Although cellular gene expression is significantly dampened by widespread mRNA degradation during lytic gammaherpesvirus infection, select transcripts escape this fate. We previously showed that there are multiple mechanisms mediating escape of mRNAs from SOX-induced depletion, including absence of a SOX targeting element and transcriptional upregulation (35
). Here, we reveal that the IL-6 mRNA is protected by a unique mechanism: it contains a sequence within its 3′ UTR that inhibits cleavage by SOX. This sequence contains a SOX-resistant element (SRE) that helps confer protection even when fused to a heterologous transcript and thus prevents SOX targeting via a novel, dominant protective mechanism. One component of the IL-6 SRE, termed SRE1, maps to a regulatory region of the transcript that contains several AU-rich elements (AREs) previously shown to recruit host factors primarily involved in mRNA destabilization (24
). Indeed, we confirmed that this element complexes with host proteins, including the ARE binding proteins AUF1 and HuR. However, our data suggest that in SOX-expressing cells, these interactions contribute to protection of the IL-6 mRNA, rather than mediating its degradation (). Although we do not yet know the mechanism(s) by which protection is conferred, it could, for example, be through recruitment of a SOX inhibitor, steric hindrance of SOX binding or cleavage, or shunting of the mRNA to a location or decay pathway inaccessible to SOX. Collectively, our findings demonstrate that RNA-protein interactions can directly influence the susceptibility of cellular mRNAs to cleavage by the gammaherpesviral SOX protein, even when they occur distal to the actual cleavage site.
Model. IL-6 contains an element in its 3′ UTR that protects it from cleavage by the KSHV protein SOX. This element is bound by a complex containing AUF1 and HuR, both of which contribute to the escape of IL-6 from SOX-mediated cleavage.
AREs are among the most common determinants of mRNA stability in mammalian cells and play a prominent role in regulating turnover of the IL-6 message. AREs influence transcript stability through the recruitment of specific ARE binding proteins, often leading to enhanced mRNA deadenylation and decay. These elements are enriched in 3′ UTRs of many unstable mRNAs encoding tightly regulated proteins such as transcription factors, proto-oncogenes, and cytokines. They can be grouped into at least three classes depending on their sequence and rates of decay (36
); IL-6 contains a series of nonclustered class I-like AREs. Presumably, the type and magnitude of the effect exerted by an ARE are determined by the cohort of ARE binding proteins recruited by that element. Several ARE binding proteins mediate mRNA destabilization by recruiting the degradation machinery, although some enhance mRNA stability (26
The IL-6 AREs have been shown to bind AUF1, TTP, and KSRP, proteins that generally promote mRNA degradation (24
), but IL-6 mRNA stability is also been shown to be indirectly positively regulated by HuR (28
). Using a targeted RNA pulldown approach, we found that two of these proteins, AUF1 (also known as hnRNP D) and HuR, associate with the SRE1 core. Although AUF1 is predominantly nuclear, it is a shuttling protein and binds to target mRNAs in the cytoplasm where it enhances their decay (33
). The mechanisms underlying its translocation remain incompletely understood and are complicated by the fact that AUF1 has 4 alternative splicing isoforms that may hetero-oligomerize (38
). HuR is also a shuttling protein that primarily resides in the nucleus, though its cytoplasmic translocation is instead associated with stabilization and translational regulation of its mRNA targets (42
). Interestingly, both AUF1 and HuR have been shown to interface with RNA viruses. AUF1 is relocalized to the cytoplasm upon poliovirus infection, whereupon it is cleaved by the viral 3CD proteinase (43
). HuR is prominently relocalized during Sindbis virus infection and binds and stabilizes the viral mRNAs, and it has also been shown to bind RNAs of other alphaviruses and hepatitis C virus in a manner important for viral replication (44
). In KSHV-infected cells, both proteins retain their prominent nuclear localization but are detectable in the cytoplasm as well. Thus, KSHV does not cause a robust translocation of either protein, although it may result in a modest increase in the cytoplasmic levels of AUF1. These observations argue against cytoplasmic relocalization of these proteins as a primary contributor to the stabilization phenotype during KSHV infection. Nonetheless, the fact that depletion of either protein significantly reduced the protective effect of the IL-6 3′ UTR in SOX-expressing cells argues that their binding to this region of the RNA contributes importantly to the escape mechanism. Presumably, the cytoplasmic concentrations of AUF1 and HuR are sufficient to mediate these effects, although it is also formally possible that they assemble onto the transcript while in the nucleus. We did not observe an additive effect on IL-6-mediated stabilization upon codepletion of AUF1 and HuR (S. Hutin and B. Glaunsinger, unpublished observations), suggesting that both proteins are operating in the same pathway.
Although the IL-6 escape element contains AREs, it cannot be generalized that ARE-bearing mRNAs are protected from SOX-induced degradation. In fact, a previous analysis of RNA features associated with escape from SOX found a negative correlation between the presence of an ARE and protection from SOX-induced decay (35
). Furthermore, ARE-bearing mRNAs such as the granulocyte-macrophage colony-stimulating factor (GM-CSF) transcript are degraded by SOX, arguing against a simple failure of SOX to target unstable transcripts (6
). We instead hypothesize that proteins associated with the IL-6 ARE, including AUF1 and HuR, coordinate with other factors or sequences within the escape element to confer protection from SOX. That said, in the context of lytic KSHV infection, there is an enrichment of ARE-bearing mRNAs in the escapee population (7
). However, in this setting, the viral Kaposin B and vGPCR proteins are expressed (both of which have been shown to activate MK2 signaling), leading to stabilization of ARE mRNAs (16
). It is thus likely that multiple viral factors contribute to the overall manipulation of host gene expression during KSHV infection.
While the majority of protein complex binding to the IL-6 mRNA occurs within SRE1, additional flanking sequences are required to confer maximal protection from SOX. This is highlighted by the observation that fusion of SRE1 alone to the GFP reporter increases its abundance in SOX-expressing cells by approximately 2-fold, whereas inclusion of an additional 98 nt bordering the 5′ end of the SRE1 core leads to an ~4-fold increase. It is possible that neighboring sequences assist with RNP architecture or assembly in vivo
or that they contribute to the protection via an independent mechanism. For example, the IL-6 mRNA is regulated by a series of miRNA binding sites, including miR-26 and let-7 (49
). Most of the predicted miRNA target sides are surrounding SRE1, while miR-26 partially overlaps at the 3′ end. It has also been shown that the KSHV ORF57 protein contributes to IL-6 mRNA stability by interacting with a binding site for miR-608 (53
), although this site is within the coding region of IL-6 and is thus distinct from the mechanism of protection against SOX. Given the partial overlap between miR-26 or potentially other miRNAs and the SRE1 core, an important future direction will be to determine whether they similarly influence targeting by SOX. In addition, we are currently working toward identifying additional proteins associated with the SRE1.
Collectively, our findings demonstrate that specific mRNA regulatory elements, together with their associated ribonucleoproteins, can render transcripts inaccessible to SOX. Presumably, such additional elements exist, and their discovery and characterization are anticipated to reveal new insight into the mechanisms by which cis-acting RNA elements function to control transcript stability.