m6A is the most widespread mRNA modification and is primarily implicated in controlling mRNA stability. Fundamental questions pertaining to m6A are the extent to which it is dynamically modulated within cells and across stimuli, and the forces underlying such modulation. Prior work has focused on investigating active mechanisms governing m6A levels, such as recruitment of m6A writers or erasers leading to either ‘global’ or ‘site-specific’ modulation. Here, we propose that changes in m6A levels across subcellular compartments and biological trajectories may result from passive changes in gene-level mRNA metabolism. To predict the intricate interdependencies between m6A levels, mRNA localization, and mRNA decay, we establish a differential model ‘m6ADyn’ encompassing mRNA transcription, methylation, export, and m6A-dependent and independent degradation. We validate the predictions of m6ADyn in the context of intracellular m6A dynamics, where m6ADyn predicts associations between relative mRNA localization and m6A levels, which we experimentally confirm. We further explore m6ADyn predictions pertaining to changes in m6A levels upon controlled perturbations of mRNA metabolism, which we also experimentally confirm. Finally, we demonstrate the relevance of m6ADyn in the context of cellular heat stress response, where genes subjected to altered mRNA product and export also display predictable changes in m6A levels, consistent with m6ADyn predictions. Our findings establish a framework for dissecting m6A dynamics and suggest the role of passive dynamics in shaping m6A levels in mammalian systems.
Overall design: These sereies comprises different experimental series with varying treatments. There is: WT and Mettl3 KO cytoplasmic and nucelar fraction followed by RNA-seq. Cytoplasmic and nucelar fractionation experiment of NIH3T3 cells followed by m6A-seq2. Cytoplasmic and nucelar fraction of pMEFs cells followed by m6A-seq2. CPT treatement followed by m6A-seq2 in MCF7 cells. Actinomycin D timecourse followed by m6A-seq2 in A9 cells. YTHDF1-3 triple knock-down experiment via siRNA followed by m6A-seq2 in NIH3T3 cells. YTHDF1-3 triple knock-down experiment via siRNA followed cytoplasmic and nucelar fraction followed by RNA-seq in NIH3T3 cells. Heat stress timecourse followed by m6A-seq2 in WT primary MEFs. Heat stress timecourse followed by m6A-seq2 in HSF1 KO primary MEFs. Cytoplasmic and nucelar fractionation experiment followed by RNA-seq after heat stress in WT primary MEFs. Cytoplasmic and nucelar fractionation experiment followed by RNA-seq after heat stress in HSF1 KO primary MEFs.
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