M.tuberculosis-induced Alternative Processing of IL12RB1 mRNA

描述(由申请人提供)：IL12R？TM(亚型2)是一种可供选择的IL12受体亚型，在暴露于结核分枝杆菌(Mtb)后由小鼠白细胞表达。异构体2缺乏IL12R？1(异构体1)的跨膜区，具有与异构体1不同的定位模式，与最初的预测相反，在体外增强了IL12依赖的活性。虽然很明显，结核分枝杆菌激活人类白细胞也会通过选择性剪接/多聚腺苷作用刺激异构体2的产生，但两者之间存在差距。 在我们理解异型体2所需的因素、替代RNA加工以及这一途径对控制体内结核分枝杆菌感染的意义方面。这一知识鸿沟很重要 因为在被填满之前，对IL12活性进行治疗性操作的途径仍然遥不可及。我们的中心假设是，免疫原性信号通过mRNA中的顺式元件诱导选择性剪接/Polya产生异构体2，这是一种通过促进IL12依赖的TH1分化来调节体内结核病控制的蛋白质。我们的长期目标是了解结核分枝杆菌如何刺激信号以诱导IL12RB1替代的mRNA处理，并操纵异构体2的产生来影响T细胞生物学和结核分枝杆菌的控制。本应用的目的是确定IL12RB1前mRNA中作为信号和中介异构体2产生的靶标的顺式元件，并使用人和鼠系统测试异构体2对结核病控制的意义。该建议的基本原理是，识别IL12RB1前-mRNA中介导替代RNA加工的信号响应顺式元件，以及异构体2在结核病背景下的功能，将导致改变通路活性的治疗靶点。这一假说将通过两个特定的目的来检验：1)确定介导异构体2 RNA替代加工的顺式元件和反式因子；2)确定IL12RB1异构体2影响结核分枝杆菌免疫的程度。AIM 1将利用IL12R？1微型基因，将其核重组到T细胞中，以确定IL12RB1 mRNA中的顺式元件，这些元件介导病原体诱导的RNA替代处理。候选加工因子的作用(基于受调控的PolyA位点附近的潜在结合位点)将为异构体2替代加工进行评估。Aim 2将使用依赖于IL12的生物测定来测试人类淋巴细胞中异构体2沉默的影响，以及使用新产生的小鼠品系来确定在没有异构体2的情况下实验性结核病的结果。这项工作是创新的，因为它关注于人类中完全不同水平的IL12R？1活性调节-信号介导的转录后选择性RNA剪接/多聚腺苷化。这项工作意义重大，因为它是为了解人类细胞中异构体2产生的早期事件所做的初步努力，这将揭示下游信号通路，并导致操纵异构体2水平的策略，从而改善T细胞功能。

DESCRIPTION (provided by applicant): IL12R¿1?TM (Isoform 2) is an alternative IL12 receptor isoform that is expressed by mouse leukocytes following exposure to M. tuberculosis (Mtb). Isoform 2 lacks the transmembrane domain of IL12R¿1 (Isoform 1), has a localization pattern that is distinct from that of Isoform 1, and in contrast to the initial predictions, enhancs IL12-dependent activities in vitro. While it is clear than the activation of human leukocytes with Mtb also stimulates Isoform 2 production via alternative splicing / polyadenylation, there is a gap in our understanding of the factors required for Isoform 2 alternative RNA processing and the significance of this pathway for controlling Mtb infection in vivo. This knowledge gap is important because until filled, avenues for therapeutic manipulation of IL12 activity will remain out of reach. Our central hypothesis is that immunogenic signaling induces alternative splicing/polyA via cis elements in the mRNA to generate Isoform 2, a protein that regulates TB control in vivo by promoting IL12-dependent TH1 differentiation. Our long-term goal is to understand how Mtb stimulates signaling to induce IL12RB1 alternative mRNA processing, and to manipulate Isoform 2 production to influence T cell biology and Mtb control. The objectives in this application are to identify the cis-elements in IL12RB1 pre-mRNA that are the targets of signaling and mediate Isoform 2 production and to test, using human and mouse systems, the significance of Isoform 2 to TB control. The rationale for the proposal is that identification of signal-responsive cis elements in IL12RB1 pre-mRNA that mediate alternative RNA processing, as well as the function of Isoform 2 in the context of TB, will lead to therapeutic targets to modiy pathway activity. The hypothesis will be tested through two specific aims: 1) Identify the cis-elements and trans-factors that mediate Isoform 2 RNA alternative processing and 2) determine the extent to which IL12RB1 Isoform 2 influences Mtb immunity. Aim 1 will utilize IL12R¿1 minigenes that will be nucleofected into T cells to identify cis-elements within the IL12RB1 mRNA that mediate pathogen-induced RNA alternative processing. Roles for candidate processing factors (based on potential binding sites near the regulated polyA site) will be assessed for Isoform 2 alternative processing. Aim 2 will use an IL12-dependent bioassay to test the affect of Isoform 2 silencing in human lymphocytes, as well use a newly generated mouse strain to determine the outcome of experimental TB in the absence of Isoform 2. The work is innovative because it focuses on a completely different level of IL12R¿1 activity regulation - signal-mediated post-transcriptional alternative RNA splicing / polyadenylation - in humans. This work is significant because it is the initial effort towards understanding the earlies events in Isoform 2 production in human cells, which should reveal downstream signaling pathways and lead to strategies for manipulating Isoform 2 levels and thus improving T cell function.