The m⁶A landscape of polyadenylated nuclear (PAN) RNA and its related methylome in the context of KSHV replication

Abstract

Polyadenylated nuclear (PAN) RNA is a non-coding transcript involved in Kaposis sarcoma-associated herpesvirus (KSHV) lytic reactivation and regulation of cellular and viral gene expression. We have shown that PAN RNA has a dynamic secondary structure and protein binding profiles that can be influenced by the epitranscriptomic modifications. N6-methyladenosine (m6A) is an abundant signature found in viral and virus-encoded RNAs. Here, we combined an antibody-independent next generation mapping with direct RNA sequencing to elucidate the m6A landscape of PAN RNA during the KSHV latent and lytic stages of infection. Using a newly developed method, termed Selenium-modified deoxythymidine triphosphate reverse transcription and Ligation Assisted PCR analysis of m6A (SLAP), we gained insight into the fraction of modification at identified sites. Using comprehensive proteomic approaches, we identified writers, erasers, and readers that regulate the m6A status of PAN. We verified the temporal and spatial subcellular availability of the methylome components for PAN modification by performing confocal microscopy analysis. Additionally, the RNA biochemical probing outlined structural alterations invoked by m6A in the context of full-length PAN RNA. This work represents the first comprehensive overview of the dynamic interplay between the cellular epitranscriptomic machinery and a specific viral RNA.

The m6A reader YTHDC2 is essential for escape from KSHV SOX-induced RNA decay

“…The m 6 A-eCLIP confirmed previous results seen where upon reactivation there is an overall decrease in methylation on host transcripts and a massive increase in methylation of viral transcripts [35,36,42]. Furthermore, we observed a 5' UTR hypomethylation and a concomitant 3' UTR hypermethylation following KSHV reactivation from latency.…”

“…Recent transcriptome-wide m 6 A mapping of multiple viruses [35][36][37][38][39][40] have brought to the forefront research concerning a complex interplay between the m 6 A pathways and viral replication success. Previous research has shown that KSHV can hijack this system to deposit m 6 A onto its own transcripts, including KSHV ORF50 (RTA), the master latent-to-lytic switch protein and on the multifunction long-noncoding RNA (lncRNA) PAN [36,[41][42][43]. While there is strong evidence that the m 6 A landscape is reshaped during KSHV infection, and that this shifts ultimately promotes the progression of KSHV infection, it remains unclear whether this m 6 A repurposing also affects the fate of host transcripts.…”

Highlights from the ‘Noncoding RNA world: From Mechanism to Therapy’ conference, July 21-23, 2021

“…A large number of discoveries on ncRNAs have been recently demonstrated in various fields such as oncology, cardiology, virology, and others (1)(2)(3)(4)(5)(6). ncRNAs are divided into different classes based on their size, architecture and mechanisms of action.…”