Comprehensive Profiling of N6-methyladnosine (m6A) Readouts Reveals Novel m6A 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.