Comprehensive profiling of N6-methyladnosine (m6A) readouts reveals novel m6A readers that regulate human embryonic stem cell differentiation

N6-methyladenosine (m6A) methylation has emerged as a prevalent RNA modification that extensively impacts various physiological and pathological processes via various post-transcriptional readout effects in mammals. High-throughput methylome profiling has outlined the landscape of m6A modification sites, but their downstream readouts require comprehensive investigation. To this end, we systematically assessed the effects of m6A on mRNA half-life, translation efficiency, and alternative splicing across five cell lines (A549, HEK293T, HUVEC, JURKAT, and human embryonic stem cells (hESCs)) using actinomycin D-disrupted temporal transcriptome sequencing, ribosome sequencing, and ultra-high-depth transcriptome sequencing, respectively. Our analysis, coupled with the integration of public and re-profiled m6A methylome data, revealed high cell type specificity in m6A readouts where m6A level alone is insufficient to predict m6A readouts. Machine learning models focused on the RNA binding protein (RBP) binding context of m6A sites demonstrated substantial predictive ability of m6A readouts while prioritizing putative m6A readers from their informative RBP features. Four novel m6A readers (DDX6, FUBP3, FXR2, and L1TD1) were identified and validated through m6A RNA pull-down assays and transcriptome-wide RBP binding site mapping. Notably, FUBP3, FXR2 and L1TD1 were found to regulate hESC differentiation without impairing self-renewal, underscoring their critical roles in stem cell biology. Together, this study bridges the gap in understanding m6A functional readouts and lays the groundwork for future research on m6A-mediated stem cell fate decisions.