Nature Biotechnology's Wang Jing team developed a m6A single-base quantitative sequencing detection method

On October 27, 2022, the team of Wang Jing, Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University, and the State Key Laboratory of Natural Medicines and Biomimetic Drugs, together with the Yichengji team of the School of Life Sciences of Peking University, were in the international top academic journal Nature Biotechnology The results of the study entitled "Absolute quantification of single-base m⁶A methylation in the mammalian transcriptome using GLORI
。

^m6Ais the most abundant modification within the mRNA of higher eukaryotes, with ^m6A modification present in approximately 0.
2-0.
6% of adenosine
。 ^m6Arelies on modifying enzymes, demodifiers, and binding proteins to perform regulatory functions
.
At present, ^m6 A has been foundto regulate mRNA splicing, nucleation, stability and protein translation, and can participate in the functional regulation
of various physiological and pathological processes such as development, gamete generation, cell reprogramming, biorhythm, and disease.
In order to better study m6A biological function and clinicopathology research, the development of ^m6Ahigh-throughput sequencing technology has been m ⁶ Hot spots
in the field of A.

Although a variety of^m6Adetection and sequencing methods have been developed, there are several important limitations of existing technologies: (1) based on m Detection methods for ^6Aantibodies do not have 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 can providesingle-base information and quantify m6A, third-generation sequencing has problems such as high cost, low detection accuracy, and cannot achieve absolute quantification of ^m6A
。 To date, the unpreferred detection and absolute quantification of the whole transcriptome of ^M6A remains unresolved
.

The m ⁶A detection technology "GLORI" (Glyoxal and nit ri) developed by Wang Jing's team Te-mediated deamination of unmethylated adenosine) breaks through the limitations of the above technologies and achieves for the first time true high efficiency, high sensitivity, Highly specific, unpreferred, single-base^m6Asite detection and^m6 The modification level of site A is absolutely quantified
.
The core of GLORI's technology is to independently rely on antibodies, through chemical reaction combination screening to find glyoxal and nitrite catalytic systems, efficient deamination of unmethylated adenosine to form inosine (A-to-I, > 98%), inosine is read as guanosine (G) during sequencing and formed A-to-G conversion; After sequencing, m6A is still read as A, thus realizing the ^m6A Single base recognition
.
GLORI achieves absolute quantification of the single base m6A by detecting the proportion of A in the sequenced read sequence.

Therefore, the GLORI technique is conceptually similar to the method used to quantify the genome 5mC with bisulfite.

Figure 1: Detection principle
of GLORI.
a , GLORI realizes the conversion
of A-to-I.
b, the chemical reaction process
of GLORI.
C, LC-MS/MS ANALYSIS
OF GLYOXAL AND NITRITE-MEDIATED DEAMINATION BEFORE AND AFTER (TOP) AND (BOTTOM) IN GLORI.
Example of
GLORI technology for quantitative detection of m6A locus on gene MRPS26.

Subsequently, Wang Jing's team and Yichengji team of the School of Life Sciences of Peking University identified 17 6,6 42 m in the HEK293T transcriptome ^6Alocus, and it was found that detectable^m6A sites remain unsaturated as sequencing depth increases, the results extend to mRNA on mRNA ⁶A Awareness
of the existence of quantity.
In addition, GLORI technology enables accurate quantification of m6A: even ^m6A (5%) with low levels of modification GLORI is also capable of accurate detection
.
Further functional analysis of mRNAs containing different levels of ^m6 A modification found that the overall mRNA was m ⁶ The modification level of A is negatively correlated with
the transcription level and translation efficiency of RNA.
So far, we have achieved the ^m6 A quantitative map of the whole transcriptome for the first time using GLORI technology, and also completed the quantitative evaluation
of gene expression and translation regulation.

Figure 2: Quantitative detection of m6A
within the GLORI transcriptome.
a , sites detected by GLORI in HEK293
.
b, GLORI is able to accurately detect the modification level
of m 6A in spike-in.
c, correlation
of modification levels at m6A sites detected by GLORI between technical replicates.
d,Overall distribution of
m6A modification levels in m6A in HEK293.
e, transcription levels of mRNA containing different ^m6Amodification levels
.
f.
Comparison
of translation efficiency of mRNA containing different m6A modification levels.

In addition, Wang Jing's team also found that a class of m6A loci (^m6A clusters) with clustering distribution appeared in specific regions of some genes
.
Compared with genes that do not have such m6A clusters, the genes of such ^m6Aclusters significantly reduce the transcription level and translation efficiency of genes, so as to exert The role
of negatively regulating gene expression.

Figure 3: Discovery and function
of M6A cluster.
a Specific regions of the SPEN gene have clusters of^m6Aclusters
.
b, the ^m6A sites involved in the formation of ^m6A clustershave significantly higher levels of
modification.
mRNA with ^m6Aclusters has significantly lower transcription levels
.
mRNA with ^m6Aclusters has significantly lower translation efficiency
.

Finally, Wang Jing's team applied GLORI technology to two stress conditions of heat shock and hypoxia in HeLa and MEF cell lines The dynamic regulation of m6A was observed and a quantitative map
of m6A on stress condition stress on the transcriptome was provided.
The results show that there are dynamic changes at about 4.
8-11%^m6A sites under both pressure systems, and ^m6 is adjusted up and down The A site showed different enrichment patterns: in the absence of oxygen, the up- and down-regulated ^m6A sites were mainly enriched near the 5′UTR and stop codons, while in the heat shock system, this enrichment showed the opposite Results
.
Different from the negative regulation of gene expression by ^m6A in wild-type cells, the translation efficiency of mRNA with elevated overall modification level of ^m6A was significantly upregulated under heat shock conditions , and this upregulation is more pronounced
in mRNA with m6A clusters.
The results suggest that m6 A has specific regulation of gene expression in different environments, and also provides a powerful technical tool for subsequent research on the biological function of ^m6A dynamic regulation
。

Figure 4: GLORI detects dynamic changes in^m6A
.
a , Hypoxia-induced mRNA distribution atlas
at m6A sites.
b.
Heat shock-induced mRNA distribution atlas
at the m6A site.
c.
Box plot and dot plot
of upregulation of translation efficiency of m6A-related genes in MEF cells before and after heat shock.
d, boxplot
of translation efficiency of genes with or without m6A cluster after heat shock.

In summary, this study demonstrates the characteristics of GLORI technology with high sensitivity and high specificity for the absolute quantitative detection of m6A without preference single base, which overcomes the bottleneck of current ^m6A quantitativesequencing technology
。 Based on its excellent performance in detecting m6A, GLORI will help promote and solve the problem of ^m6Ain cell differentiation, Functional research and core biological issues in various fields, such as embryonic development and clinical testing, are expected to become the "gold standard"
of quantitative m6A sequencing technology.

Postdoctoral fellow Yi Yunpeng directly Shen Weiguo

Professor Wang Jing of Peking University School of Pharmacy and Professor Yi Chengji of School of Life Sciences, Peking University are the corresponding authors
of this research paper.
2 018 postdoctoral fellow Yi Yunpeng (outbound) and 2018 direct doctoral student Shen Weiguo, School of Pharmacy, Peking University, and Liu Cong, a postdoctoral fellow at the School of Life Sciences, Peking University, and doctoral student Sun Hanxiao, and Li Kai (graduated) are the 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.

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(State Key Laboratory of Natural Medicines and Biomimetic Drugs, School of Pharmacy)