methylation at internal and 5' sites. , 2014, Cell reports.

[14]  H. Ploegh,et al.  Recent advances in sortase-catalyzed ligation methodology. , 2016, Current opinion in structural biology.

[15]  Samir Adhikari,et al.  Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase , 2014, Cell Research.

[16]  J. Iwahara,et al.  Practical aspects of (1)H transverse paramagnetic relaxation enhancement measurements on macromolecules. , 2007, Journal of magnetic resonance.

[17]  Zhaolei Zhang,et al.  developmental regulators in embryonic stem cells , 2014 .

[18]  D. Torchia,et al.  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 , 1993, Protein science : a publication of the Protein Society.

[19]  Yang Shi,et al.  m6A RNA methylation regulates the UV-induced DNA damage response , 2016, Nature.

[20]  Tingting Zou,et al.  Human m6A writers: Two subunits, 2 roles , 2017, RNA biology.

[21]  G. Marius Clore,et al.  Using Xplor‐NIH for NMR Molecular Structure Determination , 2006 .

[22]  Samie R. Jaffrey,et al.  m6A RNA methylation promotes XIST-mediated transcriptional repression , 2016, Nature.

[23]  Tao Pan,et al.  Dynamic RNA Modifications in Gene Expression Regulation , 2017, Cell.

[24]  G. Wagner,et al.  Solution Structure of the Cuz1 AN1 Zinc Finger Domain: An Exposed LDFLP Motif Defines a Subfamily of AN1 Proteins , 2016, PloS one.

[25]  P. V. Konarev,et al.  ATSAS 2.8: a comprehensive data analysis suite for small-angle scattering from macromolecular solutions , 2017, Journal of applied crystallography.

[26]  Yan Wang,et al.  Visualizing an ultra-weak protein-protein interaction in phosphorylation signaling. , 2014, Angewandte Chemie.

[27]  K. Tomczak,et al.  The Cancer Genome Atlas (TCGA): an immeasurable source of knowledge , 2015, Contemporary oncology.

[28]  Miao Yu,et al.  A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation , 2013, Nature chemical biology.

[29]  Yang Wang,et al.  N6-methyladenosine modification destabilizes developmental regulators in embryonic stem cells , 2014, Nature Cell Biology.

[30]  Qiang Wang,et al.  Structural basis of N6-adenosine methylation by the METTL3–METTL14 complex , 2016, Nature.

[31]  Ian R Kleckner,et al.  An introduction to NMR-based approaches for measuring protein dynamics. , 2011, Biochimica et biophysica acta.

[32]  F. Allain,et al.  Structure determination and dynamics of protein-RNA complexes by NMR spectroscopy. , 2011, Progress in nuclear magnetic resonance spectroscopy.

[33]  Jie Wu,et al.  RMBase: a resource for decoding the landscape of RNA modifications from high-throughput sequencing data , 2015, Nucleic Acids Res..

[34]  Chun Tang,et al.  Transient protein-protein interactions visualized by solution NMR. , 2016, Biochimica et biophysica acta.

[35]  P. Blackshear,et al.  RNA-binding proteins in immune regulation: a focus on CCCH zinc finger proteins , 2016, Nature Reviews Immunology.

[36]  L. Aravind,et al.  Adenine methylation in eukaryotes: Apprehending the complex evolutionary history and functional potential of an epigenetic modification , 2015, BioEssays : news and reviews in molecular, cellular and developmental biology.

[37]  Kenneth M Merz,et al.  Structural Survey of Zinc Containing Proteins and the Development of the Zinc AMBER Force Field (ZAFF). , 2010, Journal of chemical theory and computation.

[38]  M. Peng,et al.  Toward a comprehensive characterization of a human cancer cell phosphoproteome. , 2013, Journal of proteome research.

[39]  Gideon Rechavi,et al.  Gene expression regulation mediated through reversible m6A RNA methylation , 2014, Nature Reviews Genetics.

[40]  D. Patel,et al.  Structure of DNMT1-DNA Complex Reveals a Role for Autoinhibition in Maintenance DNA Methylation , 2011, Science.

[41]  Alexa B. R. McIntyre,et al.  N6-Methyladenosine in Flaviviridae Viral RNA Genomes Regulates Infection , 2016, Cell host & microbe.

[42]  Stefan Canzar,et al.  Temporal Control of Mammalian Cortical Neurogenesis by m6A Methylation , 2017, Cell.

[43]  Michel Herzog,et al.  MTA Is an Arabidopsis Messenger RNA Adenosine Methylase and Interacts with a Homolog of a Sex-Specific Splicing Factor[W][OA] , 2008, The Plant Cell Online.

[44]  Kathi Zarnack,et al.  Recognition of distinct RNA motifs by the clustered CCCH zinc fingers of neuronal protein Unkempt , 2015, Nature Structural &Molecular Biology.

[45]  Chengqi Yi,et al.  Chemical Modifications to RNA: A New Layer of Gene Expression Regulation. , 2017, ACS chemical biology.

[46]  J. Thornton,et al.  AQUA and PROCHECK-NMR: Programs for checking the quality of protein structures solved by NMR , 1996, Journal of biomolecular NMR.

[47]  Maili Liu,et al.  Noncovalent Dimerization of Ubiquitin** , 2011, Angewandte Chemie.

[48]  L. Kay,et al.  Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy: application to staphylococcal nuclease. , 1989, Biochemistry.

[49]  H. Dyson,et al.  Structural Basis for Interaction of the Tandem Zinc Finger Domains of Human Muscleblind with Cognate RNA from Human Cardiac Troponin T , 2017, Biochemistry.

[50]  M. Kupiec,et al.  Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq , 2012, Nature.

[51]  Michael Sattler,et al.  Integrated structural biology to unravel molecular mechanisms of protein-RNA recognition. , 2017, Methods.

[52]  Jian Li,et al.  Efficient segmental isotope labeling of multi-domain proteins using Sortase A , 2015, Journal of biomolecular NMR.

[53]  Charles D Schwieters,et al.  Using small angle solution scattering data in Xplor-NIH structure calculations. , 2014, Progress in nuclear magnetic resonance spectroscopy.