Fine-tuning m6A and METTL3 levels have profound impact on cellular proliferation and protein synthesis

This study investigates how partial loss of the m6A RNA methyltransferase METTL3 alters the transcriptomic landscape and translational regulation in Triple Negative Breast Cancer cells. m6A is the most abundant internal RNA modification in eukaryotic mRNAs and long non-coding RNAs and plays important roles in RNA metabolism, translation, and gene expression regulation. Dysregulation of the m6A pathway has been implicated in multiple cancers, including triple negative breast cancer (TNBC), where reduced METTL3 expression has been associated with poor prognosis and increased metastatic potential. To investigate how reduced METTL3 dosage affects RNA methylation and downstream cellular processes, we generated CRISPR/Cas9-engineered METTL3 heterozygous knockout (HKO) cell lines in the human TNBC cell line MDA-MB-468. These cells exhibit reduced METTL3 expression and modest global decreases in m6A levels, allowing investigation of the effects of partial m6A depletion, which may better reflect physiologically relevant perturbations observed in cancer. Using Oxford Nanopore Direct RNA Sequencing, we performed transcriptome-wide profiling of m6A modifications in wild-type and METTL3 heterozygous knockout cells. Computational approaches were applied to identify differential m6A sites and estimate modification stoichiometry across transcripts. These data were integrated with functional analyses including proliferation assays, quantitative proteomics, phosphoproteomics, and global protein synthesis measurements to assess the biological consequences of altered m6A levels. The study aims to determine how partial reductions in METTL3 activity reshape the m6A landscape and influence gene expression programs, particularly those related to translational control, growth signalling, and cancer cell proliferation. Our findings demonstrate that modest decreases in global m6A preferentially affect transcripts involved in translational regulation and are associated with increased translational output and proliferative capacity, whereas stronger pharmacological inhibition of METTL3 suppresses translation and cell growth. Overall, this work provides insight into the dose-dependent effects of METTL3 and m6A RNA methylation on gene regulation, highlighting how partial disruption of the m6A machinery can reprogram translational pathways and contribute to tumour progression.