Nature Biotechnology's Wang Jing research group developed m6A single-base quantitative sequencing detection method

On October 27, 2022, the Department of Chemical Biology, School of Pharmacy, Peking University, and Wang Jing's Research Group of the State Key Laboratory of Natural Medicines and Biomimetic Drugs joined forces with Yi Chengji Research Group of the School of Life Sciences of Peking University in top international academic journals The journal Nature Biotechnology published a title titled "Absolute quantification of single-basem⁶A.
" methylation in the mammalian transcriptome using GLORI"
.

m6A is the most abundant modification within the mRNA of higher eukaryotes, with approximately 0.
2-0.
6% adenosine present m⁶A retouching
.
^m6A relies on modifying enzymes, demodifiers, and binding proteins to perform regulatory functions
.
At present, ^m6A has been found to regulate mRNA splicing, nucleation, stability and protein translation, and can participate in development, gametogenesis, cell reprogramming, biorhythm, Functional regulation
of various physiological and pathological processes such as diseases.
To better study m6A biofunction and clinicopathological research, ^m6A was developed 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, there are several important limitations of existing technologies: (1) based on Detection methods for ^m6A antibodies are not available for their high-resolution site information
.
(2) Restriction enzyme-based detection techniques can only detect ^m6A containing ACA
motifs.
(3) Methods
based on third-generation sequencing and machine learning.
Although this method provides single-base information and quantifies ^m6A, third-generation sequencing has problems such as high cost, low detection accuracy, and does not allow m6 to be ^realized Absolute quantification
of A.
To date, the unpreferred detection and absolute quantification of the whole transcriptome of ^M6A remains unresolved
.

Glyoxal and nitrite-mediated deamination of unmethylated adenosine (GLORI) (Glyoxal and nitrite-mediated deamination of unmethylated adenosine) developed by Wang Jing's research group Breaking through the limitations of the above technologies, it has achieved the first true sense of high efficiency, high sensitivity, high specificity, and no preference for single-base m⁶A site detection The modification level of the^m6A site is absolutely quantified
.
The core of GLORI's technology is to independently rely on antibodies, and the catalytic system of glyoxal and nitrite is discovered by chemical reaction combination screening, and the unmethylated adenosine is efficiently deamined to form inosine (A-to-I, > 98%).
Inosine is read as guanosine (G) during sequencing to form the conversion of A-to-G; After sequencing, ^m6A is still read as A, thus realizing the ^{m6 pair} Single base recognition
of A.
GLORI achieves single-base ^m6A by detecting the proportion of A in the sequenced read sequence Absolute quantification
.
Therefore, GLORI technology is conceptually similar to the method of quantifying the
genome 5mC using bisulfites.

Figure 1: Detection principle
of GLORI.
a, GLORI realizes the conversion
of A-to-I.
b, the chemical reaction process
of GLORI.
c, glyoxal and nitrite in GLORI before and after (top) and (bottom) before and after adioxal and nitrite-mediated deamination LC-MS/MS analysis
.
Example of
GLORI technology for quantitative detection of m6A locus on gene MRPS26.

Subsequently, we collaborated with the Yichengji research group of the Peking University Academy of Biosciences to identify 176,642 m6 in the HEK293T transcriptome A sites, and it was found that detectable ^m6A sites remained unsaturated as sequencing depths increased, and the results extended to people on mRNA m^6A Awareness of
the existence of quantities.
In addition, GLORI technology enables accurate quantification of ^m6A: even at low levels of modification ⁶ A (5%), GLORI is also able to achieve accurate detection
.
Further functional analysis of mRNAs containing different levels of ^m6A modification found that the overall mRNA was m The modification level of ⁶A was negatively correlated with
the transcription level and translation efficiency of RNA.
So far, we have achieved the ^m6A 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 levels of m6A modification.
f, comparison of translation efficiency of
mRNA containing different m6A modification levels.

In addition, we found that a class of clustered m6A loci (m6A) has occurred in specific regions of some genes Cluster)
.
Compared to genes that do not have such m6A clusters, this type of ^m6A The cluster gene significantly reduces the transcription level and translation efficiency of the gene, thereby playing the role
of negatively regulating the expression of the gene.

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

Finally, we applied GLORI technology to two stress conditions of heat shock and hypoxia in HeLa and MEF cell lines for further observation The dynamic regulation of m6A and the quantitative mapping of ^m6A to stressful conditions on the transcriptome are provided
。 The results show that about 4.
8-11% ^m6A sites have dynamic changes under the two pressure systems, and the up-regulated and down-regulated ^m6A sites show different enrichment modes: up-regulated and down-regulated m in the absence of oxygen ^{The 6}A locus is mainly enriched near the 5′UTR and stop codon, while this enrichment shows the opposite result
in the heat shock system.
Different from the negative regulation of gene expression by ^m6A in wild-type cells: under heat shock, the translation efficiency of mRNA with elevated overall modification level of ^m6A was significantly upregulated.
And this upregulation is more pronounced
in mRNA with ^m6Aclusters.
The results suggest that m6A has specific regulation of gene expression in different environments, and also provides a powerful technical tool for subsequent research on the biological function of ^m6Adynamic regulation
。

Figure 4: GLORI detects dynamic changes in ^m6A
.
a.
A.
Hypoxia-induced mRNA distribution atlas
at m6A sites.
b, heat shock-induced mRNA distribution atlas
at the m6A site.
Boxplot and dot plot
of up-regulation 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 high-sensitivity and high-specificity of GLORI technology for the absolute quantitative detection of ^m6A without preference single base, which overcomes the current m ⁶A bottleneck of
quantitative sequencing technology.
Based on its excellent performance in detecting m6 A, GLORI will help to promote and solve ^m6 A's functional research and core biological issues in various fields such as cell differentiation, embryonic development and clinical detection are expected to become quantitative ^m6A sequencing technology "Gold standard"
.