Many cellular noncoding RNAs contain chemically modified nucleotides that are essential for their function. The Epstein-Barr virus expresses two highly abundant noncoding RNAs called EBV-encoded RNA 1 (EBER1) and EBER2. To examine whether these viral RNAs contain modified nucleotides, we purified native EBERs from EBV-infected cells and performed mass spectrometry analysis. While EBER2 contains no modified nucleotides at stoichiometric amounts, EBER1 was found to carry 5-methylcytosine (m 5 C) modification. Bisulfite sequencing indicated that a single cytosine of EBER1 is methylated in ∼95% of molecules, and the RNA methyltransferase NSUN2 was identified as the EBER1-specific writer. Intriguingly, ablation of NSUN2 and thus loss of m 5 C modification resulted in an increase in EBER1 levels. We further found that EBER1 is a substrate for the RNase Angiogenin and cleavage in vivo is dependent on the presence of m 5 C, providing an explanation as to why loss of m 5 C increases EBER1 levels. Taken together, our observations indicate that m 5 C, a modification previously shown for tRNAs to oppose Angiogenin-mediated degradation, can also adversely affect RNA stability.

Epstein-Barr virus (EBV) expresses two highly-abundant noncoding RNAs called EBV-encoded RNA 1 (EBER1) and EBER2, which are preserved in all clinical isolates of EBV, thus underscoring their essential function in the viral life cycle. Recent epitranscriptomics studies have uncovered a vast array of distinct RNA modifications within cellular as well as viral noncoding RNAs that are instrumental in executing their function. Here we show that EBER2 is marked by pseudouridylation, and by employing HydraPsiSeq the modification site was mapped to a single nucleotide within the 3′ region of EBER2. The writer enzyme was identified to be the snoRNA-dependent pseudouridine synthase Dyskerin, which is the catalytic subunit of H/ACA small nucleolar ribonucleoprotein complexes, and is guided to EBER2 by SNORA22. Similar to other noncoding RNAs for which pseudouridylation has a positive effect on RNA stability, loss of EBER2 pseudouridylation results in a decrease in RNA levels. Furthermore, pseudouridylation of EBER2 is required for the prolific accumulation of progeny viral genomes, suggesting that this single modification in EBER2 is important for efficient viral lytic replication. Taken together, our findings add to the list of RNA modifications that are essential for noncoding RNAs to implement their physiological roles.