A Ribonucleoprotein Complex Protects the Interleukin-6 mRNA from Degradation by Distinct Herpesviral Endonucleases

Abstract

During lytic Kaposi’s sarcoma-associated herpesvirus (KSHV) infection, the viral endonuclease SOX promotes widespread degradation of cytoplasmic messenger RNA (mRNA). However, select mRNAs escape SOX-induced cleavage and remain robustly expressed. Prominent among these is interleukin-6 (IL-6), a growth factor important for survival of KSHV infected B cells. IL-6 escape is notable because it contains a sequence within its 3’ untranslated region (UTR) that can confer protection when transferred to a SOX-targeted mRNA, and thus overrides the endonuclease targeting mechanism. Here, we pursued how this protective RNA element functions to maintain mRNA stability. Using affinity purification and mass spectrometry, we identified a set of proteins that associate specifically with the protective element. Although multiple proteins contributed to the escape mechanism, depletion of nucleolin (NCL) most severely impacted protection. NCL was re-localized out of the nucleolus during lytic KSHV infection, and its presence in the cytoplasm was required for protection. After loading onto the IL-6 3’ UTR, NCL differentially bound to the translation initiation factor eIF4H. Disrupting this interaction, or depleting eIF4H, reinstated SOX targeting of the RNA, suggesting that interactions between proteins bound to distant regions of the mRNA are important for escape. Finally, we found that the IL-6 3’ UTR was also protected against mRNA degradation by the vhs endonuclease encoded by herpes simplex virus, despite the fact that its mechanism of mRNA targeting is distinct from SOX. These findings highlight how a multitude of RNA-protein interactions can impact endonuclease targeting, and identify new features underlying the regulation of the IL-6 mRNA.

Nuclease escape elements protect messenger RNA against cleavage by multiple viral endonucleases

“…Given that both proteins are also bound by the G-SRE, we evaluated whether they were similarly important for G-SRE-mediated escape. [27] (gray nodes) overlaid with the set of proteins that were also recovered by ChIRP-MS for the IL-6 and GADD45B 3' UTRs (purple nodes) (C) 293T were transfected with the GFP-GADD45B 3'UTR reporter, then 24h later they were subjected to ChIRP analysis and protein samples were western blotted. (D) Crosslinked lysates of 293T cells were subjected to RNA immunoprecipitation (RIP) with control IgG, anti-NCL, or anti-HuR antibodies and the level of co-purifying endogenous GADD45B mRNA was quantified by RT-qPCR.…”

“…Studying these 'escapees' in aggregate is complicated, however, by the fact that multiple mechanisms can promote apparent escape. These include lack of a targeting motif, indirect transcriptional effects, and active evasion of cleavage [22,[24][25][26][27][28]. This latter phenotype, termed dominant escape, is particularly notable as it involves a specific RNA element whose presence in the 3' UTR of an mRNA protects against SOX cleavage, regardless of whether the RNA contains a targeting motif.…”

C19ORF66 Broadly Escapes Virus-Induced Endonuclease Cleavage and Restricts Kaposi’s Sarcoma-Associated Herpesvirus

“…SOX is conserved throughout the herpesvirus family, but only gammaherpesviral SOX homologs display RNase activity in cells (8)(9)(10), and studies indicate that SOX activity is important for the in vivo viral life cycle (11,12). Although SOX targets a degenerate RNA motif present on most mRNAs (13)(14)(15), multiple studies have shown that some transcripts robustly escape SOX-induced decay (16)(17)(18)(19)(20)(21). Studying these "escapees" in aggregate is complicated, however, by the fact that multiple mechanisms can promote apparent escape.…”

“…These include the lack of a targeting motif, indirect transcriptional effects, and active evasion of ribonucleolytic cleavage (16,(20)(21)(22)(23)(24). The latter phenotype, termed "dominant escape," is particularly notable, as it involves a specific RNA element whose presence in the 3= untranslated region (UTR) of an mRNA protects against SOX cleavage, regardless of whether the RNA contains a targeting motif (19)(20)(21). This protective RNA element was termed SRE (for SOX resistance element), but we recently showed that the SRE is also effective against a broad range of viral endonucleases.…”

“…Little is currently known about these types of RNA elements, how widespread they may be in the genome, and how they may contribute to the overall viral-host arms race for the control of resources. To date, only two SRE-bearing dominant escapees are known: the host interleukin-6 (IL-6) (18,20,21) and the growth arrest and DNA damage-inducible 45 beta (GADD45B) (19) transcripts. Both the IL-6 and GADD45B SREs were mapped to their 3= UTRs and were shown to protect against an array of viral-but not host-RNases.…”