How to Investigate m6A Methylation in Livestock

N6-methyladenosine(m6A) is the most prevalent type of RNA modification. The regulation and functional modulation of m6A methylation involve the coordinated actions of m6A methyltransferases (writers), demethylases (erasers), and m6A-binding proteins (readers). Currently, research on m6A methylation within the field of animal husbandry is expanding, focusing primarily on animal development, animal nutrition, and animal disease prevention and control.

Approaches for m6A Omics Research

The investigation begins by detecting the overall levels of m6A using techniques such as dot blotting and liquid chromatography-mass spectrometry (LC-MS). Subsequently, the expression of m6A-related enzymes is analyzed through methods including Western blotting (WB) and quantitative polymerase chain reaction (qPCR). Thereafter, m6A sequencing (m6A-seq) is employed to screen for target genes, which are then subjected to downstream validation to elucidate their molecular mechanisms of action.

Primary Diseases Studied

• Various animal epidemics, such as classical swine fever.

Main Types of Samples

• Muscle tissues.
• Cell samples post viral infection.

Principal Research Areas

• Animal development.
• Animal nutrition.
• Animal disease prevention and control.

m6A Research Strategies in Livestock

Investigations into N6-methyladenosine (m6A) modifications within animal husbandry require a progressive logic centered around "global level detection-enzyme expression analysis-target screening-functional validation." Experimental systems should be designed considering the biological characteristics of livestock. This article systematically elucidates the technical pathways, experimental design principles, and case applications across three dimensions.

I. Core Considerations in the Design of Treatment and Control Groups

1. Genetic Background Control

To minimize interference from genetic heterogeneity, randomized block designs are employed, assigning offspring from the same parent or litter to treatment and control groups. For example, six piglets from the same sow can be randomly divided into three groups. This method has been applied in studies on m6A and fat deposition in pigs.

2. Standardization of Environmental Variables

• Feeding Conditions: Variables such as temperature, photoperiod, and feed formulation must be identical across groups. For instance, broiler experiments utilize iso-thermal environmental controls.
• Sampling Time: Physiological cycles of livestock (e.g., estrous cycles, circadian rhythms) significantly impact m6A dynamics; therefore, unified sampling time windows are essential.

3. Multiple Control Settings

In nutritional intervention studies, a tripartite control system is often adopted:

4. Disease Model Construction

In viral challenge experiments, the following groups are established:

• Healthy Control Group: Uninfected animals.
• Infected Untreated Group: Natural course control.
• Therapeutic Intervention Group: Treated with antiviral agents.

For example, in the Porcine Epidemic Diarrhea Virus (PEDV) infection model in piglets, the regulatory network mediated by ALKBH5 involving IFIT3/HERC5 was validated using this design.

II. Selection of Techniques for Global m6A Level Detection

1. Comparison of Methods and Applicable Scenarios

2. Dynamic Monitoring Scheme

Sampling at multiple time points during key developmental stages of livestock (e.g., intramuscular fat deposition period in cattle):

• Neonatal Stage (0 days old) → Rapid Growth Stage (3 months old) → Maturity Stage (12 months old)

Using LC-MS/MS to plot m6A abundance curves reveals correlations between methylation levels and production traits.

III. Technical System for Analyzing Expression of m6A-Related Enzymes

1. Key Regulatory Enzyme Spectrum

2. Multidimensional Expression Validation

• mRNA Level:

  Extraction of total RNA using the Trizol method, designing primers spanning exons to avoid genomic DNA interference, and performing absolute quantification using SYBR Green (standard curve method).

• Protein Level:

  Preparation of tissue lysates requires the addition of protease inhibitors. The use of enhanced chemiluminescence (ECL) reagents is recommended. For example, bovine muscle tissue requires extended homogenization times up to 5 minutes.

3. Construction of Spatiotemporal Expression Profiles

Taking Qinchuan cattle as an example:

Data Source: Dynamic expression profiles detected in 12 tissues of neonatal and adult cattle using qPCR.

IV. Technical Workflow for MeRIP-seq Target Screening

1. Standardized Operating Procedures

MeRIP-seq Technical Workflow:

1. RNA Extraction: Obtain high-quality total RNA from samples.
2. Fragmentation: Chemically fragment RNA to an optimal size (~100 nt).
3. Immunoprecipitation: Enrich m6A-modified RNA fragments using specific antibodies.
4. Library Preparation: Convert enriched RNA into a sequencing library.
5. High-Throughput Sequencing: Perform next-generation sequencing.
6. Data Analysis: Identify m6A peaks and differential methylation sites.

Outline of MeRIP-Seq protocol and distribution of sequencing reads. (Joseph Martin et al,. 2018)

2. Key Quality Control Indicators

• Immunoprecipitation Efficiency: The ratio of input RNA to IP product should be greater than 5%.
• Peak Width: Qualified peak widths should range between 100–200 nucleotides.
• Differential Peak Screening: Criteria of |log₂FC| ≥ 1 and false discovery rate (FDR) < 0.05.

3. Functional Enrichment Strategy

Combined KEGG and GO analyses are employed, focusing on:

• Lipid Metabolism Pathways: PPAR signaling, fatty acid degradation.
• Muscle Development Pathways: mTOR signaling, myogenesis.
• Microbial Interaction Pathways: Butanoate metabolism.

V. Repository of Downstream Functional Validation Methods

1. Gene Editing Technologies

2. Phenotypic Association Analysis

• Cellular Level: Oil Red O staining to quantify lipid droplet area (model of broiler preadipocyte differentiation).
• Tissue Level: Near-infrared spectroscopy to determine intramuscular fat content (sampling of bovine longissimus dorsi muscle).
• Whole-Organism Level: Dual-energy X-ray absorptiometry (DEXA) scanning to measure body composition (live pigs for fat/muscle ratio determination).

3. Molecular Mechanism Elucidation

• m6A–Transcriptome Association: Polysome profiling to validate changes in translation efficiency, such as YTHDF2-dependent degradation of bovine TM4SF1.
• Epigenetics–Metabolomics Integration: LC-MS to detect microbial metabolites like butyrate and folate, analyzing their regulatory effects on m6A modifications.

Research Directions of m6A Methylation in Livestock

m6A methylation plays a pivotal role in various biological processes in livestock. By modulating the function of messenger RNA (mRNA) and non-coding RNA (ncRNA), m6A modification influences muscle development, fat deposition, reproductive traits, and disease responses. This article reviews the primary research directions of m6A methylation in livestock, highlighting key findings and molecular mechanisms.

Muscle Development and Regulation

Role of m6A in Myogenesis

m6A modification is critical in the development of livestock muscle tissue. It regulates mRNA and ncRNA functions, affecting skeletal muscle differentiation, cardiac remodeling, and adipogenesis. For example, the stability and expression levels of m6A-modified IGF2BP1 and GADD45B mRNAs are controlled to promote myocyte differentiation.

In goat skeletal muscle development, m6A reader proteins such as YTHDF2, IGF2BP1, and IGF2BP3 are involved in the regulation of myofiber formation. These findings reveal the molecular mechanisms by which m6A modification influences muscle development in livestock.

Fat Deposition and Metabolism

Impact of m6A on Adipogenesis

m6A modification significantly affects fat deposition in domestic animals. For instance, the demethylase ALKBH5 regulates the stability of LCAT mRNA through m6A modification, influencing adipogenesis in chickens.

Research on fat deposition in broiler chickens has demonstrated substantial regional differences in m6A modification distribution. Genes with unique m6A modifications in fat and lean broiler breeds exhibit significant disparities in functional enrichment analyses.

Reproductive Traits and Fertility

Influence of m6A on Gametogenesis

m6A modification plays an essential role in animal reproductive traits. It participates in spermatogenesis by affecting the fate determination of spermatogonia and chromosomal histone modifications.

In studies of fat deposition in yaks, m6A modification is implicated in the sex-specific regulatory mechanisms governing fat accumulation in grazing yaks.

Disease and Health

Role of m6A in Disease Resistance

The significance of m6A modification in animal diseases has attracted considerable attention. The absence of m6A modification can markedly reduce viral loads in mouse lungs, nasal turbinates, and brains, alleviating histopathological damage.

In porcine sperm cryopreservation studies, m6A modification has been associated with RNA stability following freezing, suggesting its role in maintaining sperm viability.

Technologies and Methods

Advances in m6A Research Techniques

Emerging technologies such as methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) have been extensively applied in m6A modification studies. For example, MeRIP-seq combined with high-throughput sequencing has been utilized to investigate the regulatory mechanisms of m6A modification in avian fat deposition.

Artificial intelligence (AI) technologies have also been employed to predict m6A sites, enhancing the accuracy of predictive models and facilitating the identification of m6A modifications across the genome.

m6A methylation is a crucial epigenetic modification influencing vital physiological processes in livestock. Understanding its role in muscle development, fat metabolism, reproductive traits, and disease resistance provides valuable insights for advancing animal husbandry practices. Continued research utilizing advanced technologies will further elucidate the molecular mechanisms of m6A modification, potentially leading to novel strategies for improving livestock productivity and health.

Research Case Study

Direction 1: Animal Development

Title: FTO-Mediated Demethylation of GADD45B Promotes Myogenesis Through the Activation of the p38 MAPK Pathway

Published in: Molecular Therapy Nucleic Acids

Impact Factor: 8.8

Publication Year: 2021

DOI: 10.1016/j.omtn.2021.06.013

Sample Selection

Samples: Goat skeletal muscle tissues at different developmental stages.

Research Techniques

m6A-seq and colorimetric quantification assays.

Background

Skeletal muscle is an indispensable component of the body, playing a critical role in locomotion, metabolism, and the maintenance of homeostasis. Understanding the molecular mechanisms underlying skeletal muscle development is essential for advancing livestock muscle growth and meat production.

Objective

To investigate the role of N6-methyladenosine (m6A) modification in the development of goat skeletal muscle and to elucidate the molecular mechanisms involved in myogenesis.

Research Strategy

m6A modification has been recognized as a key regulator in mammalian development. In this study, the global m6A levels in goat skeletal muscle were quantified at different developmental stages, including embryonic and neonatal periods, using colorimetric assays. The results indicated a decrease in m6A levels during myogenic differentiation.

Through m6A-seq analysis, Growth Arrest and DNA Damage-Inducible Beta (GADD45B), a gene with reduced m6A methylation, was identified as a target gene. The expression of GADD45B increased during myoblast differentiation. Knockdown of GADD45B was found to inhibit myogenic differentiation and mitochondrial biogenesis.

Further investigation revealed that GADD45B regulates the expression of Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-Alpha (PPARGC1A) by activating the p38 MAPK signaling pathway. Moreover, silencing of the m6A demethylase Fat Mass and Obesity-Associated Protein (FTO) led to increased m6A modification of GADD45B mRNA, resulting in reduced mRNA stability and impaired myogenic differentiation.

Conclusion

The study demonstrates that FTO-mediated demethylation of GADD45B mRNA enhances skeletal muscle differentiation by activating the p38 MAPK pathway. These findings provide a molecular mechanism for the regulation of myogenesis through RNA methylation, offering potential targets for improving muscle development in livestock.

General representation of the degradome sequencing approach, miRNA target mimicry, and silencing of miRNAs. (Júlio Lima et al,. 2012)

Direction 2: Animal Nutrition

Case 1: ALKBH5 Regulates Chicken Adipogenesis by Mediating LCAT mRNA Stability via m6A Modification

Published in: BMC Genomics

Impact Factor: 3.9

Publication Year: 2024

DOI: https://doi.org/10.1186/s12864-024-10537-2

Sample Selection

Samples: Abdominal adipose tissue from 100-day-old female Sanhuang chickens.

Research Techniques

MeRIP-seq, RNA sequencing (RNA-seq), LCAT gene knockdown and overexpression studies.

Background

Excessive abdominal fat deposition in poultry leads to feed wastage and metabolic diseases. The role of N6-methyladenosine (m6A) modification in adipogenesis remains unclear.

Objective

To investigate the regulatory mechanism by which the m6A demethylase ALKBH5 influences adipose deposition in chickens.

Research Strategy

m6A Modification of LCAT Gene: The m6A modification level of the LCAT gene was significantly downregulated in the low-fat group. Overexpression of ALKBH5 reduced m6A methylation of LCAT mRNA, enhancing its stability.

Activation of Cholesterol Metabolism Pathway: ALKBH5 activated the cholesterol metabolism pathway by regulating LCAT expression, thereby inhibiting the differentiation of preadipocytes.

Effects of ALKBH5 Knockout: Knockout of ALKBH5 led to degradation of LCAT mRNA, resulting in reduced fat deposition.

Conclusion

ALKBH5 regulates chicken adipogenesis by modulating the stability of LCAT mRNA in an m6A-dependent manner. These findings provide a novel target for nutritional interventions aimed at controlling adipose formation in poultry.

Analysis of MeRIP-seq in chicken abdominal fat tissues between he high-fat and low-fat groups.

Direction 3: Animal Disease Prevention and Control

Title: Classical Swine Fever Virus Non-Structural Protein 5B Hijacks Host METTL14-Mediated m6A Modification to Counteract Host Antiviral Immune Response

Published in: PLoS Pathogens

Impact Factor: 6.7

Publication Year: 2024

DOI: 10.1371/journal.ppat.1012130

Sample Selection

Samples: PK-15 porcine kidney cells with knockdown of METTL14.

Research Techniques

m6A-seq

Background

Classical swine fever (CSF), caused by the classical swine fever virus (CSFV), results in significant economic losses for the global swine industry. The virus's capacity to evade the host's innate immune response leads to persistent infections, posing substantial challenges for disease control.

Objective

To investigate the role of m6A methylation in the mechanism by which CSFV circumvents host immune surveillance, thereby facilitating persistent infection.

Research Strategy

Elevation of m6A Modification Post-Infection:

Following CSFV infection, an increase in m6A modifications was observed both in vitro and in vivo.

Notably, the expression of the m6A methyltransferase METTL14 was upregulated.

Regulation by CSFV Non-Structural Protein 5B:

CSFV non-structural protein 5B was found to interact with the E3 ubiquitin ligase HRD1.

This interaction inhibits the degradation of METTL14, leading to elevated m6A methylation levels.

Identification of METTL14 Target Gene-TLR4:

Methylated RNA immunoprecipitation sequencing (MeRIP-seq) analysis identified Toll-like receptor 4 (TLR4) as a target of METTL14-mediated m6A modification.

Increased m6A modification of TLR4 mRNA resulted in its destabilization.

YTHDF2-Mediated mRNA Degradation:

The m6A reader protein YTHDF2 promoted the degradation of m6A-modified TLR4 mRNA.

This degradation reduced TLR4 expression levels.

Suppression of the NF-κB Signaling Pathway:

The decrease in TLR4 impaired the TLR4/NF-κB signaling pathway.

As a result, the host antiviral immune response was suppressed, facilitating viral persistence.

Conclusion

The study reveals a novel mechanism by which CSFV evades the host immune response by manipulating the m6A methylation machinery. CSFV non-structural protein 5B stabilizes METTL14 expression by inhibiting its degradation via HRD1. Elevated METTL14 levels increase m6A modification of TLR4 mRNA, leading to its degradation through YTHDF2. This process suppresses the TLR4/NF-κB signaling pathway, allowing the virus to counteract the host antiviral immune response. Targeting METTL14 presents a potential antiviral strategy to inhibit CSFV infection.

m6A modification compares PK-15 cells transfected with siCtrl or siMETTL14 by MeRIP-seq.

Technological Advances and Interdisciplinary Integration

Innovations in Detection Technologies

The integration of MeRIP-seq with single-cell sequencing-exemplified in studies of porcine embryonic skeletal muscle-has significantly enhanced the precision of m6A site detection. Additionally, the development of direct nanopore sequencing by the Oxford team has further improved the accuracy and efficiency of identifying m6A modifications. Furthermore, artificial intelligence (AI) technologies have been employed to predict m6A sites, enhancing the predictive accuracy of computational models.

Multi-Omics Integrative Analysis

The combined analysis of the epitranscriptome and metabolome in broiler chicken fat deposition research has unveiled the m6A–LCAT–cholesterol metabolism axis. This integration has provided deeper insights into the regulatory mechanisms of adipogenesis. The application of spatial transcriptomics technology, such as 10x Genomics Visium, in muscle development studies has enabled the simultaneous resolution of gene expression patterns and tissue localization. This advancement facilitates a more comprehensive understanding of the spatial dynamics of gene expression during muscle development.

Functional Validation Using Gene Editing

The utilization of CRISPR-Cas9-mediated METTL3 knockout models in bovine skeletal muscle research and conditional knockout mice in immunological studies has provided reliable tools for functional validation of m6A-related genes. These gene editing technologies allow for precise manipulation of specific genes, thereby elucidating their roles in various biological processes and advancing the understanding of m6A methylation functions.

Future Development Directions

Precision Breeding Applications

Leveraging m6A modification profiles to screen for high-quality breeding livestock presents a promising avenue for precision breeding. For instance, regulating LCAT expression through ALKBH5 can improve fat deposition efficiency in broiler chickens. This approach may enhance desirable traits in livestock, contributing to increased productivity and efficiency in animal husbandry.

Development of Disease Targets

Targeting regulatory factors such as WTAP and YTHDF2 offers potential for the development of small-molecule inhibitors for antiviral or antitumor therapies. By modulating the activity of these m6A-related proteins, it may be possible to interfere with disease processes, providing novel therapeutic strategies for veterinary medicine.

Research on Environmental Interactions

Investigating the dynamic effects of nutritional stress-such as low-protein diets-on m6A modifications and their epigenetic regulatory mechanisms is essential for understanding environmental interactions. This research can elucidate how nutritional factors influence gene expression and epigenetic landscapes, ultimately affecting animal growth, development, and health.

Conclusion

Research on m6A methylation in the field of animal husbandry has established a comprehensive continuum from fundamental mechanisms to industrial applications. The deep integration of multi-omics technologies and gene editing holds promise for breakthrough advancements in molecular breeding, disease prevention and control, and nutritional regulation. These developments are poised to provide critical technological support for the sustainable development of the animal husbandry industry, fostering innovations that enhance productivity and animal welfare.

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