m6A modification regulates cell proliferation via reprogramming the balance between glycolysis and pentose phosphate pathway
N6-methyladenosine (m6A) stands as the predominant modification in eukaryotic mRNA and is involved in various biological functions. Aberrant m6A has been implicated in abnormal cellular phenotypes, including defects in stem cell renewal and tumorigenesis. However, the precise effects of m6A on cell proliferation and the underlining mechanism of metabolic gene regulation remain incompletely understood. Here, we established a cellular environment with low-m6A levels and observed a severe impairment of cell proliferation. Mechanistic studies revealed that the depletion of m6A on TIGAR mRNA led to increased expression, subsequently inhibiting glycolysis while promoting the pentose phosphate pathway (PPP). A genome-wide CRISPR-Cas9 screen identified extensive genes involved in cell proliferation that are affected by m6A modification, with G6PD emerging as a key regulator. Integration of gene expression and survival data from LIHC patients suggested specific subsets with elevated G6PD expression may exhibit enhanced responsiveness to tumor growth inhibition through m6A suppression. Our findings elucidate the critical role of m6A in cellular metabolic regulation, particularly in the metabolic reprogramming of cell proliferation, offering new insight for cancer therapy. Overall design: Comparative gene expression profiling analysis of RNA-seq data for HEK293T cells treated by DMSO and 5uM STM2457 for 72h respectively. To investigate whether m6A inhibitor STM2457 has any preference on specific gene, we utilized MeRIP-seq assay to determine the m6A alteration patterns in HEK293T cells. We devised a comprehensive CRISPR-centric genetic screen to identify genes that govern cell growth suppression in a low m6A environment. Using lentivirus delivery, we introduced a single guide RNA (sgRNA) library targeting 18,360 protein-encoding genes into HEK293T cells constitutively expressing Cas9. Following a two-week treatment period with either DMSO or STM2457, we employed next-generation sequencing (NGS) to uncover the variation in sgRNA distribution between samples. To verify the identified genes from our CRISPR screen, we initiated a validation process for a subset. Dual sgRNAs for each gene were transfected into HEK293T cells, and PCR amplification of editing sites followed by NGS sequencing confirmed the editing efficiency.