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

Several prior reports have demonstrated that the epitranscriptomic addition of m6A to viral transcripts promotes the replication and pathogenicity of a wide range of viruses yet the underlying mechanism(s) causing this positive effect has remained unclear. It is known that m6A function is largely mediated by cellular m6A binding proteins or readers, however, how these m6A reader proteins contribute to the regulation of HIV-1 gene expression has remained controversial. Here, we confirm that m6A indeed enhances HIV-1 gene expression. We demonstrate that this effect is collectively mediated by the cytoplasmic reader YTHDF2, which increases HIV-1 transcript stability, and the nuclear m6A reader YTHDC1, which binds HIV-1 RNA at 7 distinct m6A methylated sites, regulating viral transcript alternative splicing. Splicing Assays run with 4 primer sets (1.8 KB, 4 KB, Random Reverse, Common K-mer) on 293T cell knockdowns of YTHDC1 using a control (siC) or YTHDC1 siRNA (siD); or overexpressed YTHDC1 (plusD). Experiments were done in triplicate