Epitranscriptomic addition of m⁶A regulates HIV-1 RNA stability and alternative splicing

Previous work has demonstrated that the epitranscriptomic addition of m⁶A to viral transcripts can promote the replication and pathogenicity of a wide range of DNA and RNA viruses, including HIV-1, yet the underlying mechanisms responsible for this effect have remained unclear. It is known that m⁶A function is largely mediated by cellular m⁶A binding proteins or readers, yet how these regulate viral gene expression in general, and HIV-1 gene expression in particular, has been controversial. Here, we confirm that m⁶A addition indeed regulates HIV-1 RNA expression and demonstrate that this effect is largely mediated by the nuclear m⁶A reader YTHDC1 and the cytoplasmic m⁶A reader YTHDF2. Both YTHDC1 and YTHDF2 bind to multiple distinct and overlapping sites on the HIV-1 RNA genome, with YTHDC1 recruitment serving to regulate the alternative splicing of HIV-1 RNAs. Unexpectedly, while YTHDF2 binding to m⁶A residues present on cellular mRNAs resulted in their destabilization as previously reported, YTHDF2 binding to m⁶A sites on HIV-1 transcripts resulted in a marked increase in the stability of these viral RNAs. Thus, YTHDF2 binding can exert diametrically opposite effects on RNA stability, depending on RNA sequence context.