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

Xiaoxu Cui,Kersti Nilsson,Stefan Schwartz,Naoko Kajitani

doi:10.1007/s11262-022-01889-6

Xiaoxu Cui, Kersti Nilsson + Show 2 more

Open Access

https://doi.org/10.1007/s11262-022-01889-6

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Journal: Virus genes	Publication Date: Feb 21, 2022
Citations: 10	License type: open-access

Affiliation: Lund University, Uppsala University

Abstract

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.

Highlights

Human Papillomaviruses (HPVs) are small DNA viruses associated with 99% of all cervical cancers, and a growing number of head and neck cancers [1, 2]
First we analyzed the effect of fat mass and obesity-associated protein (FTO) and alkB homolog 5 RNA demethylase (ALKBH5) on alternatively spliced human papillomavirus type 16 (HPV16) E6/E7 mRNAs (Fig. 1C)
Unlike the general pattern of m6A on cellular mRNAs, which clusters around start codons and in the 3’-UTR [19, 40], m6A on HPV16 mRNAs are predicted to cluster around HPV16 splice sites (Supplementary Fig. S3)

Summary

Introduction

Human Papillomaviruses (HPVs) are small DNA viruses associated with 99% of all cervical cancers, and a growing number of head and neck cancers [1, 2]. HPV16 replicates in the nucleus of keratinocytes and viral gene expression is linked to the differentiation program of the cell [4, 5]. The HPV16 E2 protein E2 regulates HPV gene expression. Cellular mRNAs carry various chemical modifications that contribute to the regulation of RNA processing. M6A is believed to be the most common modification of mRNAs in eukaryotic cells. The m6A modification is reversible and is catalyzed by a methyltransferase complex consisting of methyltransferase-like-3 (METTL3), methyltransferaselike-14 (METTL14), and additional factors such as Wilms tumor 1- associated protein (WTAP), RNA-binding motif protein 15 (RBM15), KIIA1429, and zinc-finger CCCHtype 13 containing (CZ3H13) [14, 15]. METTLL3 is the catalytic subunit, while other factors such as

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