RNA-seq and Ribo-seq analysis of the effects of METTL3/14 depletion on gene expression during the interferon response in Huh7 cells

The RNA modification N6-methyladenosine (m6A) regulates gene expression through various transcript-specific effects. The overall goal of these experiments was to determine the effects of the m6A methyltransferase complex proteins METTL3 and METTL14 on the expression of interferon stimulated genes. First, we depleted METTL3 and METTL14 in Huh7 cells using siRNAs and treated with Mock or IFN-β to analyze the regulatory effects of METTL3/14 on interferon-stimulated genes' transcript abundance. We then used Ribo-seq following METTL3/14 siRNA depletion and IFN treatment to quantify its effect on the translation of ISGs and other genes. Overall, we found that METTL3/14 exerts transcript-specific regulatory effects on a subset of interferon-stimulated genes. Overall, METTL3/14 appeared to enhance the translation of a subset of interferon-stimulated genes. We characterized the molecular effects of METTL3/14 and m6A on certain ISGs and determined that m6A augments the antiviral effects of the interferon response. siRNA transfections were performed for 36 hours in Huh7 cells prior to IFN-β treatment (50 units/mL) for 8 hours, after which cells were harvested and RNA-seq or Ribo-seq protocols were performed. For RNA-seq, 3 replicates of each condition were performed and for Ribo-seq, 4 replicates for each condition were performed.