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Identification of Alternative Splicing and Polyadenylation in RNA-seq Data
Published on: June 24, 2021
Integrative MeRIP-Seq and RNA-Seq Analyses Reveal Innate Immune and Infection-Related Transcriptomic Changes upon
Qian Tang1,2, Yong Hu2, Lin Zhu2
1School of Life Sciences and Medical Engineering, Anhui University, Hefei 230601, China.
Abstract:
Background: As a major regulator, methyltransferase-like 3 (METTL3) catalyzes N6-methyladenosine (m6A) modification in mRNA. The m6A modifications mediated by METTL3 influence RNA splicing, nucleocytoplasmic distribution, stability, and other functions, thereby playing a vital and indispensable role in genetic regulatory network. Although several studies have shown its critical role in mRNA fate, the global pattern of mRNA methylation alteration driven by METTL3 remain unclear. Methods: Here, a HEK293T cell line with METTL3 depletion was constructed, and RNA sequencing (RNA-seq) and methylated RNA Immunoprecipitation Sequencing (MeRIP-seq) were implemented. Additionally, quantitative Reverse Transcription PCR (qRT-PCR) technology was used to confirm some of the differentially expressed genes. Result: The mRNA methylation alteration landscape was clarified and the regions altered by m6A modification due to METTL3 deletion that was annotated and characterized, with 5763 hypomethylated/269 hypermethylated genes after METTL3 silence. Several methylation-related innate anti-infection immune genes, including MYD88, RIG-1, CYLD and IRF9, were exposed through comprehensive analysis to MeRIP-seq and RNA-seq data, and these genes were principally enriched in pathogen infection and innate immune response pathways such as Shigellosis, Yersinia infection, and the HIV-1 viral life cycle. Conclusion: Our study discovered that the METTL3 association with differentially expressed genes, suggested that METTL3 and the genes it regulates might serve as targets for defense against infection.
Insights
Methyltransferase-like 3 (METTL3) regulates mRNA methylation, impacting gene expression. Depleting METTL3 altered thousands of genes, revealing its role in innate immunity and potential as an anti-infection target.
Area of Science:
- Molecular Biology
- Epigenetics
- Immunology
Background:
- Methyltransferase-like 3 (METTL3) is a key enzyme catalyzing N6-methyladenosine (m6A) mRNA modifications.
- METTL3 influences RNA splicing, stability, and distribution, playing a crucial role in gene regulation.
- The global impact of METTL3 on mRNA methylation patterns remains incompletely understood.
Purpose of the Study:
- To elucidate the global landscape of mRNA methylation alterations driven by METTL3.
- To identify specific genes and pathways regulated by METTL3-mediated m6A modification.
- To explore the role of METTL3 in innate immune responses against pathogens.
Main Methods:
- Constructed a HEK293T cell line with METTL3 depletion.
- Performed RNA sequencing (RNA-seq) and methylated RNA Immunoprecipitation Sequencing (MeRIP-seq).
- Utilized quantitative Reverse Transcription PCR (qRT-PCR) for gene expression validation.
Main Results:
- Characterized the mRNA methylation alteration landscape upon METTL3 depletion.
- Identified 5763 hypomethylated and 269 hypermethylated genes after METTL3 silencing.
- Discovered METTL3-regulated innate immune genes (e.g., MYD88, RIG-1, CYLD, IRF9) enriched in pathogen infection and immune response pathways.
Conclusions:
- METTL3 significantly impacts the global mRNA methylation profile.
- METTL3-regulated genes are involved in crucial innate immune pathways, including responses to Shigellosis, Yersinia, and HIV-1.
- METTL3 and its regulated genes represent potential therapeutic targets for combating infections.
