Jing Wang's research team in the School of Pharmacy developed a single-base quantitative sequencing detection method for m6A

On October 27, 2022, the research group of researcher Wang Jing, Department of Chemical Biology and State Key Laboratory of Natural Medicines and Biomimetic Drugs, School of Pharmacy, Peking University, and Professor Yi Chengji's research group of Peking University School of Life Sciences, published a title entitled "Absolute quantification of single-base m⁶" in the top international academic journal Nature Biotechnology A methylation in the mammalian transcriptome using GLORI
".

Screenshot of the article

^m6Ais the most abundant modification inside mRNA in higher eukaryotes, with^m6Amodifications
present in 0.
2-0.
6% of adenosine.
m6A-dependent modifying enzymes, demodifying enzymes, and binding proteins perform regulatory functions.

At present, it has been found that^m6A has the functions of regulating mRNA splicing, nucleation, stability and protein translation, and can participate in the functional regulation
of various physiological and pathological processes such as development, gametogenesis, cell reprogramming, biorhythm, and disease.
In order to better study the biological function of m6A and clinical pathology research, the development of m6A high-throughput sequencing technology has always been a hot spot
in the field of^m6A.

Although a variety of m6A detection and sequencing methods have been developed, existing technologies still have several important limitations: (1)^m6A-baseddetection methods cannot obtain high-resolution site information; (2) Restriction enzyme-based detection techniques can only detect^m6Acontaining ACA motifs; (3) Methods
based on third-generation sequencing and machine learning.
Although this method provides single-base information of m6A and enables quantification, third-generation sequencing is expensive, inaccurate and does not enable absolute quantification
of^m6A.
To date, preference-free detection and absolute quantification of the^m6Awhole transcriptome remains unresolved
.

The m6 A detection technology "GLORI" (Glyoxal and nitrite-mediated deamination of unmethylated adenosine) developed by Wang Jing's research group breaks through the limitations of the above technology and achieves for the first time the real meaning of highefficiency, high sensitivity, high specificity, and no preference single base m6 A site detection, and^m6 The modification level of the A site is absolutely quantified
.
The technical core of GLORI is not dependent on antibodies, and the catalytic system of glyoxal and nitrite is found through chemical reaction combination screening, and the unmethylated adenosine is efficiently deaminated to form inosine (A-to-I, > 98%), and inosine is read as guanosine (G) during sequencing to form the transformation of A-to-G; ^M6Ais still read as A after sequencing, thus achieving single-base recognition
of^M6A.
GLORI achieved absolute quantification
of single-base^m6Aby detecting the proportion of A in the sequenced read sequence.
Therefore, the GLORI technique is conceptually similar to the method of quantifying the
genome 5mC using bisulfites.

The detection principle
of GLORI.
a.
GLORI achieves A-to-I transformation; b.
The chemical reaction process of GLORI; c.
LC-MS/MS analysis of glyoxal and nitrite-mediated deamination in GLORI before and after (top) and (bottom); d.
Example of the quantitative detection of^m6Aloci by GLORI technology on the gene MRPS26

In collaboration with Yichengji's group, Wang Jing's group identified 176,642 m6A sites in the HEK293T transcriptome, and found that the detectable m6A sites were still unsaturated with the increase of sequencing depth, which expanded people's understanding
of the amount of^m6Aon mRNA.
In addition, GLORI technology enables accurate quantification of^m6A: GLORI enables accurate detection
^{even at low}levels of modification (5%).
Further functional analysis of mRNA containing different m6A modification levels showed that the overall^m6Amodification levelon mRNA was negatively correlated
with the transcription level and translation efficiency of RNA.
So far, the research team has used GLORI technology to achieve the^m6Aquantitative map of the whole transcriptome for the first time, and also completed the quantitative evaluation
of gene expression and translation regulation.

GLORI transcriptome quantitative detection^m6A
.
a.
Sites detected by GLORI in HEK293; b.
GLORI was able to accurately detect the modification level of the^m6Asite in spike-in; c.
Correlation between technical replicates of modification levels at^m6Asites detected by GLORI; d.
Overall distribution of modification levels at^m6Asite in HEK293; e.
Transcription levels of mRNA containing different levels of^m6Amodification; Comparison of translation efficiency of mRNAs containing different levels of^m6Amodification

In addition, Wang Jing's group also found that a class of m6A sites (^m6Aclusters)with aggregation and distribution appeared in specific regions of some genes
.
Compared with genes that do not have such m6A clusters, genes in such^m6Aclusters significantly reduce the transcription level and translation efficiency of genes, thereby playing a negative
regulatory role in the expression of genes.

Discovery and function
of M⁶A cluster.
a.
Specific regions of the SPEN gene have clustered^m6Aclusters; b.
M6A sites involved in the formation of^m6Aclusters have significantly high levels of modification; mRNA with^m6Aclusters with significantly low transcription levels; mRNA with^m6Aclusters has significantly low translation efficiency

Jing Wang's group applied GLORI technology to the two stress conditions of heat shock and hypoxia in HeLa and MEF cell lines, further observed the dynamic regulation of m6A, and provided a quantitative map
of^m6Astress on the transcriptome.
The results show that 4.
8-11% m6A sites have dynamic changes in the two stress systems, and the up-regulated and down-regulated^m6Asites exhibit different enrichment patterns: in hypoxia, up-regulated and down-regulated^m6Asites are mainly enriched around 5′UTR and stop codons, while this enrichment shows the opposite result
in heat shock systems.
Different from the negative regulation of gene expression by m6A in wild-type cells: under heat shock conditions, the translation efficiency of mRNA with an elevated overall modification level of m6A was significantly upregulated, and this upregulation was more pronounced
in mRNA with^m6Aclusters.
The results suggest that m6A has specific regulation of gene expression in different environments, and also provide a powerful technical tool
for subsequent studies of the biological function of^m6Adynamic regulation.

GLORI detects dynamically changing^m6A
.
mRNA distribution map of hypoxia-induced^m6Asite; b.
mRNA distribution map of the^m6Asite induced by heat shock; c.
Boxplots and dotplots of upregulation of the translation efficiency of^m6A-relatedgenes in MEF cells before and after heat shock; d.
Boxplot of the translation efficiency of genes with or without^m6Aclusters after heat shock

In summary, this study shows the characteristics of GLORI technology with high sensitivity and high specificity of absolute single-base detection of m6A, and overcomes the bottleneck
of current^m6Aquantitative sequencing technology.
Based on its excellent performance in detecting m6A, GLORI will help promote and solve the functional research and core biological problems of m6A in various fields such as cell differentiation, embryonic development and clinical detection, and is expected to become the "gold standard"
for quantitative^m6Asequencing technology.

Yi Yunpeng

Shen Weiguo

Wang Jing and Yi Chengji are the corresponding authors
of this research paper.
Yunpeng Yi (outbound), a 2018 postdoctoral fellow at Peking University School of Pharmacy, Shen Weiguo, a 2018 direct doctoral student, and Liu Cong, a postdoctoral fellow at Peking University School of Life Sciences, and doctoral students Sun Hanxiao and Li Kai (graduated) are co-first authors
of the paper.
This work was supported
by the Key R&D Program of the Ministry of Science and Technology and the National Natural Science Foundation of China.