Nature Biotechnology: m6A single-base quantitative sequencing detection method

On October 27, 2022, Professor Yi Chengji's research group of the School of Life Sciences of Peking University and the research group of Wang Jing, a researcher from the School of Pharmacy of Peking University Health Science Center, jointly published an online publication entitled "Absolute quantification of single-base m⁶" in the journal Nature Biotechnology A methylation in the mammalian transcriptome using GLORI"
.

Screenshot of the paper

^m6Ais the most abundant modification in mRNA in higher eukaryotes, and^m6Ais
present in about 0.
2-0.
6% of adenosine.
^m6Arelies 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 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 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 the following limitations: (1)^m6A-baseddetection methods cannot obtain their high-resolution site information
.
(2) Restriction enzyme-based detection technology can only detect^m6Acontaining ACA
motifs.
(3) Methods based on third-generation sequencing and machine learning are expensive, have low detection accuracy, and cannot achieve absolute quantification
of^m6A.
To date, the unpreferred detection and absolute quantification of the whole transcriptome of^M6Aremains unresolved
.

The newly developed^m6A detection technology "GLORI" breaks through the above technical limitations, and for the first time realizes the true detection of high-efficiency, high-sensitivity, high-specificity, and non-preferential single-base m 6 A sites, and absolutely quantifies
the modification level of m⁶A sites 。 The core of GLORI's technology is to independently rely on antibodies, and the catalytic system of glyoxal and nitrite is found through chemical reaction combination screening, and the unmethylated adenosine is efficiently deamined to form inosine (A-to-I, > 98%), which is read as guanosine (G) during the sequencing process to form the conversion of A-to-G; m⁶A is still read as A
.
GLORI achieves absolute quantification
of the single base^m6Aby detecting the proportion of A in the sequenced read sequence.
Therefore, the GLORI technique is conceptually similar to the quantitative analysis of the genome 5 mC content
using bisulfites.

Figure 1.
GLORI's detection principle
.
GLORI realizes the transformation
of A-to-I.
b.
Chemical reaction process
of GLORI.
C.
LC-MS/MS analysis
before and after (top) and (bottom) glyoxal and nitrite-mediated deamination in GLORI.
Example of quantitative detection of^m6Alocus on gene MRPS26 by GLORI technology

Subsequently, the research team identified 176,642 m6A sites in the HEK293T transcriptome and found that detectable m6A sites can be further increased with the increase of sequencing depth, which expands people's understanding of
the number of^m6Aon mRNA.
In addition, GLORI technology enables accurate quantification of m6 A: even with low levels of modification^m6A (5%), GLORI enables accurate detection
.
Subsequently, functional analysis of mRNA containing different m6 A modification levels found that the overall^m6Amodification level on the mRNA showed a negative correlation
with the transcription level and translation efficiency of RNA.
Thus, GLORI has achieved the^m6Aquantitative mapping of the whole transcriptome for the first time, and has also completed the quantitative evaluation
of gene expression and translation regulation.

Figure 2.
Quantitative detection of^m6A
within the GLORI transcriptome.
Sites detected by GLORI in HEK293
.
GLORI is able to accurately detect the modification level
of the m^6Asite in spike-in.
Correlation
of modification levels at^m6Asites detected by GLORI between technical replicates.
Overall distribution of
modification levels at^m6Alocus in HEK293.
Transcription levels
of mRNA containing different levels of^m6Amodification.
Comparison of translation efficiency of mRNAs containing different levels of^m6Amodifications

In addition, the team found that a class of clustered m6A loci (^m6Aclusters)
in specific regions of some genes emerged.
Compared with genes without such m6A clusters, the genes of such^m6Aclusters significantly reduce the transcription level and translation efficiency of genes, thereby negatively regulating gene expression
.

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

This study further applied GLORI technology to observe the dynamic regulation of m6A in two pressure systems of heat shock and hypoxia, and two cell lines of HeLa and MEF, and provided a quantitative map
of^m6Aon stress conditions on the transcriptome.
It was found that about 4.
8-11%^m6Asites had dynamic changes under the two pressure systems, and the up-regulated and down-regulated^m6A sites showed different enrichment patterns: in the absence of oxygen, the up-regulated and down-regulated sites were distributed around the 5′UTR and stop codons, while the enrichment in the heat shock system showed the opposite trend
.
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^m6Awas significantly upregulated under heat shock conditions, and this upregulation was more significant
in mRNA with^m6Aclusters.
The results suggest that^m6Ahas specific regulation
of gene expression in different environments.

Figure 4.
GLORI detects dynamically changing^m6A
.
A.
Hypoxia-induced mRNA distribution atlas
at the^m6Asite.
Heat shock-induced mRNA distribution atlas
at the^m6Asite.
Boxplot and dot plot
of up-regulation of translation efficiency of^m6A-relatedgenes in MEF cells before and after heat shock.
Boxplot of translation efficiency of genes with or without^M6Acluster 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, breaking through 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"
of quantitative^m6Asequencing technology.

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