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

Area of Science:

• Molecular Biology
• Virology
• Epigenetics

Background:

• Human papillomavirus type 16 (HPV16) is a major cause of cancer.
• RNA methylation, specifically N-methyladenosine (m6A), is a critical regulator of gene expression.
• The role of m6A in HPV16 gene expression, particularly in cancer, remains incompletely understood.

Purpose of the Study:

• To investigate the role of m6A modification and associated proteins in regulating HPV16 mRNA splicing and production.
• To determine how m6A 'writers,' 'erasers,' and 'readers' affect HPV16 gene expression in tonsillar cancer cells.

Main Methods:

• Overexpression and knockdown of m6A-modifying enzymes (METTL3, ALKBH5) and an m6A-binding protein (YTHDC1) in cells containing HPV16.
• Analysis of HPV16 mRNA splicing patterns, including intron retention and exon skipping.
• Detection of m6A modification on HPV16 mRNAs in tonsillar cancer cells.
• RNA immunoprecipitation to confirm binding of YTHDC1 to HPV16 mRNAs.

Main Results:

• Overexpression of ALKBH5 (m6A-demethylase) promoted intron retention in HPV16 E6 mRNAs and altered L1 mRNA splicing.
• Knockdown of ALKBH5 reversed these splicing effects.
• Overexpression of METTL3 (m6A-methylase) induced intron-containing HPV16 E1 mRNAs and affected L1 splicing oppositely to ALKBH5.
• Overexpression of YTHDC1 (m6A-reader) enhanced E6 intron retention and E6 mRNA production, with YTHDC1 binding to HPV16 mRNAs.
• HPV16 mRNAs were found to be m6A-methylated in tonsillar cancer cells.

Conclusions:

• HPV16 mRNAs are subject to m6A methylation in HPV16-infected tonsillar cancer cells.
• m6A 'writers' (METTL3), 'erasers' (ALKBH5), and 'readers' (YTHDC1) significantly influence HPV16 mRNA splicing.
• m6A modification plays a crucial role in the HPV16 gene expression program within cancer cells.