The Ma Jinbiao team of Fudan University and the Kaiming team of Zhang Kaiming of the University of Science and Technology of China cooperated to analyze the cryo-electron microscopy structure of human m6A methylase complex

On September 27, 2022, the Ma Jinbiao team of Fudan University and the Zhang Kaiming team of the University of Science and Technology of China published an article entitled "Cryo-EM structures of human m6 A writer complexes" online in Cell Research magazine, which analyzed two sets of human source m⁶ with a resolution of 3.
0 degrees through cryoelectron microscopy A methylase regulates the high-resolution structure of the core region of the subunit complex, as well as the overall structure
of the human source^m6Amethylase complex with an overall resolution of 4.
4 ?.
The research team combined protein-protein and protein-RNA cross-linking mass spectrometry and GST pull-down biochemical experiments and structural analysis to propose a reasonable model
for the identification and modification of RNA substrates by^m6Amethylase complex.

Figure 1.
MAC-MACOM activity test and the overall structure
of the MACOM complex.
a, A working model
of MAC-MACOM for RNA^m6Amethylation modification.
b, c, MAC, HWVZ, MAC-HWVZ complex size exclusion chromatographic peak diagram and corresponding SDS-PAGE electrophoresis map
.
d, Actb-1 RNA was used to compare the^m6Amethylation 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
.

There are many modifications on RNA, among which adenine^N-6 methylation (^m6A) modification is the most abundant and widely distributed RNA modification in mammals
.
It has been found that^m6Amodification 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 complex consisting of multiple protein members, where the catalytic subunit consisting of the MT-A70 family proteins METTL3 and METTL14 formingheterodimers is called the^m6A-METTLcomplex (^m6 A-METTL complex, abbreviated as MAC); WTAP, VIRMA (KIAA1942), ZC3H13 and HAKAI mainly play a regulatory role, and there is a significant interaction, and the regulatory subunit they are composed of is called^m6A-METTL associated complex (MACOM
).
In the absence of MACOM, MAC only shows relatively low in vitro m6A modification activity, so MACOM has an important regulatory effect
on the^m6Amethylase activity of MAC.
Although the catalytic domain of METTL3/14 that constitutes the MAC core and the zinc finger structure of METTL3 have been resolved, there is still a lack of any structural information for the regulatory complex MACOM and its member proteins, which greatly hinders the study
of its function and the regulatory mechanism of the catalytic subunit MAC activity.

The newly analyzed MACOM composite cryo-EM structure resembles a "war horse in a saddle"
.
WTAP forms a compact homodimer by four segments of α-coiled-coil H1-H4, forming 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 spiral (Figure 1f-g
).
The close interaction between the WTAP dimer and VIRMA acts as a rigid skeleton of the subunit regulated by the entire^m6Amethylase complex
.
ZC3H13 is deeply bound to the central part of VIRMA by a C-terminal fragment and thus anchored to the
complex.
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?, and built the relative spatial relationship between the two subunits in the MAC-MACOM complex by means of the resolved METTL3/14 core crystal structure and the zinc finger solution structure (Video 1), and then analyzed the cross-linked mass spectrometry results of the s4U-labeled RNA substrate and the MAC-MACOM complex.
The binding site of RNA substrate on MAC-MACOM complex is obtained, and a possible^m6Amethylase complex modifies the action model of RNA substrate (Figure 2).

2.
Interaction between MAC and MACOM complex 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, high threshold density plot on the left and low threshold density plot
on the right.
c, Based on the crosslinking mass spectrometry results of s4U-RNA and MAC-MACOM complexes, the model of the action between the^m6Amethylase complex and the RNA substrate is proposed
.

Video 1.
Structural model
of the MAC-MACOM complex.

In summary, this study is the first to analyze the high-resolution cryo-EM structure of the MACOM complex and the core structure model of the whole enzyme complex (MAC+MACOM) in the process of^m6A modification, and further propose 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 m6 A modification in addition to MAC-catalyzed subunit complexes to achieve potential therapeutic purposes
for human diseases such as cancers associated with RNA^m6modification.

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.
Liu Jianzhao's team from the Department of Polymers at Zhejiang University and Peng Chao from the National Protein Science Center (Shanghai) also participated in the research work
.
The Center for Cryo-EM of the University of Science and Technology of China supported data collection and processing, and the electron microscopy platform 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.

Paper Link: https://doi.
org/ 10.
1038/s41422-022-00725-8