Glioblastoma represents the most aggressive type of human brain cancer with dismal prognosis. Recent studies have revealed extensive intratumoral heterogeneity in transcript expression and increased plasticity of human glioma stem cells, which are thought to contribute to treatment failure and tumor recurrence. The role of m6A RNA and ALKBH5 in the regulation of self-renewal and maintenance of tumorigenicity of glioma stem cells has been recently demonstrated. Limited knowledge exists with regards to the role of m6A RNA methylation in the regulation of translation efficiency in human cancer cells. Moreover, the role of m6A RNA methylation machinery that includes m6A ?writers? (methyltransferases e.g. Mettl14, Mettl3) and ?erasers? (demethylases e.g. ALKBH5, FTO), in the regulation of translation during the transition of human glioma stem cells to differentiated glioma cells has not been studied. We hypothesize that alterations at the level of m6A RNA methylation influences the rate of translation of certain transcripts during the transition of human glioma stem cells to differentiated cells. We propose that loss of m6A RNA methylation increases the rate of translation of transcripts with sequence motifs of their m6A peak regions that are complementary to the seed sequences of specific miRNAs. These miRNAs interact and regulate the m6A RNA machinery in human glioma cells.
The specific aims of our proposal will provide a comprehensive and subtype specific insight into the role of m6A RNA in the regulation of translation during the transition between hGSCs and differentiated GCs: -SA1: Perform RNA sequencing, MeRIP sequencing, Ribo-sequencing and computational analysis of 10 additional human glioma stem cells and differentiated glioma cells -SA2: Determine the functional significance of the miRNA binding within the m6A RNA methylation peaks and how the miRNAs regulate RNA methylation machinery. Our proposal constitutes a functional exploration of the role of m6A RNA methylation in the regulation of translation during the transition of hGSCs to differentiated cells. In addition, it introduces the role of specific miRNAs as regulators of hGSC translation through modulation of the RNA methylation machinery. Since the reversible transition of hGSCs to differentiated cells constitutes a critical denominator of tumor recurrence and aggression, identification of molecular regulators of this process could provide novel targets for therapeutic interventions.
Our proposal is the first attempt to study the role of m6A RNA methylation in regulating translation efficiency during the differentiation of human glioma stem cells (hGSCs). Moreover, we propose that miRNAs mediate target-specific loss of m6A RNA and concomitant increase in translation during hGSC differentiation by modulating the RNA methylation machinery.