Overexpression of m6A-factors METTL3, ALKBH5, and YTHDC1 alters HPV16 mRNA splicing.

We report that overexpression of the m6A-demethylase alkB homolog 5 RNA demethylase (ALKBH5) promoted production of intron retention on the human papillomavirus type 16 (HPV16) E6 mRNAs thereby promoting E6 mRNA production. ALKBH5 also altered alternative splicing of the late L1 mRNA by an exon skipping mechanism. Knock-down of ALKBH5 had the opposite effect on splicing of these HPV16 mRNAs. Overexpression of the m6A-methylase methyltransferase-like protein 3 (METLL3) induced production of intron-containing HPV16 E1 mRNAs over spliced E2 mRNAs and altered HPV16 L1 mRNA splicing in a manner opposite to ALKBH5. Overexpression of the nuclear m6A-"reader" YTH domain-containing protein 1 (YTHDC1), enhanced retention of the E6-encoding intron and promoted E6 mRNA production. We also show that HPV16 mRNAs are bound to YTHDC1 in human cells and that YTHDC1 affected splicing of HPV16 E6/E7 mRNAs produced from the episomal form of the HPV16 genome. Finally, we show that HPV16 mRNAs are m6A-methylated in tonsillar cancer cells. In summary, HPV16 mRNAs are methylated in HPV16-infected tonsillar cancer cells and overexpression of m6A-"writer" METTL3, m6A-"eraser" ALKBH5 and the m6A-"reader" YTHDC1 affected HPV16 mRNA splicing, suggesting that m6A plays an important role in the HPV16 gene expression program, at least in cancer cells.