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

Abstract

The role of N6-methyladenosine (m⁶A) modifications has increasingly been associated with a diverse set of roles in modulating viruses and influencing the outcomes of viral infection. Here, we report that the landscape of m⁶A deposition is drastically shifted during Kaposi's sarcoma-associated herpesvirus (KSHV) lytic infection for both viral and host transcripts. In line with previous reports, we also saw an overall decrease in host methylation in favor of viral messenger RNA (mRNA), along with 5' hypomethylation and 3' hypermethylation. During KSHV lytic infection, a major shift in overall mRNA abundance is driven by the viral endoribonuclease SOX, which induces the decay of greater than 70% of transcripts. Here, we reveal that interlukin-6 (IL-6) mRNA, a well-characterized, SOX-resistant transcript, is m⁶A modified during lytic infection. Furthermore, we show that this modification falls within the IL-6 SOX resistance element, an RNA element in the IL-6 3' untranslated region (UTR) that was previously shown to be sufficient for protection from SOX cleavage. We show that the presence of this m⁶A modification is essential to confer SOX resistance to the IL-6 mRNA. We next show that this modification recruits the m⁶A reader YTHDC2 and found that YTHDC2 is necessary for the escape of the IL-6 transcript. These results shed light on how the host cell has evolved to use RNA modifications to circumvent viral manipulation of RNA fate during KSHV infection.

Keywords: IL-6; RNA decay; herpesvirus; m6A; m6A readers.

Fig. 1.

Examining iSLK m⁶A epitrascriptome during KSHV lytic reactivation. (A) Schematic of m⁶A eCLIP set up. iSLK.WT cells were either left latent or lytically reactivated with doxycycline and sodium butyrate for 48 h. Total RNA was collected then incubated with an m⁶A antibody. Samples were ultraviolet cross-linked before being reverse transcribed then attached with 3′ adapters in part of library preparation. Finally, m⁶A-enriched samples were sequenced. (B) Most significant DRACH motifs with m⁶A peaks identified by HOMER in latent and lytic cells. (C) Heat map of a metagene plot depicting the average number of sites mapped to certain genomic regions. The number of sites is calculated for each region of every gene, the lengths of the regions are then normalized, and the average number of sites for a set number of positions along the regions are calculated. (D) Heat map of the most significant m⁶A-enriched functional pathways in latent and lytic cells calculated through an enrichment analysis preformed using the R package clusterProfiler. (E) m⁶A PureCLIP scores of lytically reactivated KSHV genes aligned over an annotated KSHV genome. PureCLIP is the log posterior probability ratio of the m⁶A cross-link sites over the input samples.

Fig. 2.

IL-6 SRE contains an m⁶A site that is necessary for viral endonuclease protection. (A) PureCLIP scores of the 3′ UTR IL-6 gene in latent and lytic (48 hpr) iSLK.WT cells. Schematic to the right illustrates an IL-6 gene (its 5′ UTR, coding region "CDS" and 3′ UTR): the DRACH motif identified through m⁶A-eCLIP is in blue and the methylated adenosine in red. (B) Cells were transfected with WTSRE or mutSRE GFP reporter, and total RNA was harvested 24 h later and subjected to meRIP followed by RT-qPCR using GFP primers. Fold enrichment was determined by calculating the fold change of the IP to control Ct values that were normalized through the input. (C) 293T cells transfected with one of three viral endonucleases, as indicated along with the indicated GFP reporters. RNA was collected and quantified using RT-qPCR. ****P < 0.0001; ns, not significant.

Fig. 3.

YTHDC2 is necessary for IL-6’s evasion of SOX. (A) HEK293T cells were transfected with Flag-tagged YTHDC2 and a GFP-WTSRE reporter as indicated. Cells were cross-linked and IP using Flag-coated beads. RNA fraction was collected and used for RT-qPCR. (B) 293TΔYTHDC2 cells were obtained by stably expressing and single-cell–selecting 293TCas9 cells expressing a YTHDC2-targeting guide RNA. Cells’ clones were tested for knockout efficiency by Western blot using a YTHDC2 antibody and GAPDH as a loading control. YTHDC2 expression in these cells was rescued by transfecting Flag-tagged YTHDC2 on a plasmid. (C) 293TΔYTHDC2, 293TΔYTHDC2+Flag YTHDC2 or WT cells were transfected with SOX (or mock), along with a GFP-WTSRE reporter. RNA was then collected and used for RT-qPCR. *P < 0.05; ns, not significant.