Our research focuses on the regulation of gene expression, in particular the mechanisms controlling cellular and viral mRNA expression. Analyses of retroviral regulatory systems, pioneered by research on HIV-1, have shed light into important aspects of nuclear mRNA export and provided critical insights into mechanisms governing cellular mRNA and protein transport. The dissection of the mechanisms of posttranscriptional control and nucleocytoplasmic trafficking of macromolecules is relevant to understanding the processes involved in cellular gene expression as well as virus expression. Understanding the basic mechanisms of mRNA expression led to the development of RNA/codon optimization as a key strategy to improve gene expression, which also led to the development of efficient expression vectors for both HIV/SIV antigens and cytokines. Retroviral model systems, and in particular the regulation of HIV-1, have led to major discoveries in the field of mRNA metabolism. HIV-1 Rev was the first identified viral mRNA export factor, and its discovery was instrumental in the discovery of molecular mechanisms mediating posttranscriptional control of gene expression as well as our development of methods to increase the expression of viral proteins, i.e. development of RNA optimization (also referred to as codon optimization), which is presently a key technology for many gene therapy applications, including HIV vaccines. The study of Rev also prompted us to derive the concept that all retroviruses and retroelements use posttranscriptional control mechanisms essential for their replication. These controls require a combination of viral RNA elements and cellular/viral factors able to efficiently link the viral mRNA to the nuclear export pathways and to promote translation. The study of retrovirus and retroelement export could provide important tools to understand essential and complex steps in cellular gene expression. In fact, this strategy resulted in our past identification and characterization of NXF1, which is the key nuclear receptor for the export of cellular mRNAs. We identified the mRNA export requirement of the simian type D retroviral transcript, which is mediated by the cis-acting RNA export element (CTE) and its binding partner, the cellular protein NXF1. CTE is a highly conserved RNA element located next to the 3LTR in SRV-1, SRV-2, MPMV/SRV-3, and the recently discovered SRV-4 as well as in some murine LTR-retroelements. CTE is essential for the expression and mobility of these retroviruses. We discovered that cellular NXF1 protein acts as the key nuclear receptor for cellular mRNAs and that this function is conserved in metazoa and is essential. We also identified that the expression and mobility of a different subclass of murine LTR-retroelements depends on the presence of a distinct cis-acting RNA transport element, RTE, which acts like CTE but does not share its sequence or structural features. Thus, despite a complex evolutionary history, retroelements and retroviruses share a dependency on posttranscriptional regulation, but the detailed molecular mechanisms are distinct. We identified the mechanism that promotes the export of the RTE RNA export and reported that the RNA binding motif 15 (RBM15) protein acts as the cellular factor that binds and exports RTE-containing mRNAs via the NXF1 export pathway. RBM15, a novel mRNA export factor, belongs to the SPEN family of proteins and is conserved among metazoa. We recently reported that another SPEN protein, OTT3, acts as an RNA export co-factor like RBM15. Biochemical and subcellular localization studies showed that OTT3 and RBM15 also interact with each other in vivo, further supporting a shared function. Genetic knock-down of RBM15 in mouse is embryonic lethal, indicating that OTT3 cannot compensate for the RBM15 loss, which supports the notion that these proteins, in addition to sharing similar activities, likely have distinct biological roles. Why have vertebrates evolved to maintain variants of these otherwise similar factors? Since RBM15 and OTT3 are factors of the NXF1 pathway, which controls the export of general mRNA, we speculated that they may be part of a developmental or/and tissue-specific switch that controls mRNA export rates or/and specificity. NXF1 is a key nuclear receptor for cellular and retroviral transcripts. How does NXF1 guide the transcripts through the nuclear pore complex (NPC)? The DEAD-box ATPase DBP5, located at the NPC, is thought to mediate the directional passage of mRNA through the NPC via an unknown mechanism, since it does not bind to NXF1. We recently reported that RBM15 provides the missing link, since it binds to both DBP5 and NXF1 and thus, it acts as molecular link to the NXF1 export pathway. Together, comparative studies of different viral models provide unique tools to address the complex network of molecular steps controlling viral and cellular mRNA expression and this has led to major discoveries on the factors regulating cellular nuclear export mechanisms. We also studied regulation of expression of some cytokine genes, which are highly regulated at several posttranscriptional as well as posttranslational steps. The use of cytokine DNAs (IL-12 and IL-15) as molecular vaccine adjuvants was found to improve the quantity and alter the quality of the immune responses. To optimally use these cytokines, we are studying their regulation and have found that IL-15/IL-15Ra as well as the IL-12 cytokine family use similar posttranscriptional and posttranslational regulation strategies. As a result, the formation and secretion of the subunits and heterodimers are highly regulated steps. Using this knowledge, we have generated optimized expression vectors, which allow their efficient use in animal models. We previously reported that a combination of posttranscriptional and posttranslational modification led to the efficient expression of several hundred-fold higher levels of bioactive IL-15. We have studied the function of long signal peptide (LSP) and short signal peptide (SSP) variants of IL-15, produced by alternatively spliced transcripts. We recently reported that similar to LSP IL-15, SSP IL-15 is stabilized and secreted efficiently upon coexpression of IL-15Ralpha. Co-expression of both LSP IL-15 and SSP IL-15 in the presence of IL-15Ralpha results in lower levels of bioactive IL-15, indicating that LSP and SSP IL-15 compete for the binding to IL-15Ralpha when expressed in the same cell. Since the SSP IL-15 interaction to IL-15Ralpha leads to a less stable complex, SSP IL-15 functions as competitive inhibitor of LSP IL-15. The data suggest that the use of alternative splicing is an additional level of control on IL-15 activity. Expression of both the SSP and LSP forms of IL-15 appears to be conserved in many mammals, suggesting that SSP may be important for expressing a form of IL-15 with a lower magnitude and duration of biological effects. Like IL-15, the expression of the family of the IL-12 cytokines is subject to complex posttranscriptional and posttranslational regulation. Using the expression of individual subunits alone or in combinations, we found that one of these subunits was relatively unstable and limiting;expression of the other subunit in the same cell increased the stability and secretion of the unstable partner and the levels of the secreted heterodimer. This allowed us to generate optimized vectors to produce such cytokines for in vivo application. In a cancer model, we reported that injection of optimized IL-27 DNA, together with IL-2, successfully abolished neuroblastoma metastasis. Together, these optimized cytokine DNAs provide us with important molecular tools to be tested as molecular adjuvants in vaccine and in cancer immunotherapy, with promising future translational applications.
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