N6-methyladenosine of the 7SK small nuclear RNA underlies RNA Polymerase II transcription regulation

N6-methyladenosine (m6A) modifications play crucial roles in RNA metabolism. However, the mechanism by which m6A regulates RNA Polymerase (Pol) II transcription remains unclear. Here, we find that 7SK small nuclear (sn) RNA, a central player in regulating Pol II promoter-proximal pausing, is highly m6A-modified in non-small cell lung cancer (NSCLC) cells. In NSCLC A549 cells, we identified eight m6A sites on 7SK and discovered METTL3 and ALKBH5 as the responsible m6A writer and eraser. When the m6A-7SK is specifically erased by a 7SK-targeting dCasRx-ALKBH5 fusion protein, A549 cell growth is significantly attenuated, due to reduction of Pol II transcription. Mechanistically, removal of m6A on 7SK leads to structural rearrangements that facilitate sequestration of the positive transcription elongation factor (P-TEFb) complex, which results in both the reduction of Serine 2 phosphorylation (Ser2P) in the Pol II C-terminal domain (CTD) and attenuation of Ser2P Pol II in the promoter proximal region. Taken together, we uncover a role of m6A modification on a non-coding RNA in regulating Pol II transcription and NSCLC tumorigenesis.