N6-Methyladenosine Landscape of Glioma Stem-Like Cells: METTL3 Is Essential for the Expression of Actively Transcribed Genes and Sustenance of the Oncogenic Signaling
Abstract
:1. Introduction
2. Materials and Methods
2.1. Neurosphere Cell Culturing
2.2. RNA Isolation, Reverse Transcription and qPCR Analysis
2.3. RNA Preparation for RIP-Sequencing
2.4. m6A RNA Immunoprecipitation Enrichment
2.5. m6A RNA Immunoprecipitation Sequencing
2.6. Transfection of Cells with Plasmids and siRNAs
2.7. RNA Stability Assay
2.8. Western Blot
2.9. RNA Immunoprecipitation of METTL3
2.10. Luciferase Reporter Assay
2.11. RNA Isolation, cDNA Synthesis and qRT-PCR
2.12. Lentivirus Preparation
2.13. RNA Editing
2.14. Differential Alternative Splicing
2.15. Alignment of RNA-Sequences
2.16. Gene Set Enrichment Analysis (GSEA)
- Genes were grouped as activated (H3K4me3) or repressed (H3K27me3, H3K27me3 + H3K4me3) in MGG8 GSC compared to NSC.
- Genes were grouped as activated (H3K4me3) or repressed (H3K27me3, H3K27me3 + H3K4me3) in MGG8 GSC compared to MGG8 DGC.
- Genes were grouped as activated (H3K27Ac) in MGG8 GSC compared to MGG8DGC.
- The activated and upregulated/repressed and downregulated genes in GSC compared to NSC based on H3K27me3/H3K4me3marks.
- The activated and upregulated/repressed and downregulated genes in GSC compared to DGC based on H3K27me3/H3K4me3marks.
- The activated and upregulated genes in GSC compared to DGC based onH3K27Ac
2.17. Pathway Analysis
2.18. RNA Immunoprecipitation Sequencing Analysis
2.18.1. Alignment of m6A RNA Immunoprecipitation Peaks
2.18.2. Peak Calling for m6A Samples
2.18.3. Peak Visualization
2.18.4. Distribution of m6A Peak Regions in 5′UTR, exon and 3′UTR
2.19. Cumulative Frequency Distribution
2.20. miRNA Target Prediction
2.21. Motif Finding
3. Results
3.1. Transcriptome-Wide Mapping of m6A Modification Landscape in Glioma Stem-Like Cells
3.2. Impact of METTL3-Mediated m6A Modification on the Glioma Stem-Like Cells Transcriptome
3.3. METTL3 Is Essential for the Expression of Epigenetically Activated Genes in Glioma Stem-Like Cells
3.4. m6A Modification Regulates RNA Editing
3.5. Functional Association of RNA Processing Factors with m6A Modification
3.6. Regulation of Long Non-Coding RNAs and miRNAs by m6A-Methylome
3.7. Integrated Pathway Analysis of Regulated Transcriptome Implies an Oncogenic Role for METTL3
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
List of Abbreviations
m6A | N6-methyladenosine |
METTL3 | methyltransferase-like3 |
METTL14 | methyl transferase-like 14 |
FTO | fat mass and obesity-associated protein |
ALKBH5 | alkB homolog 5 |
GSC | glioma stem-like cell |
DGC | differentiated glioma cell |
NSC | normal neural stem cell |
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Gene Name | Primers (5′-3′) |
---|---|
METTL3-FP | ACCTATGCTGACCATTACCAAG |
METTL3-RP | CTGTTGGTTCAGAAGGCTCTC |
SOX2-FP | AACCCCAAGATGCACAACTC |
SOX2-RP | GCTTAGCCTCGTCGATGAAC |
OLIG2-FP | CCAGAGCCCGATGACCTTTT |
OLIG2-RP | AGGACGACTTGAAGCCACTG |
POU3F2-FP | TGACGATCTCCACGCAGTAG |
POU3F2-RP | GGCAGAAAGCTGTCCAAGTC |
SALL2-FP | TAATCTCGGACTGCGAAGGT |
SALL2-RP | TAGAACATGCGTTCTGGTGG |
ATP5G-FP | CCAGACGGGAGTTCCAGAC |
ATP5G-RP | GACGGGTTCCTGGCATAGC |
DLL1-FP | GCAGCTCTTCACCCTGTTCT |
DLL1-RP | GGTGCAGGAGAAGTCGTTCA |
NOTCH1-FP | GAGGCCTGGCAGACTATGC |
NOTCH1-RP | CTTGTACTCCGTCAGCGTGA |
HES1-FP | AGTGAAGCACCTCCGGAAC |
HES1-RP | TCACCTCGTTCATGCACTC |
NOTCH2-fp | CTGTGAGTGTCTGAAGGGTTATG |
NOTCH2-rp | GGCACTGGAAACGATTGACTTT |
NOTCH3-fp | CGTGGCTTCTTTCTACTGTGC |
NOTCH3-rp | CGTTCACCGGATTTGTGTCAC |
NOTCH4-fp | CTGGGTGTCAATGGAGAGGGA |
NOTCH4-rp | GGGTGAGACGTGCCAGTTTC |
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Visvanathan, A.; Patil, V.; Abdulla, S.; Hoheisel, J.D.; Somasundaram, K. N6-Methyladenosine Landscape of Glioma Stem-Like Cells: METTL3 Is Essential for the Expression of Actively Transcribed Genes and Sustenance of the Oncogenic Signaling. Genes 2019, 10, 141. https://doi.org/10.3390/genes10020141
Visvanathan A, Patil V, Abdulla S, Hoheisel JD, Somasundaram K. N6-Methyladenosine Landscape of Glioma Stem-Like Cells: METTL3 Is Essential for the Expression of Actively Transcribed Genes and Sustenance of the Oncogenic Signaling. Genes. 2019; 10(2):141. https://doi.org/10.3390/genes10020141
Chicago/Turabian StyleVisvanathan, Abhirami, Vikas Patil, Shibla Abdulla, Jörg D. Hoheisel, and Kumaravel Somasundaram. 2019. "N6-Methyladenosine Landscape of Glioma Stem-Like Cells: METTL3 Is Essential for the Expression of Actively Transcribed Genes and Sustenance of the Oncogenic Signaling" Genes 10, no. 2: 141. https://doi.org/10.3390/genes10020141
APA StyleVisvanathan, A., Patil, V., Abdulla, S., Hoheisel, J. D., & Somasundaram, K. (2019). N6-Methyladenosine Landscape of Glioma Stem-Like Cells: METTL3 Is Essential for the Expression of Actively Transcribed Genes and Sustenance of the Oncogenic Signaling. Genes, 10(2), 141. https://doi.org/10.3390/genes10020141