Abstract
Despite recent advances in N6-methyladenosine (m6A) biology, the regulation of crucial RNA processing steps by the RNA methyltransferase-like 3 (METTL3) in glioma stem-like cells (GSCs) remains obscure. An integrated analysis of m6A-RIP (RNA immunoprecipitation) and total RNA-Seq of METTL3-silenced GSCs identified that m6A modification in GSCs is principally carried out by METTL3. The m6A-modified transcripts showed higher abundance compared to non-modified transcripts. Further, we showed that the METTL3 is essential for the expression of GSC-specific actively transcribed genes. Silencing METTL3 resulted in the elevation of several aberrant alternative splicing events. We also found that putative m6A reader proteins play a key role in the RNA stabilization function of METTL3. METTL3 altered A-to-I and C-to-U RNA editing events by differentially regulating RNA editing enzymes ADAR and APOBEC3A. Similar to protein-coding genes, lincRNAs (long intergenic non-coding RNAs) with m6A marks showed METTL3-dependent high expression. m6A modification of 3′UTRs appeared to result in a conformation-dependent hindrance to miRNA binding to their targets. The integrated analysis of the m6A regulome in METTL3-silenced GSCs showed global disruption in tumorigenic pathways that are indispensable for GSC maintenance and glioma progression. We conclude that METTL3 plays a vital role in many steps of RNA processing and orchestrates successful execution of oncogenic pathways in GSCs.
Highlights
Glioma stem-like cells (GSCs) represent a primitive form in the hierarchy of tumor cells, which can initiate and populate the tumor
To understand the role of Methyltransferase-like 3 (METTL3)-mediated m6 A modification in glioma stem-like cells (GSCs), we employed an integrated approach of whole-transcriptome and m6 A RNA-immunoprecipitation-coupled sequencing (m6 A-RIP Seq) of RNA isolated from MGG8 GSCs transduced with a non-targeting shRNA or
We found that the majority of m6A peaks seen in shNT-MGG8 GSCs were METTL3-dependent as they either showed a complete loss(peaks n=3128; genes n=1680) or reduced intensity in METTL3-depleted GSCs, when we analyzed the protein-coding genes (n = 9830) among the indirect targets of METTL3, we found that a larger subset (n = 6128; 62.34%) was downregulated compared to the number of upregulated genes (n = 3702; 37.7%) in the METTL3-silenced condition
Summary
Glioma stem-like cells (GSCs) represent a primitive form in the hierarchy of tumor cells, which can initiate and populate the tumor. GSCs are highly tumorigenic and contribute to the poor therapy response of glioblastoma (GBM) patients [1]. Dissecting the cellular mechanisms that sustain GSC growth is essential for identifying therapeutic targets. A GSC is a flexible cellular system that undergoes differentiation (differentiated glioma cell, DGC) in the presence of serum and can revert to stem-like cells through reprogramming/dedifferentiation [2]. GSCs have the potential to undergo inter-conversion of cell state with extra-cellular cues and stress conditions, such as drug treatment [3].
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