Mapping of pseudouridine residues on cellular and viral transcripts using a novel antibody-based technique

Abstract

Pseudouridine () is the most common noncanonical ribonucleoside present on mammalian noncoding RNAs (ncRNAs), including rRNAs, tRNAs, and snRNAs, where it contributes ∼7% of the total uridine level. However, constitutes only ∼0.1% of the uridines present on mRNAs and its effect on mRNA function remains unclear. residues have been shown to inhibit the detection of exogenous RNA transcripts by host innate immune factors, thus raising the possibility that viruses might have subverted the addition of residues to mRNAs by host pseudouridine synthase (PUS) enzymes as a way to inhibit antiviral responses in infected cells. Here, we describe and validate a novel antibody-based mapping technique called photo-crosslinking-assistedsequencing (PA-seq) and use it to mapresidues on not only multiple cellular RNAs but also on the mRNAs and genomic RNA encoded by HIV-1. We describe 293T-derived cell lines in which human PUS enzymes previously reported to add residues to human mRNAs, specifically PUS1, PUS7, and TRUB1/PUS4, were inactivated by gene editing. Surprisingly, while this allowed us to assign several sites of addition on cellular mRNAs to each of these three PUS enzymes, sites present on HIV-1 transcripts remained unaffected. Moreover, loss of PUS1, PUS7, or TRUB1 function did not significantly reduce the level of residues detected on total human mRNA below the ∼0.1% level seen in wild-type cells, thus implying that the PUS enzyme(s) that adds the bulk of residues to human mRNAs remains to be defined.