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

被引：52

作者：

Varshney, Dhaval ^{[1
,2
]}

Petit, Alain-Pierre ^{[3
]}

Bueren-Calabuig, Juan A. ^{[4
,5
]}

Jansen, Chimed ^{[3
]}

Fletcher, Dan A. ^{[3
]}

Peggie, Mark ^{[6
]}

Weidlich, Simone ^{[6
]}

Scullion, Paul ^{[3
]}

Pisliakov, Andrei V. ^{[4
,5
]}

Cowling, Victoria H. ^{[1
,2
]}

机构：

[1] Univ Dundee, Sch Life Sci, Ctr Gene Regulat & Express, Dow St, Dundee DD1 5EH, Scotland

[2] Univ Dundee, Sch Life Sci, MRC Prot Phosphorylat & Ubiquitylat Unit, Dow St, Dundee DD1 5EH, Scotland

[3] Univ Dundee, Sch Life Sci, Div Biol Chem & Drug Discovery, Drug Discovery Unit, Dow St, Dundee DD1 5EH, Scotland

[4] Univ Dundee, Sch Life Sci, Computat Biol, Dow St, Dundee DD1 5EH, Scotland

[5] Univ Dundee, Sch Sci & Engn, Phys, Dundee DD1 5EH, Scotland

[6] Univ Dundee, Sch Life Sci, Div Signal Transduct Therapies, Dow St, Dundee DD1 5EH, Scotland

来源：

NUCLEIC ACIDS RESEARCH | 2016年 / 44卷 / 21期

基金：

英国惠康基金; 英国医学研究理事会; 英国工程与自然科学研究理事会;

关键词：

CAPPING ENZYME READS; CAP METHYLTRANSFERASE; POLYMERASE-II; STRUCTURAL INSIGHTS; D12; SUBUNIT; MECHANISM; EVOLUTION; DOMAIN; PHOSPHORYLATION; METHYLATION;

D O I：

10.1093/nar/gkw637

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

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 alpha-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.

引用

页码：10423 / 10436

页数：14

共 35 条

[1] Human cap methyltransferase (RNMT) N-terminal non-catalytic domain mediates recruitment to transcription initiation sites [J].