N6-Methyladenosine Modulates Nonsense-Mediated mRNA Decay in Human Glioblastoma

Abstract

The N 6 -methyladenosine (m 6 A) modification influences various mRNA metabolic events and tumorigenesis, however, its functions in nonsense-mediated mRNA decay (NMD) and whether NMD detects induced carcinogenesis pathways remain undefined. Here, we showed that the m 6 A methyltransferase METTL3 sustained its oncogenic role by modulating NMD of splicing factors and alternative splicing isoform switches in glioblastoma (GBM). Methylated RNA immunoprecipitation-seq (MeRIP-seq) analyses showed that m 6 A modification peaks were enriched at metabolic pathwayrelated transcripts in glioma stem cells (GSC) compared with neural progenitor cells. In addition, the clinical aggressiveness of malignant gliomas was associated with elevated expression of METTL3. Furthermore, silencing METTL3 or overexpressing dominant-negative mutant METTL3 suppressed the growth and self-renewal of GSCs. Integrated transcriptome and MeRIP-seq analyses revealed that downregulating the expression of METTL3 decreased m 6 A modification levels of serineand arginine-rich splicing factors (SRSF), which led to YTHDC1-dependent NMD of SRSF transcripts and decreased SRSF protein expression. Reduced expression of SRSFs led to larger changes in alternative splicing isoform switches. Importantly, the phenotypes mediated by METTL3 deficiency could be rescued by downregulating BCL-X or NCOR2 isoforms. Overall, these results establish a novel function of m 6 A in modulating NMD and uncover the mechanism by which METTL3 promotes GBM tumor growth and progression.Significance: These findings establish the oncogenic role of m 6 A writer METTL3 in glioblastoma stem cells.

High expression of WTAP leads to poor prognosis of gastric cancer by influencing tumour‐associated T lymphocyte infiltration

“…m6A is the most widely distributed methylation modification in eukaryotic mRNA, and its formation may modulate a series of processes after transcription, such as splicing, transport, degradation and translation of pre‐mRNA. The formation of m6A is catalysed by a large methyltransferase complex . Our study also found that high WTAP expression in patients can affect the splicing, transport, degradation and translation of mRNAs by interacting with the DLX2, DLX5, SIX1, HOXB5, HOXC6, HOXC8, RBM48 and KRAS core genes.…”

The Emerging Roles of RNA Modifications in Glioblastoma

“…The expression of METTL3, the most important m 6 A writer, is significantly elevated in GSCs, while it is attenuated during GSCs differentiation [56]. Elevated expression of METTL3 are associated with the clinical aggressiveness of malignant gliomas [57]. An integrated analysis of m 6 A-RNA immunoprecipitation (RIP) and total RNA-Sequencing (RNA-seq) of METTL3-silenced GSCs revealed that METTL3 is the most important writer responsible for m 6 A modification in GSCs [45].…”

“…An integrated analysis of m 6 A-RNA immunoprecipitation (RIP) and total RNA-Sequencing (RNA-seq) of METTL3-silenced GSCs revealed that METTL3 is the most important writer responsible for m 6 A modification in GSCs [45]. Moreover, METTL3 silencing also reverses RasV12-mediated malignant transformation of mouse immortalized astrocytes and suppresses GBM tumor growth in vitro and in vivo [56,57]. Furthermore, METTL3 silencing in GSCs enhances their sensitivity to γ-irradiation and reduces DNA repair [56].…”

“…In addition to directly affecting the pluripotent genes, an m 6 A-specific methylated RNA immunoprecipitation with NGS (MeRIP-Seq or m 6 A-Seq) analysis also shows that m 6 A modification peaks seem to be enriched at transcripts responsible for metabolic pathways in GSCs, compared with neural progenitor cells [57]. METTL3 also causes alterations such as decrease of adenosine-to-inosine (A-to-I) and cytidine to uridine (C-to-U) RNA editing events in GSCs by downregulating adenosine deaminase RNA specific 1 (ADAR1) and apolipoprotein B mRNA editing enzyme catalytic subunit 3A (APOBEC3A), which are RNA editing enzymes [45].…”

“…Furthermore, gene ontology analysis reveals that the direct targets of METTL3 seems to be enriched in some major oncogenic pathways including NOTCH signaling, vascular endothelial growth factor (VEGF) signaling, angiogenesis, glycolysis and Hedgehog signaling pathways, whereas the indirect targets are enriched in the RAS, mitogen-activated protein kinase (MAPK), G protein-coupled receptors (GPCRs), cadherin signaling pathways and cell cycle [45]. Additionally, in GSCs, METTL3-dependent m 6 A modification also affects the levels of serineand arginine-rich splicing factors (SRSFs) through upregulating BCL-X (Bcl-2-like protein 1, BCL2L1) or nuclear receptor corepressor 2 (NCOR2) isoforms, which are important to prevent YTHDC1-dependent nonsense-mediated mRNA decay (NMD) [57]. lncRNAs with METTL3-dependent m 6 A marks is also highly expressed in GSCs, compared with that of protein-coding genes [45].…”