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Multiple classes of pharmaceuticals, including acetaminophen, aspirin, and other nonsteroidal anti-inflammatory drugs (NSAIDs), are used to relieve mild to moderate pain; however, one of the oldest classes of pharmaceuticals, opioids, remains the primary class of drugs used in the management of severe pain. For decades, the unique pharmacological profiles of opioid compounds have suggested the existence of multiple opioid receptor subtypes and, accordingly, four opioid receptors have been cloned to date; the mu (μ)-opioid receptor, the kappa (κ)-opioid receptor, the delta (δ)-opioid receptor, and the nociceptin/orphanin FQ receptor. Additionally, each receptor is encoded by its own distinct gene; the OPRM1, OPRK1, OPRD1, and OPRL1, respectively. Despite the identification and characterization of these four opioid receptor subtypes, pharmacological data, particularly from opioid receptor knockout mice, does not conform to the predications of a four opioid receptor model and instead suggests the existence of additional receptor subtypes. Additional opioid receptors have since been proposed but corresponding genes have either been unidentified or found to be genetically unrelated. Interestingly, this problem is not unique to opioid receptors, as there is a large discrepancy between the number of protein encoding genes and the repertoire of mRNA transcripts and encoded proteins they produce, with gene products far more numerous than estimates would predict. It is now understood that this discrepancy is due to the generation of multiple RNA transcripts from a single gene. Several mechanisms are utilized in order to generate mRNA transcript variants, or isoforms, from a single gene; however, the primary mechanism, known as alternative splicing, involves a complex macromolecular machine, referred to as the spliceosome, through which specific portions of the precursor mRNA (pre-mRNA) sequence are selectively removed and the remaining nucleotide sequences are ligated to form a unique mRNA transcript. Recently, multiple opioid receptor isoforms, particularly for the μ-opioid receptor, have been identified; however, both their regulation and their functional significance are poorly characterized. As such, multiple studies are needed to more precisely describe alternatively spliced μ-opioid receptor isoforms, particularly the regulation of spliceosome components that determine the splicing specificity of particular isoforms as well as the distinct signaling pathways utilized by particular isoforms both constitutively and following agonist binding. Using a model of dopaminergic neurons, this study sought to examine these questions and found that expression of a particular splice variant, MOR-1X, was up-regulated by morphine through a mechanism involving the essential splicing factor ASF/SF2. Structural comparison of this isoform to the prototypical variant MOR-1 found that the unique distal portion of C-terminal domain contains two additional PKA phosphorylation sites as well as a second agonist-induced phosphorylation motif highly conserved among opioid receptors. Functional comparison of MOR-1 and MOR-1X found distinct signaling differences, both constitutively and following morphine treatment, in MAPK signaling cascades, particularly ERK1/2. While the pharmacological significance of MOR-1X expression and signaling remains unclear, the clinical importance of this finding extends beyond a mechanism of opioid analgesic variability, as the physiological roles of opioids also include immunomodulation and have been implicated specifically in the exacerbation of HIV viral replication and pathology, particularly neurocognitive dysfunction. Accordingly, the HIV viral protein Tat was found to block morphine-mediated increases in MOR-1X expression by similarly blocking morphine-mediated increases in ASF/SF2 expression. Consequently, MOR-1X and HIV viral proteins were found to have a unique and synergistic role in the regulation of intrinsic apoptotic signaling cascades, specifically Bax expression, and in cell proliferation. Therefore, the regulation of alternative splicing events by both opioids and HIV viral proteins involves, in part, the inverse regulation of ASF/SF2 protein expression, through which the expression of the MOR-1X isoform is subsequently and significantly altered. This, in turn, may lead to functional consequences in opioid pharmacokinetics as well as in opioid-related pathology, such as the exacerbation of HIV associated neurocognitive dysfunction, as MOR-1X contains unique functional regions which may be responsible for the observed differences in MAPK and intrinsic apoptotic signaling and cellular proliferation. Collectively, these findings support previous studies that suggest alternative splicing of the MOR is altered by exogenous factors, such as morphine and HIV, identify unique signaling pathways for various opioid receptor isoforms, and are the first to suggest a potential mechanism through which pharmacological interventions could be utilized to alter opioid receptor isoform expression, thereby altering the pharmacological and physiological effects of opioids. / Biomedical Neuroscience

Retinoic acid receptor (RAR), a member of the steroid/thyroid hormone nuclear receptor superfamily, functions as a RA-dependent transcription activator bound to the RA response element (RARE) within the promoter or enhancer region of target genes. The transcriptional activity of RAR is modulated by a large number of coregulators including coactivators and corepressors. Acinus is a nuclear protein with three isoforms (Acinus-L, Acinus-S and Acinus-S'). Acinus-S' interacts with the A/B domain of RAR and represses RAR-regulated genes expression. Acinus (without isoform definition) has been identified as a component of nuclear speckles, the spliceosome and the exon junction complex (EJC), suggesting its localization in nuclear speckles and involvement in RNA processing. Acinus-S has been shown to localize in nuclear speckles. However, it is unclear whether the other two isoforms also localize in nuclear speckles. In addition, the role of Acinus in regulating pre-mRNA splicing is unclear. The goal of these studies was to examine the nuclear localization of Acinus-L and Acinus-S' and to determine the role of Acinus isoforms in RAR-dependent splicing. The sub-nuclear localization of Acinus-L and Acinus-S' was determined using fluorescence microscopy. Acinus-S' colocalizes with SC35 in nuclear speckles while Acinus-L localizes diffusely throughout the nucleoplasm. RA treatment has little effect on the sub-nuclear localization of Acinus-L and Acinus-S'. The domains/regions necessary for the distinct sub-nuclear localization of Acinus-L and Acinus-S' were identified. The speckled sub-nuclear localization of Acinus-S' is dependent on its C-terminal RS- and RD/E-rich region but is independent of the phosphorylation status of Ser-453 and Ser-604 within this region. The unique N-terminal SAP-motif of Acinus-L is responsible for its diffuse localization in the nucleus. Moreover, the sub-nuclear localization of Acinus isoforms is affected by each other, which is determined by the combinatorial effect of the more potent SAP motif of Acinus-L and the C-terminal RS- and RD/E-rich region in all Acinus isoforms. The C-terminal RS- and RD/E-rich region of Acinus mediates the colocalization of Acinus isoforms as well as with its interacting protein RNPS1. The role of Acinus isoforms in regulating pre-mRNA splicing was explored using in vivo splicing assays. Both Acinus-L and Acinus-S', with the activity of Acinus-L higher than that of Acinus-S', increase the splicing of a RA-responsive minigene containing a weak 5' splice site but not a RA-responsive minigene containing a strong 5' splice site. RA treatment further enhances the splicing activity of Acinus in a dose- and time-dependent manner, suggesting a RA-dependent activity in addition to a RA-independent activity of Acinus. The RA-independent effect of Acinus on the splicing of pre-mRNAs containing the weak 5' splice site occurs to varying degrees using minigene constructs containing several different promoters while the RA-dependent splicing activity of Acinus is specific for transcripts derived from the minigene driven by the RARE-containing promoter. This suggests that the ligand-dependent splicing activity of Acinus is related to the RA-activated RAR bound to the RARE. The ligand-dependent splicing activity of Acinus was further shown to be promoter-specific, depending on the ligand-dependent transcription activator. The RRM domain was identified to be necessary for the RA-dependent splicing activity of Acinus. The RA-independent splicing activity of Acinus is repressed by RNPS1. Unexpectedly, the C-terminal RS- and RD/E rich region is dispensable for the splicing activity of Acinus in regulating the minigene containing a weak 5' splice site. Importantly, measurement of the splicing of endogenous human RARâ and Bcl-x in vivo demonstrates that Acinus stimulates the use of the weaker alternative 5' splice site of these two genes in a RA-dependent manner for RARâ and in a RA-independent manner for Bcl-x. Taken together, these studies demonstrate the distinct sub-nuclear localization of Acinus-L and Acinus-S', and identified the domains that are responsible for their sub-nuclear localization, which shed light on possible distinct functions between Acinus isoforms. In addition, both Acinus-L and Acinus-S' have been shown to be splicing cofactors (with the activity of Acinus-L higher than that of Acinus-S') that facilitate constitutive splicing of pre-mRNAs containing a weak 5' splice site and regulate alternative splicing in favor of the isoform generated from the weaker alternative 5' splice site. Both Acinus-L and Acinus-S' have a RA-dependent splicing activity specific for RA-responsive genes, which suggests that Acinus functions in RAR-dependent splicing. / Biochemistry