m6Am sequesters PCF11 to suppress premature termination and drive neuroblastoma differentiation

N⁶,2′-O-dimethyladenosine (m⁶Am) is an abundant mRNA modification that impacts multiple diseases, but its function remains controversial because the m⁶Am reader is unknown. Using quantitative proteomics, we identified transcriptional terminator premature cleavage factor II (PCF11) as a m⁶Am-specific reader in human cells. Direct quantification of mature versus nascent RNAs reveals that m⁶Am does not regulate mRNA stability but promotes nascent transcription. Mechanistically, m⁶Am functions by sequestering PCF11 away from proximal RNA polymerase II (RNA Pol II). This suppresses PCF11 from dissociating RNA Pol II near transcription start sites, thereby promoting full-length transcription of m⁶Am-modified RNAs. m⁶Am’s unique relationship with PCF11 means m⁶Am function is enhanced when PCF11 is reduced, which occurs during all-trans-retinoic-acid (ATRA)-induced neuroblastoma-differentiation therapy. Here, m⁶Am promotes expression of ATF3, which represses neuroblastoma biomarker MYCN. Depleting m⁶Am suppresses MYCN repression in ATRA-treated neuroblastoma and maintains their tumor-stem-like properties. Collectively, we characterize m⁶Am as an anti-terminator RNA modification that suppresses premature termination and modulates neuroblastoma’s therapeutic response.