Molecular basis of RNA guanine-7 methyltransferase (RNMT) activation by RAM

Dhaval Varshney, Alain-Pierre Petit, Juan A. Bueren-Calabuig, Chimed Jansen, Dan A. Fletcher, Mark Peggie, Simone Weidlich, Paul Scullion, Andrei V. Pisliakov, Victoria H. Cowling (Lead / Corresponding author)

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Abstract

Maturation and translation of mRNA in eukaryotes requires the addition of the 7-methylguanosine cap. In vertebrates, the cap methyltransferase, RNA guanine-7 methyltransferase (RNMT), has an activating subunit, RNMT-Activating Miniprotein (RAM). Here we report the first crystal structure of the human RNMT in complex with the activation domain of RAM. A relatively unstructured and negatively charged RAM binds to a positively charged surface groove on RNMT, distal to the active site. This results in stabilisation of a RNMT lobe structure which co-evolved with RAM and is required for RAM binding. Structure-guided mutagenesis and molecular dynamics simulations reveal that RAM stabilises the structure and positioning of the RNMT lobe and the adjacent α-helix hinge, resulting in optimal positioning of helix A which contacts substrates in the active site. Using biophysical and biochemical approaches, we observe that RAM increases the recruitment of the methyl donor, AdoMet (S-adenosyl methionine), to RNMT. Thus we report the mechanism by which RAM allosterically activates RNMT, allowing it to function as a molecular rheostat for mRNA cap methylation.

Original languageEnglish
Pages (from-to)10423-10436
Number of pages14
JournalNucleic Acids Research
Volume44
Issue number21
Early online date15 Jul 2016
DOIs
Publication statusPublished - 1 Dec 2016

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Catalytic Domain
mRNA (guanine(N7))-methyltransferase
Methyltransferases
Protein Biosynthesis
Molecular Dynamics Simulation
Eukaryota
Mutagenesis
Methionine
Methylation
Vertebrates
Messenger RNA
7-methylguanosine

Cite this

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title = "Molecular basis of RNA guanine-7 methyltransferase (RNMT) activation by RAM",
abstract = "Maturation and translation of mRNA in eukaryotes requires the addition of the 7-methylguanosine cap. In vertebrates, the cap methyltransferase, RNA guanine-7 methyltransferase (RNMT), has an activating subunit, RNMT-Activating Miniprotein (RAM). Here we report the first crystal structure of the human RNMT in complex with the activation domain of RAM. A relatively unstructured and negatively charged RAM binds to a positively charged surface groove on RNMT, distal to the active site. This results in stabilisation of a RNMT lobe structure which co-evolved with RAM and is required for RAM binding. Structure-guided mutagenesis and molecular dynamics simulations reveal that RAM stabilises the structure and positioning of the RNMT lobe and the adjacent α-helix hinge, resulting in optimal positioning of helix A which contacts substrates in the active site. Using biophysical and biochemical approaches, we observe that RAM increases the recruitment of the methyl donor, AdoMet (S-adenosyl methionine), to RNMT. Thus we report the mechanism by which RAM allosterically activates RNMT, allowing it to function as a molecular rheostat for mRNA cap methylation.",
author = "Dhaval Varshney and Alain-Pierre Petit and Bueren-Calabuig, {Juan A.} and Chimed Jansen and Fletcher, {Dan A.} and Mark Peggie and Simone Weidlich and Paul Scullion and Pisliakov, {Andrei V.} and Cowling, {Victoria H.}",
note = "Research funded by Medical Research Council Senior Non-Clinical Fellowship MR/K024213/1 and Lister Prize Research Fellowship (VHC), the Scottish Universities Physics Alliance (AVP), Wellcome Trust Centre Award 097945/Z/11/Z and Wellcome Trust Strategic Award 100476/Z/12/Z, and the Division of Signal Transduction Therapy, University of Dundee, funded by AstraZeneca, Boehringer-Ingelheim, GlaxoSmithKline, Janessen, Merck-Serono and Pfizer. Funding for open access charge: Wellcome Trust and MRC.",
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Molecular basis of RNA guanine-7 methyltransferase (RNMT) activation by RAM. / Varshney, Dhaval; Petit, Alain-Pierre; Bueren-Calabuig, Juan A.; Jansen, Chimed; Fletcher, Dan A.; Peggie, Mark; Weidlich, Simone; Scullion, Paul; Pisliakov, Andrei V.; Cowling, Victoria H. (Lead / Corresponding author).

In: Nucleic Acids Research, Vol. 44, No. 21, 01.12.2016, p. 10423-10436.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Molecular basis of RNA guanine-7 methyltransferase (RNMT) activation by RAM

AU - Varshney, Dhaval

AU - Petit, Alain-Pierre

AU - Bueren-Calabuig, Juan A.

AU - Jansen, Chimed

AU - Fletcher, Dan A.

AU - Peggie, Mark

AU - Weidlich, Simone

AU - Scullion, Paul

AU - Pisliakov, Andrei V.

AU - Cowling, Victoria H.

N1 - Research funded by Medical Research Council Senior Non-Clinical Fellowship MR/K024213/1 and Lister Prize Research Fellowship (VHC), the Scottish Universities Physics Alliance (AVP), Wellcome Trust Centre Award 097945/Z/11/Z and Wellcome Trust Strategic Award 100476/Z/12/Z, and the Division of Signal Transduction Therapy, University of Dundee, funded by AstraZeneca, Boehringer-Ingelheim, GlaxoSmithKline, Janessen, Merck-Serono and Pfizer. Funding for open access charge: Wellcome Trust and MRC.

PY - 2016/12/1

Y1 - 2016/12/1

N2 - Maturation and translation of mRNA in eukaryotes requires the addition of the 7-methylguanosine cap. In vertebrates, the cap methyltransferase, RNA guanine-7 methyltransferase (RNMT), has an activating subunit, RNMT-Activating Miniprotein (RAM). Here we report the first crystal structure of the human RNMT in complex with the activation domain of RAM. A relatively unstructured and negatively charged RAM binds to a positively charged surface groove on RNMT, distal to the active site. This results in stabilisation of a RNMT lobe structure which co-evolved with RAM and is required for RAM binding. Structure-guided mutagenesis and molecular dynamics simulations reveal that RAM stabilises the structure and positioning of the RNMT lobe and the adjacent α-helix hinge, resulting in optimal positioning of helix A which contacts substrates in the active site. Using biophysical and biochemical approaches, we observe that RAM increases the recruitment of the methyl donor, AdoMet (S-adenosyl methionine), to RNMT. Thus we report the mechanism by which RAM allosterically activates RNMT, allowing it to function as a molecular rheostat for mRNA cap methylation.

AB - Maturation and translation of mRNA in eukaryotes requires the addition of the 7-methylguanosine cap. In vertebrates, the cap methyltransferase, RNA guanine-7 methyltransferase (RNMT), has an activating subunit, RNMT-Activating Miniprotein (RAM). Here we report the first crystal structure of the human RNMT in complex with the activation domain of RAM. A relatively unstructured and negatively charged RAM binds to a positively charged surface groove on RNMT, distal to the active site. This results in stabilisation of a RNMT lobe structure which co-evolved with RAM and is required for RAM binding. Structure-guided mutagenesis and molecular dynamics simulations reveal that RAM stabilises the structure and positioning of the RNMT lobe and the adjacent α-helix hinge, resulting in optimal positioning of helix A which contacts substrates in the active site. Using biophysical and biochemical approaches, we observe that RAM increases the recruitment of the methyl donor, AdoMet (S-adenosyl methionine), to RNMT. Thus we report the mechanism by which RAM allosterically activates RNMT, allowing it to function as a molecular rheostat for mRNA cap methylation.

UR - http://nar.oxfordjournals.org/content/early/2016/07/15/nar.gkw637.abstract

U2 - 10.1093/nar/gkw637

DO - 10.1093/nar/gkw637

M3 - Article

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JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

IS - 21

ER -