M.tuberculosis-induced Alternative Processing of IL12RB1 mRNA

描述（由申请人提供）：IL 12 R <$1？TM（亚型2）是一种可供选择的IL 12受体亚型，在暴露于M.结核病（Mtb）。亚型2缺乏IL 12 R？1（亚型1）的跨膜结构域，具有与亚型1不同的定位模式，与最初的预测相反，增强了IL 12体外依赖性活性。虽然很明显，用Mtb激活人白细胞也通过选择性剪接/多聚腺苷酸化刺激同种型2的产生，但存在差距。 在我们对亚型2替代RNA加工所需的因素以及该途径在体内控制Mtb感染的意义的理解中，这种知识差距很重要 因为除非得到填补，否则IL 12活性的治疗性操纵的途径将仍然遥不可及。我们的中心假设是免疫原性信号通过mRNA中的顺式元件诱导可变剪接/polyA，以产生同种型2，一种通过促进IL 12依赖性TH 1分化来调节体内TB控制的蛋白质。我们的长期目标是了解Mtb如何刺激信号传导以诱导IL 12 RB 1替代mRNA加工，并操纵同种型2的产生以影响T细胞生物学和Mtb控制。本申请的目的是鉴定IL 12 RB 1前mRNA中的顺式元件，其是信号传导的靶标并介导同种型2的产生，并使用人和小鼠系统测试同种型2对TB控制的意义。该提议的基本原理是，鉴定IL 12 RB 1前mRNA中介导替代RNA加工的信号响应性顺式元件以及同种型2在TB背景下的功能，将导致修饰途径活性的治疗靶点。将通过两个具体目的来检验该假设：1）鉴定介导亚型2 RNA替代加工的顺式元件和反式因子，和2）确定IL 12 RB 1亚型2影响Mtb免疫的程度。目的1将利用将被核转染到T细胞中的IL 12 R 1小基因来鉴定IL 12 R 1 mRNA内介导病原体诱导的RNA替代加工的顺式元件。将评估候选加工因子（基于受调控polyA位点附近的潜在结合位点）对亚型2替代加工的作用。目的2将使用IL 12依赖性生物测定来测试同种型2沉默在人淋巴细胞中的影响，以及使用新产生的小鼠品系来确定在不存在同种型2的情况下实验性TB的结果。这项工作是创新的，因为它专注于一个完全不同的水平的IL 12 R 1活性调节-信号介导的转录后选择性RNA剪接/聚腺苷酸化-在人类。这项工作是重要的，因为它是理解人类细胞中同种型2产生的早期事件的初步努力，这应该揭示下游信号通路，并导致操纵同种型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.