How MeRIP-seq Works: Principles, Workflow, and Applications Explained

RNA modifications play key roles in regulating cellular physiological and biochemical processes by affecting processes such as RNA stability, translation and splicing. m6A methylation modification, one of the most important RNA modifications in mRNAs, plays a key role in various diseases. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) is a powerful technique that can help us to study the profile of m6A modifications on RNA transcripts as well as to determine the mapping of m6A changes in diseases, etc. This article outlines the principles and methods of MeRIP-seq and the application of MeRIP-seq in gene regulation.

The Role of m6A

Before understanding MeRIP-seq, it is important to first know what m6A is. m6A refers to the methylation modification occurring at the N6 position of adenine and is the most prevalent RNA modification on eukaryotic mRNA. It is primarily enriched near the 3' UTR, around stop codons, and within long internal exons. Research indicates that m6A modifications can influence multiple processes of RNA molecules, including splicing, transport, translation, and degradation, thereby playing a significant role at the molecular, cellular, and physiological levels. Furthermore, numerous studies have shown that the distribution and variation of m6A modifications and their regulatory factors are closely associated with various diseases, such as cancer and neurodegenerative diseases, suggesting that m6A modifications may have potential functions in the treatment of various diseases and cancers. Changes in m6A levels are expected to serve as biomarkers for disease diagnosis and prognosis, making the study of m6A methylation modifications highly significant.

Principle of MeRIP-seq

MeRIP-seq is a technique for studying methylation localization on intracellular mRNAs and IncRNAs, and can detect the distribution of m6A methylation on transcripts with high precision.The basic principle of MeRIP-seq is based on the specific interaction between m6A methylation modifications present on RNAs and m6A specific antibodies, which leads to isolation and enrichment of modified RNA fragments from unmodified RNAs by immunoprecipitation methods. The modified RNA fragments are isolated and enriched. After purification and reverse transcription, the resulting cDNA libraries were subjected to high-throughput sequencing, leading to a systematic analysis of m6A-modified RNA molecules and the roles they play in biological processes. Through such specific interactions, MeRIP-seq was able to reveal regions of distribution and changes in m6A modifications on a transcriptome-wide scale.

Procedure of MeRIP-seq operation

RNA extraction

• Total RNA is extracted from cells or tissues (limited to species with reference genomes), this step ensures the integrity and purity of the RNA.

RNA fragmentation

• The extracted RNA is chemically or enzymatically cleaved into smaller fragments (usually 100-300 nucleotides) for subsequent immunoprecipitation and sequencing.

m6A enrichment

• Antibody binding: Mix the fragmented RNA with an m6A-specific antibody and incubate for a certain period of time at a suitable temperature (usually 4°C) to ensure that the antibody binds to all m6A-modified RNA.
• Immunoprecipitation: Add magnetic beads or agarose beads that can bind the antibody for immunoprecipitation, and wash away the unbound RNA to enrich the m6A-modified RNA fragments.

RNA purification and cDNA synthesis

• RNA purification: Extract enriched m6A RNA from immunoprecipitation and remove impurities.
• cDNA synthesis: Use reverse transcriptase to convert the enriched RNA into cDNA and add the splice sequence for subsequent high-throughput sequencing.

Sequencing library construction

• Sequencing libraries are constructed by PCR amplification of the transformed cDNA.

High-throughput sequencing

• Sequence the sequencing library using a high-throughput sequencing platform (e.g. Illumina) to obtain the sequence data of m6A-modified RNA.

Data analysis

• Sequence alignment: Align the obtained sequence data to a reference genome or transcriptome to determine the location of the m6A modification.
• Enrichment analysis: Assess the degree of enrichment of m6A modifications, identify m6A-modified transcripts, and perform functional analysis.
• Bioinformatics analysis: Explore the regulatory role of m6A modifications on RNA stability, translation efficiency and gene expression in conjunction with gene expression data.

You may be interested in the "MeRIP-seq Protocol" and "MeRIP-seq Analysis" .

Genome-wide DNA methylation analysis of AMI models at series of time points(Liang X et al., 2024).

Features of MeRIP-seq

• High specificity: The m6A-specific antibody enables precise enrichment of m6A-modified RNA from total RNA, providing a genome-wide picture of m6A modifications.
• High throughput: MeRIP-seq technology, combined with advanced high-throughput sequencing techniques, makes it possible to analyze m6A modifications in RNA from multiple species in a single experiment, providing comprehensive transcriptomic data.
• Diversity analysis: MeRIP-seq is able to investigate the functional impact of m6A in different RNA populations such as mRNAs and IncRNAs.
• Functional studies: By examining the dynamics of m6A modifications under various biological conditions, MeRIP-seq elucidates the critical roles these modifications play in gene expression regulation, cell fate determination, development, and disease progression.
• Data-rich: Sequence data generated by MeRIP-seq not only localizes m6A modifications, but also integrates with other data sets such as RNA expression levels and splicing patterns. This integration provides deeper biological insights and enhances our understanding of RNA function.
• Combination with other technologies: MeRIP-seq can be effectively combined with other molecular technologies, such as RNA-seq and CLIP-seq, to enable joint histological analysis and multidimensional elucidation of m6A modification mechanisms.

Applications of MeRIP-seq

Gene Expression Regulation

• MeRIP-seq can help us study the role of m6A modifications in gene expression regulation. For instance, research using MeRIP-seq has examined the distribution of m6A sites in the breast muscle of ducks at embryonic day 13 (E13) and day 19 (E19). The findings revealed that m6A peaks are primarily concentrated in genes associated with muscle development, with different pathways enriched at E13 and E19. The m6A modification participates in duck muscle differentiation by upregulating or downregulating the expression of key genes at different stages(Chen B et al., 2022).

Cell Fate Determination

• MeRIP-seq can reveal the influence of m6A modifications on cell fate, and by comparing different developmental stages or cell types, it helps us identify key regulatory factors. Analysis using MeRIP-seq and miCLIP-seq has unveiled the m6A methylation characteristics in zebrafish. The study indicates that in embryos lacking METTL3, the YTHDF2-mediated mRNA degradation of arterial endothelial genes like notch1a and rhoca is delayed, resulting in a significant decrease in m6A levels, which hinders the generation of hematopoietic stem cell precursors (HSPCs). Continuous activation of the Notch signaling pathway further impairs endogenous vasculogenesis (EHT), thereby inhibiting the earliest HSPC production(Zhang C et al., 2017).

Disease Research

• Studies have shown that m6A modifications play a critical role in tumor occurrence and development, with MeRIP-seq used to identify specific m6A modification patterns in tumor cells. In gastric cancer (GC), MeRIP-seq data indicate that most highly modified m6A genes exhibit elevated mRNA expression levels. Clinical analyses suggest that ALKBH5 acts as an upstream regulator of PKMYT1, upregulating its expression by removing m6A modifications. PKMYT1, as a downstream target of ALKBH5, promotes the invasion and migration of GC cells. Knockout of ALKBH5 or mutations in its demethylation activity lead to increased PKMYT1 expression, indicating that it regulates PKMYT1 expression in an m6A-dependent manner. In GC samples, reduced expression of ALKBH5 correlates with tumor metastasis, while its interference promotes cell migration, closely related to ALKBH5's demethylation activity(Hu Y et al., 2022).

Viral Infection

• MeRIP-seq can also study the effect of viral infections on host RNA m6A modifications. The m6A modification sites on genomic and subgenomic RNAs of two viruses, SARS-CoV-2 and HCoV-OC43, were mapped by MeRIP-seq, and it was found that the expression levels of protein factors related to m6A installation, removal and recognition remained unchanged after HCoV-OC43 infection, but the intranuclear localization of METTL3 and the cytoplasmic levels of the m6A recognition proteins YTHDF1 and YTHDF2 levels were increased. This indicates that coronavirus RNA possesses m6A modifications, and the host's m6A pathway components play a crucial role in the replication of β-coronaviruses(Burgess HM et al., 2021).

Food and Agricultural Sciences

• Crop improvement: Applying MeRIP-seq in agricultural sciences to study the role of m6A modifications in plant growth, development and response to adversity for crop improvement and yield increase. Through MeRIP-seq analysis of the whole gene transcriptome at different stages of early maize grain development, it was found that there were many and different numbers of m6 A methylation peaks at different stages of maize grain development, mainly enriched in the 3 ' -YTR region, and it was found that there were conserved m6 A modification sites that regulated genes commonly expressed during nucleolar development. Studies have shown that m6A modification is involved in the early development of maize kernels and can be used as a new target for maize molecular breeding(Wu JW et al., 2022).
• Tolerance improvement: Studies have reported that m6A methylation plays a function in drought tolerance in plants. In order to investigate the role of m6A in drought response in wheat, transcriptome-wide m6A methylation profiles of wheat under drought stress were investigated using MeRIP-seq technology, which showed that there were multiple m6A methylation peaks that differed significantly between the control and drought conditions, and that m6A-binding proteins and demethylases showed significantly increased or significantly decreased drought sensitivity in wheat. The expression of m6A-binding proteins and demethylases was up-regulated or significantly down-regulated after drought in wheat, and the loss-of-function mutant of its binding protein TaECT9 showed significantly increased drought sensitivity. The results suggest that m6A methylation plays an important role in drought tolerance in wheat and can be a potential target for improvement(Pan Y et al., 2024).

Comparative Studies Across Species

• The MeRIP-seq technology can compare m6A modification characteristics across different species. By analyzing specific positions, abundance, and functional associations of modifications, researchers can identify conserved and species-specific regions of m6A modifications, quantify changes in modification abundance, and explore their biological roles in conjunction with genomic information.

Integration with Other Omics Technologies

• Combining with other histology techniques: MeRIP-seq can be combined with other techniques such as RNA-seq or CLIP-seq to perform multi-dimensional analysis and reveal the role of m6A modification in the regulation of gene expression more precisely. For example, several examples listed in the article are almost all final data derived from the analysis of m6A modification by MeRIP-seq in combination with other technologies such as RNA-seq or CLIP-seq.

In summary, MeRIP-seq, as a powerful tool, not only provides a new perspective for the study of m6A modifications, but also lays the foundation for exploring the roles of these modifications in biological processes such as the regulation of gene expression, cell proliferation and differentiation, and also provides new research ideas and methods for disease mechanisms, clinical applications and agricultural sciences.

References:

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6. Chen B, Liu S, Zhang W, Xiong T, Zhou M, Hu X, Mao H, Liu S."Profiling Analysis of N6-Methyladenosine mRNA Methylation Reveals Differential m6A Patterns during the Embryonic Skeletal Muscle Development of Ducks."Animals (Basel).2022;12(19):2593.https://pubmed.ncbi.nlm.nih.gov/36230334/
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