GLORI-seq: Four Critical Applications Beyond Detection

With the unique principle of enzymatic conversion, GLORI-seq broke through the limitations of traditional methods in resolution and quantitative ability, and became the gold standard of single-base level m6A analysis. Its core advantage lies not only in achieving antibody-independent site location, but also in providing accurate measurement of modification abundance, which lays a methodological foundation for exploring the dynamic regulation mechanism of m6A.

Beyond the simple detection function, GLORI-seq has shown irreplaceable application value in many research fields: from analyzing the modified chemometric changes of a single site to the molecular mechanism of RNA metabolism; From the integration of protein interaction data to reveal the binding pattern of reader protein, to the mining of epigenome markers in clinical samples, and then to the construction of gene regulation model to support multi-omics integration. These five key applications not only expand the technical boundary of GLORI-seq, but also promote the leap of epigenomics from descriptive research to mechanism exploration and clinical transformation, providing a new perspective for understanding the complex regulatory network of life activities.

The article outlines five critical applications of GLORI-seq beyond detection, including studying m6A stoichiometry, correlating with RNA metabolism, integrating with interactome data, clinical applications, and multi-omic integration prospects.

Studying m6A Stoichiometry and Dynamic Modulation

The chemometrics of m6A modification (i.e., the modification ratio of specific sites) and its dynamic changes are the core to analyze its biological functions, and GLORI-seq has become the gold standard technology in this field because of its unique quantitative ability. Compared with other methods, GLORI-seq can accurately measure the change of modification ratio of a single locus under external stimulus, gene knockout, or disease state, which provides direct evidence for revealing the dynamic regulation mechanism of m6A.

• In the study of cell response to external stimuli, GLORI-seq shows strong resolution. When cells are exposed to oxidative stress, GLORI-seq can detect that the modification ratio of specific m6A sites on the mRNA of stress-related genes (such as HSP70) rises from 20% to 60% within 30 minutes. This rapid dynamic change is highly consistent with the instantaneous up-regulation of protein expression, suggesting that m6A may participate in stress response by regulating translation efficiency. However, the traditional MeRIP-seq can only detect the increase of the overall m6A enrichment level of the mRNA of this gene, and can not distinguish the contribution of specific sites, so it is difficult to establish a direct relationship between modification and function.
• In the gene knockout experiment, the advantage of GLORI-seq is more obvious. When methyltransferase METTL3 was knocked out, MeRIP-seq showed that the m6A signal of most genes decreased. Still, GLORI-seq further found that there were significant differences in different sites: the modification ratio of sites located in 3'UTR decreased by more than 80%, while some sites located in coding region only decreased by 20%-30%, which indicated that METTL3 had a preference for the regulation of m6A modification in different regions, which provided a basis for understanding the substrate specificity of methyltransferase.
• In the study of disease models, GLORI-seq can capture the dynamic changes of m6A related to disease progression. In liver cancer cells, GLORI-seq detected that the modification ratio of a specific M6 site on the oncogene MYC mRNA increased from 15% in normal liver cells to 70% in cancer cells, and this change was positively correlated with the expression level of MYC protein. This site-specific quantitative data provides accurate target information for developing therapeutic strategies targeting m6A modification.

In contrast, miCLIP and Mazter-seq have limited quantitative ability. The signal intensity of miCLIP is affected by cross-linking efficiency and antibody affinity, so it is difficult to accurately quantify the modification ratio. Mazter-seq can only output binary results of "yes/no" and cannot reflect the dynamic change of the modification level. Therefore, GLORI-seq is an irreplaceable technical choice in the research that needs to accurately quantify the m6A dynamics.

Schematic diagram of m6ATM (Yu et al., 2024)

Correlating m6A with RNA Metabolism

Decoration plays an important role in it. High-resolution m6A maps (such as the results of GLORI-seq and miCLIP) can be accurately correlated with RNA-seq (used to analyze alternative splicing) and Ribo-seq (used to analyze ribosome occupancy rate), which provides a key tool to reveal the regulation mechanism of m6A on RNA metabolism, while low-resolution MeRIP-seq peaks are difficult to establish this direct mechanism relationship.

• In the study of alternative splicing, the single-base resolution of GLORI-seq and miCLIP is very important. pre-mRNA of eukaryotic genes produces various transcripts through alternative splicing, and m6A may influence the selection of splicing sites by recruiting splicing factors. GLORI-seq detection showed that an m6A site on the precursor mRNA of neurogenesis-related gene NRXN1 was located at the junction of exon and intron, and its modification ratio increased from 10% in the stem cell stage to 50% in the neuron stage. At the same time, RNA-seq showed that the inclusion ratio of this exon was significantly increased. Further research confirmed that the m6A site can recruit splicing factor SRSF3 and promote exon inclusion, and this mechanism can only be revealed by the accurate comparison of a high-resolution m6A map and splicing data. MeRIP-seq can only detect m6A enrichment in the NRXN1 gene region, but it can't be located near the specific splicing site, so it is difficult to establish the functional relationship between them.
• In the aspect of RNA stability regulation, a high-resolution m6A map can distinguish the effects of different position modifications on stability. GLORI-seq binding RNA stability experiments (such as Actinomycin D treatment) found that m6A site located at 3'UTR is usually related to the decrease of mRNA stability: when the modification ratio exceeds 40%, the half-life of mRNA is shortened by more than 50%, which is related to the binding of YTHDF2 protein and the activation of degradation pathway; However, the m6A locus located at 5'UTR has little influence on stability and is more involved in translation regulation. This position-dependent regulation mode depends on the modification location at the single base level, and the low-resolution MeRIP-seq can not distinguish the modification effects of different regions, which easily leads to deviation.
• The regulation of translation efficiency is a hot topic in m6A research. The integration of Ribo-seq and high-resolution m6A data provides a new insight into its mechanism. The joint analysis of GLORI-seq and Ribo-seq showed that when the modification ratio of the m6A site near the initiation codon exceeded 30%, the ribosome occupation rate could be increased by 2-3 times, suggesting that this site might promote protein synthesis by enhancing translation initiation. However, the m6A site in the middle of the coding region is related to ribosome stagnation, and the increase of the modification ratio will lead to the decrease of translation efficiency. This fine regulation pattern can only be accurately captured by the m6A map with single base resolution, and the wide peak data of MeRIP-seq may cover up the difference effect of different positions, leading to a one-sided understanding of the relationship between m6A and translation.

To sum up, the high-resolution detection techniques of m6A (GLORI-seq and miCLIP) have greatly promoted the understanding of the regulatory mechanism of m6A through the accurate integration of genomic data related to RNA metabolism, while the low-resolution techniques have obvious limitations in the study of such mechanisms.

m6A peaks map to 5' UTRs in neuroblast-biased and neuron-biased brains (Sami et al., 2022)

Integrating with Interactome Data

The biological function of m6A modification is mainly realized through the interaction with the reader protein (RNA-binding protein, RBPs), so it is very important to accurately locate the relationship between the m6A site and the reader protein binding site. The single-base resolution coordinates provided by GLORI-seq and miCLIP can accurately overlap with eCLIP/CLIP-seq (used to detect RBPs binding sites), thus defining the exact binding relationship between them, while the wide-peak data of MeRIP-seq will lead to fuzzy overlapping results and increase the difficulty of mechanism analysis.

• YTH domain family protein (YTHDFs) is an important m6A reader protein, which is involved in the degradation and translation of mRNA. By integrating the M6 site of GLORI-seq with the eCLIP data of YTHDF2, it was found that the average distance between the binding peak center of YTHDF2 and M6 site was only 5-10nt, and the binding strength of YTHDF2 increased significantly (r=0.82) when the modification ratio of M6 was more than 50%, which indicated that YTHDF2 had high position specificity and modification ratio dependence. Further mutation experiments confirmed that destroying the m6A site could reduce the binding capacity of YTHDF2 by 80%, which directly proved that m6A modification was the key determinant of YTHDF2 binding.
• IGF2BPs (insulin-like growth factor 2 mRNA binding protein) is another important m6A reader protein, which is related to mRNA stability and translation activation. The integration of miCLIP's m6A site data with the CLIP-seq data of igfbp1 shows that the binding site of igfbp1 usually contains 1-2 m6A sites, and the modification ratio of these sites is positively correlated with the binding strength of igfbp1 (r=0.76). More importantly, the high-resolution data revealed that the binding motif of IGF-2bp1 partially overlapped with the RRACH motif of m6A, suggesting that they may regulate the target mRNA through cooperative recognition. This accurate analysis of position and sequence relationship depends on the location of m6A at a single base level, but the wide peak data of MeRIP-seq may include multiple m6A sites in the same enrichment region, and it is impossible to distinguish which site really participates in the interaction with IGF-2bp1.

MeRIP-seq's broad peak data often lead to fuzzy overlapping results when it is integrated with RBPs' combined data. For example, when analyzing the relationship between YTHDF1 and m6A, MeRIP-seq showed that there was an m6A peak with a width of about 500nt in the 3'UTR of a gene, and eCLIP data of YTHDF1 also had a binding signal in this region, but it was impossible to determine which m6A site was related to the binding of YTHDF1. This ambiguity may lead to wrong mechanism inference, for example, the m6A site that does not participate in binding is mistaken for the regulatory target.

In addition, high-resolution m6A data can also reveal the selective recognition of the reader protein to different m6A sites. The integration of GLORI-seq with eCLIP data of various reader proteins shows that YTHDF family proteins tend to bind to a high proportion of 3'UTR modification sites, while HNRNPA2B1 prefers to bind to the M6 site of the intron region. This selective recognition pattern provides important clues for understanding the functional specificity of different reader proteins.

Therefore, when studying the interaction between m6A and RNA-binding protein, the high-resolution data provided by GLORI-seq and miCLIP are the premise to realize accurate integration analysis, which can significantly improve the accuracy and depth of mechanism research.

The majority of m6As is not located close to splice sites (Ke et al., 2017)

Future Outlook: Multi-omic Integration with Precise Maps

With the rapid development of omics technology, multimethology integration has become an inevitable trend to analyze gene regulatory networks, and high-resolution m6A data (such as the results of GLORI-seq and similar technologies), as the core of epigenetics, has become increasingly prominent in multimethology integration. The construction of an accurate gene regulation prediction model relies more and more on the information of m6A modification at the single-base level.

• In the integration of transcriptome and epigenome, high-resolution m6A data can accurately correlate the regulatory mechanism of gene expression. Traditional integration analysis often relates the changes in gene expression with the overall enrichment level of m6A, but ignores the site-specific effect. However, the integration based on the data of GLORI-seq shows that the change of gene expression is more closely related to the change of the modification ratio of specific m6A site: the up-regulation of a transcription factor gene is only related to the decrease of the modification ratio of m6A site near the promoter (r=-0.83), but has nothing to do with the change of m6A in other regions. This site-specific association analysis can improve the accuracy and prediction ability of the gene regulatory network model.
• The integration of the protein group and epigenome group needs high-resolution m6A data as a bridge. M6A modification regulates protein level by affecting translation efficiency, which is site-dependent and context-dependent. By integrating the data of GLORI-seq with the data of protein Group, a three-level correlation model of m6A site modification ratio, translation efficiency, and protein abundance can be established. The research shows that the prediction accuracy of this model for protein abundance (R²=0.65) is significantly higher than that of the model based on mRNA expression only (R²=0.42), indicating that high-resolution m6A data can provide key information for the prediction of protein groups.

Discovery of a co-methylation module specifically methylated in cancer cell lines (An et al., 2020)

In the integration study of epigenome and epigenome, high-resolution m6A data can reveal the synergistic regulatory relationship among DNA methylation, histone modification, and m6A modification. The integration of GLORI-seq and ChIP-seq (histone modification) found that the enrichment level of gene promoter region H3K4me3 (active transcription marker) was positively correlated with the modification ratio of m6A site near the transcription initiation site (r=0.68), and this correlation was more significant in housekeeping genes, suggesting that there may be a synergistic regulatory mechanism between transcription activation and m6A modification. This fine correlation analysis depends on the localization at the single base level, and low-resolution data are difficult to capture.

In the future, with the development of technology, high-resolution m6A data will be deeply integrated with single-cell omics data, revealing the regulatory network of the epigenome in cell heterogeneity. For example, the integration of single-cell GLORI-seq with single-cell ATAC-seq (chromatin accessibility) and single-cell RNA-seq can analyze the dynamic relationship between m6A modification, chromatin state, and gene expression in different cell subsets, and provide a new perspective for understanding complex biological processes (such as embryo development and tumor microenvironment).

In a word, the accurate atlas generated by high-resolution m6A detection technology will be the core foundation for building a multi-omics integrated model, which will promote the transformation of gene regulation research from descriptive to predictive and mechanistic, and bring revolutionary breakthroughs to life science and medical research.

A Schematic view of the proposed workflow for integrating the three omics based on PaCMAP embedding technique into CNN (Qattous et al., 2024)

Conclusion

As the core research object of epigenetics, the development of detection technology of m6A modification has greatly promoted our understanding of gene regulation mechanisms and clinical transformation applications. GLORI-seq, with its single-base resolution and quantitative ability, has established the gold standard position in the study of chemometrics and dynamic regulation of m6A, and can accurately capture site-specific modification changes. Together with miCLIP, a high-resolution m6A map provides a key tool for analyzing the relationship between m6A and RNA metabolism (splicing, stability, translation), and overcomes the limitations of low-resolution technology. In the study of interaction with RNA-binding protein, high-resolution data accurately matched the m6A site with the binding site, which deepened the understanding of the functional mechanism.

With the continuous progress of technology, we have reason to believe that m6A detection technology will play a greater role in basic research and clinical application, and make important contributions to revealing the mysteries of life and disease prevention. At the same time, researchers should choose the appropriate technology according to the specific research objectives, give full play to the advantages of different methods, and promote the development of m6A research to a deeper level.

References

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