Modulation of OPRM1 alternative splicing by morphine and HIV-1 Nef

项目总结 本R01申请是对题为“艾滋病毒/艾滋病高度优先药物滥用研究”的PAS-18-915的回应。 我们的项目将研究HIV和吗啡之间的分子和功能相互作用在调节 阿片受体M1(OPRM1)前mRNA选择性剪接与增强阿片类药物的关系 在艾滋病毒携带者(PWH)中观察到依赖。临床使用的阿片类药物，如吗啡，以及 海洛因等非法药物激活G蛋白偶联受体(GPCR)成员OPRM1 一家人。OPRM1前mRNA经历了广泛的选择性剪接事件。到目前为止，人类的21个亚型 具有交替的C-末端和/或N-末端区域的OPRM1以及大鼠OPRM1的17种异构体 已确认身份。鉴于这些区域在G蛋白偶联受体(GPCR)信号中的重要性，区分 对OPRM1亚型的调控将产生功能性后果。然而，OPRM1的特征 信号转导是广义的，只有一种异构体(MOR1)被广泛研究。我们的初步数据 提示剪接调控蛋白SRSF1和MOR-1x的选择性剪接是 在暴露于吗啡的神经细胞中优先诱导。有趣的是，我们的结果还显示， 人死后脑组织中MOR-1X亚型的选择性剪接和表达 威尔斯亲王医院。这些结果表明，HIV和吗啡可能影响OPRM1的选择性剪接和 协同诱导MOR-1X亚型表达。OPRM1前-mRNA的X外显子互斥 被整合到MOR-1X基因转录本中外显子3之后的成熟转录本中。此插页 导致更长的C-末端尾巴具有潜在地与几种细胞激酶结合的新基序 这是MOR-1X亚型所独有的。我们最近报道了感染HIV-1的神经胶质细胞释放Nef 细胞外小泡(Nef-EV)中捕获的蛋白质，很容易被神经元摄取。我们进一步 评估了Nef-Evs和另外两种HIV分泌蛋白Tat和gp120在选择性剪接中的可能作用 MOR-1X。有趣的是，虽然重组的tat和gp120没有明显的效果，但用 NEF-EVS在MOR-1X选择性剪接中的诱导作用与吗啡处理相似。我们的 初步结果还显示，Nef-EVS和吗啡共同处理神经元具有协同作用 诱导MOR-1X选择性剪接。此外，在脑区诱导了MOR-1X的选择性剪接 参与了F344大鼠的奖赏通路，F344大鼠是HIV-1TG大鼠的对照株，它含有添加剂 使用吗啡进行诱导。综上所述，我们假设HIV-1对 通过放大诱导的MOR-1X选择性剪接增加PWH中阿片依赖率 用吗啡。我们建议的研究将揭示吗啡和HIV之间的新的协同作用 OPRM1前-mRNA选择性剪接导致MOR-1X亚型优先表达 对吗啡身体依赖的影响。

PROJECT SUMMARY This R01 application is in response to PAS-18-915 entitled “HIV/AIDS High Priority Drug Abuse Research”. Our project will investigate the molecular and functional interactions between HIV and morphine in regulation of alternative pre-mRNA splicing of the opioid receptor M1 (OPRM1) with implications for enhanced opioid dependence observed in the people with HIV (PWH). Clinically used opioids, such as morphine, as well as illicit drugs, such as heroin, activate OPRM1 that is a member of the G protein-coupled receptor (GPCR) family. OPRM1 pre-mRNA undergoes extensive alternative splicing events. To date, 21 isoforms of the human OPRM1 with alternative C-terminal and/or N-terminal regions and 17 isoforms of the rat OPRM1, have been identified. Given the importance of these regions in G protein-coupled receptor (GPCR) signaling, differential regulation of OPRM1 isoforms would have functional consequences. However, characterization of OPRM1 signaling is generalized, and only one isoform (MOR1) has been extensively studied. Our preliminary data suggest that expression of splicing regulatory protein SRSF1 and alternative splicing of MOR-1X is preferentially induced in neuronal cells exposed to morphine. Interestingly, our results also revealed that alternative splicing and expression of MOR-1X isoform is induced in postmortem brain tissues obtained from the PWH. These results suggested that HIV and morphine may impact OPRM1 alternative splicing and synergistically induce MOR-1X isoform expression. The mutually exclusive exon X of OPRM1 pre-mRNA is incorporated into the mature mRNA transcript following exon 3 in the MOR-1X mRNA transcript. This insertion results in substantially longer C-terminal tail having new motifs potentially binding with several cellular kinases that is unique to the MOR-1X isoform. We recently reported that glial cells infected with HIV-1 release Nef protein captured within the extracellular vesicles (Nef-EVs) which are readily taken up by neurons. We further assessed possible role of Nef-EVs and two other HIV secretory proteins, Tat and gp120, in alternative splicing of MOR-1X. Interestingly, while recombinant Tat and gp120 had no visible effects, treatment of neurons with Nef-EVs caused a comparable induction in MOR-1X alternative splicing as did treatment with morphine. Our preliminary results also revealed that co-treatment of neurons with Nef-EVs and morphine synergistically induced MOR-1X alternative splicing. Additionally, alternative splicing of MOR-1X was induced in brain regions involved in the reward pathways of the F344 rat that is the control strain of HIV-1Tg rat that has an additive induction with the exposure to morphine. Taken all together, we hypothesize that HIV-1 contributes to the increased rate of opioid dependence in the PWH by amplifying the rate of MOR-1X alternative splicing induced by morphine. Our proposed studies will reveal a novel synergistic interaction between morphine and HIV on alternative splicing of OPRM1 pre-mRNA leading to preferential expression of MOR-1X isoform with implications in physical dependence of morphine.