Epitranscriptomic Regulation of HIV-1 Gene Expression by m ⁵C and the Novel m ⁵C Reader MBD2

Abstract

How the covalent modification of individual mRNA nucleotides, termed epitranscriptomic modification, alters mRNA function remains unclear. One road block has been the difficulty of quantifying epitranscriptomic modifications. Using purified HIV-1 genomic RNA, we show that this RNA bears more epitranscriptomic modifications than cellular mRNAs, with 2’O-methyl and 5-methylcytosine (m⁵C) modifications being particularly high. The primary writer for m⁵C on HIV-1 RNAs was identified as NSUN2 and inactivation of NSUN2 inhibits HIV-1 gene expression without affecting HIV-1 RNA levels. We identify MBD2 as an m5C reader and demonstrate that MBD2 not only has a higher affinity for m5C modified RNAs but also that MBD2 binding sites on the HIV-1 genome coincide with m⁵C addition sites. Loss of MBD2 function phenocopies loss of NSUN2 in that both reduce HIV-1 gene expression and perturb alternative splicing of HIV-1 RNAs. These data identify m⁵C as a post-transcriptional regulator of both mRNA splicing and function.