Comprehensive Profiling of <i>N</i> ⁶ ‐methyladnosine (m ⁶ A) Readouts Reveals Novel m ⁶ A Readers That Regulate Human Embryonic Stem Cell Differentiation

N⁶-methyladenosine (m⁶A) modification constitutes a crucial layer of post-transcriptional regulations, but the landscape of its downstream readout effects remains less comprehensively understood. Therefore, we systematically assess the readout 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, ribosome sequencing, and ultra-high-depth transcriptome sequencing, respectively. Our analysis, coupled with the integration of public and newly profiled m⁶A methylome data, reveals high cell type specificity in m⁶A readouts where m⁶A level alone is insufficient to predict m⁶A readouts. Nonetheless, machine learning models focusing on RNA-binding protein (RBP) binding context can effectively predict the readouts and prioritize four novel m⁶A-associated proteins (FUBP3, FXR2, L1TD1, and DDX6). Their m⁶A-binding ability is validated by m⁶A RNA pull-down, transcriptome-wide binding site mapping, and electrophoretic mobility shift assay, while FUBP3 and L1TD1 are further suggested as m⁶A readers regulating mRNA stability based on half-life profiling of knockout cells. Finally, FUBP3, FXR2, and L1TD1 are demonstrated to regulate hESC differentiation without affecting self-renewal. 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.