N6-methyladenosine in 7SK small nuclear RNA underlies RNA polymerase II transcription regulation

N6-methyladenosine (m6A) modifications play crucial roles in RNA metabolism. How m6A regulates RNA po-lymerase II (RNA Pol II) transcription remains unclear. We find that 7SK small nuclear RNA (snRNA), a regu-lator of RNA Pol II promoter-proximal pausing, is highly m6A-modified in non-small cell lung cancer (NSCLC) cells. In A549 cells, we identified eight m6A sites on 7SK and discovered methyltransferase-like 3 (METTL3) and alkB homolog 5 (ALKBH5) as the responsible writer and eraser. When the m6A-7SK is specifically erased by a dCasRx-ALKBH5 fusion protein, A549 cell growth is attenuated due to reduction of RNA Pol II transcrip-tion. Mechanistically, removal of m6A leads to 7SK structural rearrangements that facilitate sequestration of the positive transcription elongation factor b (P-TEFb) complex, which results in reduction of serine 2 phos-phorylation (Ser2P) in the RNA Pol II C-terminal domain and accumulation of RNA Pol II in the promoter -prox-imal region. Taken together, we uncover that m6A modifications of a non-coding RNA regulate RNA Pol II transcription and NSCLC tumorigenesis.