Deciphering the Role of METTL3-Dependent m6A-epitranscriptome in Glioma Stem-like Cells

Abstract

The major roadblocks in treatment for GBM are resistance to therapy and recurrence of GBM cells. Regardless of various treatment strategies, the average survival of GBM patients is poor and incidence of recurrence remains high. The presence of GSCs, a dynamic cellular system within GBM contributes to chemo/radio resistance and recurrence. The plasticity of GSCs is supported by reversible biological processes including DNA methylation, histone modifications and RNA modifications. m6A is a reversible mRNA methylation which regulates various steps of RNA processing. In this thesis, we attempted to elucidate the METTL3- mediated m6A as a molecular mechanism behind the dynamic nature of GSCs. It comprises of two parts: Part 1- The landscape of METTL3-dependent m6A-epitranscriptome in GSCs and the functional orchestration of m6A targets Part 2- Essential role of METTL3 mediated m6A modification in glioma stem-like cells maintenance and radioresistance In first part, we demonstrate levels of m6A modified RNAs and METTL3 are maintained high in GSCs and they are attenuated during serum induced differentiation. Other members involved in m6A modification are either down regulated or unregulated in GSCs which confirms the dependency of GSCs in METTL3 for creating m6A marks. In addition, previous reports provide support that solely METTL3 carry catalytic domain for SAMbinding. Based on these facts, we elucidate the METTL3-dependent global m6A modification in GSCs and the impact of METTL3 targets in coordinating various functions. By comprehensive analysis of m6A-RIP seq and whole transcriptome post METTL3 silencing in GSCs, we identified the direct and indirect targets for METTL3-mediated m6A modification. The genes which preserve stem cell properties and aid in tumorigenesis were predominantly inhibited by METTL3 silencing in GSCs suggesting a universal oncogenic role of METTL3 in GBM. Large subset of genes was down regulated after METTL3 silencing suggesting a global destabilization of transcripts. The enrichment of m6A peaks near stop codon and 3’UTR indicates functional importance of METTL3 in RNA stabilization and translation termination. In addition to protein coding genes, METTL3 fine-tunes the expression of non coding RNAs which includes lncRNAs, anti-sense RNAs etc. Interestingly, we identified m6A peaks which encompass miRNA target sites and we hypothesize that m6A modification may scrutinize the binding affinity of miRNAs. We further determined the inter-play between chromatin remodeling with m6A epitranscriptome. Genes which carry active chromatin marks and transcription factor binding are further stabilized by METLT3- dependent m6A modification. The altered secondary/tertiary structure induced by m6A may act as loading site for various RBPs and achieve various RNA processing functions. Our analysis deduced RBPs HuR and QKI preferentially bind to m6A marked RNAs and helps in enhancing the expression of m6A modified targets. Together, this study provides a panoramic view on global m6A modification mediated by METTL3 in GSCs. In second part, we examined the crucial role of METTL3 in glioma stem cell physiology. Inhibition of METTL3 hinder the neurosphere formation and stem cell properties of GSCs. Anti-METTL3 RIP studies combined with m6A RIP-seq results identified SOX2 as a key m6A mediator of METTL3 and the m6A marks created by METTL3 sustains SOX2 transcript stability. The exogenous over expression of 3’UTR-less SOX2 significantly alleviated the inhibition of neurosphere formation observed in METTL3 silenced GSCs. METTL3 interaction and m6A modification in vivo required intact three METTL3/m6A sites present in the SOX2- 3’UTR. Further, we found that HuR recruitment to m6A modified RNA is essential for SOX2 mRNA stabilization by METTL3 and at global level HuR-RNA interaction prefers the m6A modified transcripts. METTL3 silenced GSCs showed enhanced sensitivity to γ-irradiation due to reduced DNA repair. Exogenous overexpression of 3’UTRless SOX2 in METTL3 silenced GSCs rescues efficiency of DNA repair and specifically homologous recombination repair. It also resulted in the significant rescue of neurosphere formation from METTL3 silencing induced radiosensitivity. GBM tumors have elevated METTL3 transcripts and silencing METTL3 in GSCs inhibited tumor growth and prolonged mice survival. METTL3 transcript levels predicted poor survival in GBMs which are enriched for GSC-specific signature. Thus our study reports the importance of m6A modification in GSCs and uncovers METTL3 as a potential molecular target in GBM therapy