Kaiming Zhang's team at the University of Science and Technology of China analyzed the cryo-electron microscopy structure of human m6A methylase complex

There are many modifications on RNA, among which adenine N6-position methylation (m6A) modification is the most abundant and widely distributed RNA modification in mammals
.
It was found that m6A modification is enriched in the coding region of mRNA and the 3'-UTR region, and is involved in the regulation
of various physiological and pathological processes.
The m6A modification of RNA relies on the m6A methylase complete complex (Holo-complex) composed of multiple protein members, where the catalytic subunit composed of the MT-A70 family proteins METTL3 and METTL14 form heterodimers is called the m6A-METTL complex (MAC); WTAP, VIRMA (KIAA1942), ZC3H13 and HAKAI mainly play a regulatory role, and there is a significant interaction, and the regulatory subunit composed of them is called m6A-METTL associated complex (MACOM) (Figure 1a).

In the absence of MACOM, MAC only shows relatively low in vitro m6A modification activity (Figure 1c), so MACOM has an important regulatory effect
on MAC's m6A methylase activity.
Although the catalytic domain of METTL3/14 that makes up the MAC core and the zinc finger structure of METTL3 have been resolved 1-4, there is still a lack of any structural information for the regulation of the complex MACOM and its member proteins, which greatly hinders the study
of its function and the regulatory mechanism of the catalytic subunit MAC activity.

Figure 1.
MAC-MACOM activity test and the overall structure
of the MACOM complex.
a, Working model
of MAC-MACOM for RNA m6A methylation modification.
b,c, MAC, HWVZ, MAC-HWVZ complex size exclusion chromatographic peak diagram and corresponding SDS-PAGE electrophoresis diagram
.
(d) Actb-1 RNA was used to compare the m6A methylation activity
of MAC alone and its combination with different component MACOM complexes.
e, schematic diagram
of the domain of the human-sourced MACOM complex used.
The dotted line represents an area
that is not visible in the electron microscope structure.
f, Schematic diagram
of electron microscopy density (left) and structure (right) of HWVZ complex.
g, Electron microscopy density (left) and structure (right) of HWV complex
.

On September 27, 2022, the team of Zhang Kaiming of the Department of Life Sciences and Medicine of the University of Science and Technology of China and the team of Ma Jinbiao of the School of Life Sciences of Fudan University published an article entitled "Cryo-EM structures of human m6A writer complexes" online in the journal Cell Research, which first obtained MACOM and MAC complexes in vitro (Figure 1b-c).
The atomic level structure of the core region of the human m6A methylase regulatory subunit complex MACOM was then resolved by cryo-EM (Figure 1f-g), and the overall structure
of the human source m6A methylase MAC-MACOM complex with an overall resolution of 4.
4 ° 。 The research team combined protein-protein and protein-RNA cross-linking mass spectrometry and pull-down biochemical experiments and structural analysis to elucidate the composition and structural details of the core region of the MACOM complex structure, and proposed a reasonable model
of MAC-MACOM methylation modified RNA.

The overall shape of the analyzed MACOM composite cryo-EM structure resembles a "war horse in a saddle"
.
WTAP forms a compact homodimer by four α-coiled-coil H1-H4, and there are three linker regions between H1-H4 and twisted (Figure 2a), so that the WTAP dimer forms a saddle-like shape
.
VIRMA uses 20 ARM-like repeating structural units to form a half-horse-shaped structure by means of a long connection helix (Figure 2b
).
The interaction interface of more than 5000 −2 between WTAP dimer and VIRMA and the conserved, multi-mode, multi-region interaction make the two tightly bound as the rigid skeleton of the subunit regulated as the entire m6A methylase complex (Figure 2c
).
ZC3H13 is deeply bound to the center of the VIRMA by a C-terminal fragment (1492-1643aa) to anchor to the complex, and VIRMA is "propped out" by the opening of about 6 degrees (Figure 2d-e).

It is worth noting that although HAKAI has been present during the purification of the complex, it has not been observed in the structure of both MACOM complexes (Figure 1), so HAKAI does not constitute the core structure of the regulatory subunit and is likely to bind in the peripheral flexible region
.

Figure 2.
Structure and relationship of
WTAP, VIRMA, ZC3H13.
a, Schematic diagram of the dimer structure of WTAP
.
The axis of the α-spiral curl spiral is indicated
by a black line.
b, Schematic diagram
of the structure of VIRMA.
c, Schematic diagram of sequence conservatism of VIRMA
.
The area where WTAP interacts with VIRMA is marked with a
box.
Both WTAP and ZC3H13 are translucent representations
.
d,e, conformational changes
of HWV complexes after ZC3H13 binding, based on the Belly and Chest domains of VIRMA.
e, Model
of conformational changes in HWV complexes caused by α6 and α7 binding of ZC3H13.

In order to obtain the structural information of the m6A methylated whole enzyme complex, the researchers further analyzed the MAC-MACOM complex structure of 4.
4 ?, of which the MACOM complex part is clear, while the MAC part is less dense and cannot be clearly identified
.
However, in the WTAP-H4/H4' region, a high occupancy α-helix density (Figure 3a) was observed, as demonstrated by protein crosslinking mass spectrometry and pull-down experiments, where the density is Leader Helix at the METTL3 N-terminus, consistent with previously reported biochemical results5
。 By lowering the cryo-EM density threshold, the relative spatial relationship of the two subunits in the MAC-MACOM complex was constructed by means of the resolved METTL3/14 core crystal structure and zinc finger solution structure 1-4 (Figure 3b, and the binding region of the RNA substrate on the MAC-MACOM complex was determined by analyzing the cross-linking mass spectrometry results of the s4U-labeled RNA substrate and the MAC-MACOM complex.
A possible m6A methylase complex modifies the action model of the RNA substrate (Figure 3c)
is proposed.

Figure 3.
MAC-MACOM complex interaction and overall substrate binding pattern diagram
.
a, the structure of the MACOM complex is placed in the electron microscopy density of the MAC-MACOM composite at a high threshold, and the excess density is circled with a dashed
coil.
b, MAC-MACOM composite model based on electron microscopy density and biochemical results, the left figure is a high threshold density map, and the right figure is a low threshold density map
.
c, the model
of the action of m6A methylase complex with RNA substrate proposed based on cross-linking mass spectrometry results of s4U-RNA and MAC-MACOM complexes.

In summary, this study is the first to analyze the high-resolution cryo-EM structure and the core structure model of the whole enzyme complex (MAC+MACOM) of the MACOM complex that has important regulatory role in the process of m6A modification, and further proposes a reasonable model of the action of
the m6A methylase complex modified RNA substrate.
This provides a structural basis for the development of inhibitors or drugs for m6A modification in addition to MAC-catalyzed subunit complexes to achieve potential therapeutic purposes
for human diseases such as cancers associated with RNA m6A modification.

Dr.
Su Shichen from the School of Life Sciences of Fudan University, Shanshan Li from the School of Life Sciences and Medicine of the University of Science and Technology of China, and Deng Ting, a doctoral student from the School of Life Sciences of Fudan University, are the co-first authors of this paper, and Professor Ma Jinbiao of the School of Life Sciences and the State Key Laboratory of Genetic Engineering of Fudan University and Kaiming Zhang, Faculty of Life Sciences and Medicine, University of Science and Technology of China, are co-corresponding authors
of this paper 。 The Key Laboratory of Cell Dynamics and the Center for Cryo-EM of the University of Science and Technology of China supported data collection and processing, and the electron microscopy platforms of the School of Life Sciences and the State Key Laboratory of Genetic Engineering of Fudan University supported
the initial screening of samples.
The research work was funded
by the National Key Research and Development Program, the National Natural Science Foundation of China, the Scientific Research Initiation Fund of the University of Science and Technology of China, and the Merit-Based Fund of the Chinese Academy of Sciences Leading the Action to Attract Talents.

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(Faculty of Life Sciences and Medicine, Faculty of Scientific Research)