m6Am sequesters PCF11 to suppress premature termination and drive neuroblastoma differentiation [fPAR-CLIP]

N6,2'-O-dimethyladenosine (m6Am) is an abundant mRNA modification that impacts multiple diseases but its function remains controversial since the m6Am reader is unknown. Using quantitative proteomics, we identified transcriptional terminator PCF11 as a m6Am-specific reader. Direct quantification of mature versus nascent RNAs reveals m6Am does not regulate mRNA stability but promotes nascent transcription. Mechanistically, m6Am functions by sequestering PCF11 away from proximal RNA Pol II. This suppresses PCF11 from dissociating 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, a repressor of the neuroblastoma biomarker MYCN. Depleting m6Am suppresses MYCN-repression in ATRA-treated neuroblastoma and maintains their tumour stem-like properties. Collectively, we characterize m6Am as an anti-terminator RNA modification that suppresses premature termination and modulates neuroblastoma therapeutic response. Overall design: UV crosslinking of s4U labelling of RNA to protein, followed by immunoprecipitation and RNA sequencing