MeRIP-seq: Comprehensive Guide to m6A RNA Modification Analysis

RNA modifications play crucial roles in the regulation of gene expression, determination of the fate of RNA molecules, cell differentiation and development, environmental adaptation, and disease genesis. By affecting RNA stability and translational efficiency, these modifications can rapidly respond to external stimuli, modulate cellular function, and exhibit aberrant patterns in a variety of diseases, including cancer. In-depth study of the dynamics of RNA modifications and mapping of RNA modifications in cells can help us explore the pathogenesis of diseases and develop new therapeutic targets. m6A methylation is an important epigenetic modification in RNA modification, which is prevalent in eukaryotes, prokaryotes, and viruses, and has been shown to regulate several physiological processes in the human body and to be associated with a variety of diseases. Therefore, we need to accurately study the profile of m6A modification in cells and find the intersection of m6A modification changes and disease association. MeRIP-seq (Methylated RNA Immunoprecipitation Sequencing) technology is currently one of the most widely used techniques for the study of m6A modification, the following let us together to understand the principle of MeRIP-seq and operation procedures and other information.

What is the MeRIP-seq?

MeRIP-seq is a powerful technique for studying m6A methylation in various types of RNA. The use of a specific anti-m6A antibody that binds specifically to m6A allows us to isolate RNA transcripts modified by m6A from total RNA. After immunoprecipitation, the enriched RNAs were then analyzed using high-throughput sequencing to explore the distribution, dynamics and function of m6A modifications throughout the transcriptome.

MeRIP-seq detects at the gene level, is capable of detecting the presence of m6A on a genome-wide scale, identifies the location of m6A peaks, quantifies the level of modification, and is capable of clarifying the level of modification for each gene for group-to-group comparisons.Currently, this technique has been widely used to explore the involvement of m6A in various biological processes, such as cell development, differentiation, regulation of environmental changes, and the effects of m6A on cancer and other diseases.

Principle of MeRIP-seq

The basic principle of MeRIP-seq is to specifically extract and enrich m6A-modified RNA molecules from the RNA pool by immunoprecipitation based on m6A-specific antibody recognition and binding to m6A on RNA, and then perform high-throughput sequencing on these RNAs. Combined with bioinformatics technology, the distribution map of m6A modification sites in the whole transcriptome can be drawn to determine the genes related to m6A modification. It is also possible to compare transcriptome data under different cell states or environmental conditions to study how m6A modification affects gene expression and functional changes. At present, the application of MeRIP-seq has been extended to many biological fields, including developmental biology, immunology and oncology.

For a more in-depth understanding of the technical principles underlying MeRIP-seq, refer to "Principle and Applications of MeRIP-seq."

Characteristics of MeRIP-seq

Specificity

• Antibody selection: MeRIP-seq uses specific antibodies ( such as m6A antibody ) to effectively identify and bind m6A methylation-labeled RNA, ensuring enrichment to the target region.IP enrichment is highly reproducible, minimizing antibody enrichment bias.
• Avoiding non-specific binding: In the process of immunoprecipitation, optimizing the experimental operating conditions such as prolonging the incubation time and optimizing the washing conditions can reduce the non-specific binding of RNA and improve the specificity of RNA.
• Validation experiments: MeRIP-qPCR and other techniques were used to verify specific methylation sites to ensure that the identified methylation signals were reliable.

High throughput

• A wide range of genomes: MeRIP-seq can support us to analyze thousands of RNA molecules at the same time to obtain methylation information of the entire genome.
• Data Depth: MeRIP-seq uses high-throughput sequencing to capture a large amount of information from thousands of RNA molecules, which helps us to identify subtle changes in m6A modification patterns under different cell conditions or environmental factors.
• Automated and efficient: With the advancement of sequencing technology, many steps in the MeRIP-seq workflow, such as RNA extraction, m6A immunoprecipitation, library preparation, and sequencing, now require very little labor.

 Widely used

• MeRIP-seq can detect m6A in animals, plants, cells or tissues, and has been widely used in research areas such as tissue development, stem cell differentiation and renewal, environmental response to stress, cancer onset and progression, and drug discovery.

Workflow of MeRIP-seq

The MeRIP-seq method combines immunoprecipitation with high-throughput sequencing to selectively enrich and analyze RNA molecules containing m6A modifications. This technique enables researchers to map the distribution of m6A across the transcriptome, revealing insights into the regulatory roles these modifications play in gene expression and cellular function.

Key Steps in the MeRIP-seq Method

RNA Extraction

• Extracting total RNA from a biological sample (e.g., cells or tissues), and must be using RNAase-free reagents to ensure that the RNA presented is complete and of high quality.

Immunoprecipitation

• The extracted RNA was immunoprecipitated using m6A-specific m6A antibody. The antibody binds specifically to m6A methylation on the RNA molecule and selectively enriches m6A-modified RNA from total RNA.

Washing and Elution

• After incubating with the antibodies, the mixture is washed to remove non-specifically bound RNA, typically requiring multiple washes to reduce background noise. This step ensures that only the m6A-enriched RNA is retained. Subsequently, the m6A-modified RNA is eluted from the antibody complex, preparing it for further analysis.

Library Preparation

• Eluted RNA is converted to cDNA using reverse transcriptase with the addition of specific primers and then amplified by PCR to construct libraries for sequencing. However, use column purification or magnetic bead-based methods to remove primer dimers and unreacted reagents to ensure the purity and quality of the library prior to high-throughput sequencing.

Sequencing

• High-throughput sequencing: After the libraries have passed the assay, load different libraries onto a sequencing platform (e.g., Illumina or Ion Torrent) for high-throughput sequencing according to the effective concentration and target downstream data volume.
• Data Output: Download raw sequencing data and perform quality checks such as removing junctions and low-quality bases to ensure the accuracy and reliability of the sequences.

Data Analysis

• Read Processing: Bioinformatics software is used to process the sequencing data, including trimming adapter sequences, quality control, and filtering.
• Peak Identification: Through alignment analysis, the sequences are mapped to a reference genome to identify peaks of m6A modification.
• Quantitative Analysis: The levels of m6A modification on each gene or transcript are quantified, comparing different conditions for differential methylation.
• Functional Annotation: Multiple datasets are integrated to analyze the effects of m6A modification on gene expression, splicing, and translation, exploring its roles in biological processes. Various statistical analyses help identify differentially methylated transcripts under varying conditions.

MeRIP-seq reveals correlation between m6A and cell adhesion, and ALKBH5 is associated with GC
prognosis(Hu Y et al., 2022)

For detailed instructions on conducting a MeRIP-seq experiment, refer to the "MeRIP-seq Protocol."

MeRIP-seq Analysis

MeRIP-seq/m6A seq is currently one of the most widely used techniques for studying m6A modification.The MeRIP-seq method introduced earlier provides the data foundation, while MeRIP-seq analysis is the process of processing and interpreting these data. Bioinformatics provides essential computational tools and methods for analysis. The integrity and effectiveness of this process depend on the close integration of three links.
MeRIP-seq data analysis

• Data processing: Using tools such as FastQC to control and filter the quality of the downloaded raw sequencing data.This step helps identify low-quality reads and prune adapters or low-quality bases in the dataset.
• Comparison: Use comparison tools such as STAR or HISAT2 to align the processed reads to the reference genome or transcriptome to determine their position in the genome. Accurate comparison is crucial for reliable identification of m6A modification sites.
• Identifying methylation sites: Using algorithms such as MACS to compare the coverage of immunoprecipitated samples with control samples and identify significantly enriched methylation sites. This helps distinguish between real m6A sites and background noise, thereby enhancing the robustness of the results.
• Differential analysis: After obtaining the gene expression of each sample, differential gene analysis is performed to identify significant changes in m6A modification patterns by comparing the treated and control groups to determine which genes have undergone large changes in expression. This process can be performed using tools such as DESeq2 or edgeR.
• Function annotation: KEGG and GO pathway analysis of identified differential m6A peaks to link m6A modifications to specific biological functions and signaling pathways.
• Visualization: Use visualization tools (e.g. Integrated Genome Viewer [IGV], Circos) to present data in heatmaps, Venn diagrams, etc. Effective visual representation helps to generate hypotheses and explain complex relationships.

In conclusion,MeRIP-seq analysis is the field of applying computational tools and methods to analyze, understand, and visualize MeRIP-seq data.

• Algorithm development: Develop new calculation methods and software tools to improve the accuracy and reliability of identifying methylation sites.
• Statistical analysis: Statistical models were used to analyze the differential patterns of methylation under different conditions and to evaluate the significance of the results.
• Data integration: MeRIP-seq data can be combined with other types of biological data ( such as RNA-seq, ChIP-seq, etc. ) to obtain more comprehensive modification data.
• Visualization tools: Use heat maps, Manhattan maps and other graphical tools to display the analysis results, so that we can understand and share the data.

More information about MeRIP-seq data analysis, refer to "MeRIP-seq Analysis".

MerRIP-seq and m6A

The common modifications of  mRNA include N6-methyladenosine ( m6A ), N1-methyladenosine ( m1A ), 5-methylcytidine ( m5C ) and pseudouridine ( PD ). After these modifications occur, they are embedded in RNA transcripts, and their information is attached to the base sequence, which can affect RNA function. m6A is a methylation modification of the N6 site of adenylate, and its enzyme system includes methyltransferase, demethylase, etc. These structures are involved in the regulation of RNA metabolism, including translation, splicing, degradation, etc., thereby regulating a variety of cellular processes, including self-repair, differentiation, invasion and apoptosis.

With the development of next-generation sequencing ( NGS ) technology, the structure and function of m6A have been gradually understood. MeRIP-seq is a technique for immunoprecipitation of m6A-modified RNA fragments using m6A-specific antibodies, followed by sequencing to explore the function of m6A modification. More than 12,000 highly conserved m6A methylation peaks have been found in both coding and non-coding RNAs. By jointly analyzing multiple MeRIP-seq sequencing data, it was found that m6A modification generally occurred in the common motif RRACH ( R = G / A ; h = A / C / U ). In the RRACH motif, m6 A often occurs in the coding sequences ( CDS ) and 3'-untranslated regions ( 3'-UTRs ), especially near the stop codon.

For example，Systemic lupus erythematosus ( SLE ) is an autoimmune disease of unknown etiology. Since m6A has been shown to be involved in various immune processes, such as interferon production and immune cell regulation, in order to explore the role of m6A in SLE immune response disorders, researchers collected PBMCs from SLE patients and used MeRIP-seq technology to detect the m6A modification map of patients. MeRIP-seq and RNA-Seq were also combined to comprehensively explore the location and dynamic changes of m6A modification. Using MeRIP-seq data to detect the obvious abnormal methylation characteristics of m6A in SLE patientsFurther analysis revealed the immune role of m6A in SLE(Liu Y et al,2024).

Fever with thrombocytopenia syndrome virus (SFTSV) is an infectious agent with high lethality. It has been shown that m6A modifications can play a role in viral infection. However, the interaction between m6A modifications and SFTSV infection remains poorly understood. Using MeRIP-seq, researchers analyzed E-JS2013-24-infected HeLa cells to explore how m6A modifications on SFTSV RNA are distributed and how they change.By MeRIP-seq, it was found that the m6A modification on SFTSV RNA interacts with YTHDF1, which reduces the stability of SFTSV RNA and the translational efficiency of SFTSV protein. It was also found that SFTSV virulence factors NSs can inhibit YTHDF1 thereby promoting YTHDF1 m6A-dependent degradation of SFTSV mRNAs(Liu B et al,2024).

To investigate how m6A modification plays a role in porcine muscle development, researchers extracted genome-wide RNA from several male piglets and analyzed the m6A modification pattern of porcine muscle tissues by MeRIP-seq. WFS1, a muscle-specific periplasmic transmembrane protein, represses normal genes in the process of myogenesis and thus promotes muscle differentiation, and the MeRIP-seq data showed that the m6A modification in combination with YTHDF2 accelerated the degradation of WFS1. Significant differences in m6A modification were also observed in different types of piglets(Gu H et al,2024).

The distribution characteristics and range of m6A methylation in oral squamous cell carcinoma (OSCC) are not clear. To investigate the relationship between m6A and OSCC, the researchers determined the m6A methylation levels of four groups of OSCC and neighboring normal tissues by using the MeRIP-seq technique and combined with GO analysis and KEGG analysis, and found that there were differentially methylated m6A peaks in OSCC, and These differentially methylated m6A sites were associated with aberrantly expressed genes in OSCC, suggesting a potential link between OSCC and m6A(Liu Y et al,2023).

In conclusion, MeRIP-seq is a powerful tool to explore m6A modification profiles in organisms including animals, plants, and viruses, and it can also be utilized to analyze m6A modifications in diseases to map RNA modifications in human diseases, to search for new therapeutic targets, or to formulate precision medicine in a wide range of human diseases.

References:

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3. Liu Y, Wang X, Huang M, Luo A, Liu S, Cai M, Li W, Yuan S, Zheng Z, Liu X, Tang C. "METTL3 facilitates kidney injury through promoting IRF4-mediated plasma cell infiltration via an m6A-dependent manner in systemic lupus erythematosus." BMC Med.2024;22(1):511.https://pubmed.ncbi.nlm.nih.gov/39501302/
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7. Nie S, Zhang L, Liu J, Wan Y, Jiang Y, Yang J, Sun R, Ma X, Sun G, Meng H, Xu M, Cheng W."ALKBH5-HOXA10 loop-mediated JAK2 m6A demethylation and cisplatin resistance in epithelial ovarian cancer."J Exp Clin Cancer Res.2021;40(1):284.https://pubmed.ncbi.nlm.nih.gov/34496932/
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