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

N-methyladenosine (mA) modifications play crucial roles in RNA metabolism. How mA regulates RNA polymerase II (RNA Pol II) transcription remains unclear. We find that 7SK small nuclear RNA (snRNA), a regulator of RNA Pol II promoter-proximal pausing, is highly mA-modified in non-small cell lung cancer (NSCLC) cells. In A549 cells, we identified eight mA sites on 7SK and discovered methyltransferase-like 3 (METTL3) and alkB homolog 5 (ALKBH5) as the responsible writer and eraser. When the mA-7SK is specifically erased by a dCasRx-ALKBH5 fusion protein, A549 cell growth is attenuated due to reduction of RNA Pol II transcription. Mechanistically, removal of mA 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 phosphorylation (Ser2P) in the RNA Pol II C-terminal domain and accumulation of RNA Pol II in the promoter-proximal region. Taken together, we uncover that mA modifications of a non-coding RNA regulate RNA Pol II transcription and NSCLC tumorigenesis.