Translational Silencing in Monocytes: Role of L13a

描述（由申请人提供）：动脉粥样硬化是血管壁驻留细胞不受控制的炎症的一种致病结果。通过细胞模型，我们发现核糖体蛋白l13a依赖的翻译沉默途径可以终止一组炎症基因的表达。在这个提议中，我们将测试这种机制是否可以解决炎症。我们已经证明，IFN-3诱导单核细胞中铜蓝蛋白（Cp）和一组其他炎症蛋白的合成是在翻译控制下的。此外，我们发现核糖体蛋白L13a及其从60S核糖体释放在IFN- γ活化翻译抑制剂（步态）复合物形成中的关键作用。翻译沉默机制依赖于靶mrna的3<未翻译区（UTR）中存在的步态元件对l13a依赖性步态复合物的识别。这是基于我们的观察，表明通过RNA沉默消耗L13a可以克服这些靶mrna的翻译抑制。令我们惊讶的是，L13a的缺失对整体蛋白质合成没有影响，但一些内部核糖体进入位点（IRES）依赖的翻译被抑制。本研究旨在验证这样一种假设，即l13a依赖性翻译沉默这些编码炎症蛋白簇的靶mrna可能作为一种内源性防御机制，抵抗不受控制的炎症和动脉粥样硬化。另一方面，L13a的作用可能仅限于核糖体的特化功能，而不是核糖体的整体功能。我们将通过以下三个具体目标来验证我们的假设：(1)分析IFN-3激活单核细胞中L13a调控和炎症反应的转录后操纵子。在这些目标中，我们将通过评估从头翻译，鉴定顺式活性元件和新目标mrna的这些元件识别的核糖核蛋白复合物，对新目标mrna的转录后操纵子成员进行全面分析。(2)步态复合物介导的翻译沉默的生理作用研究。我们已经产生了携带L13a条件空等位基因（floxed等位基因）的小鼠。LysMCre小鼠与floxed小鼠杂交会产生巨噬细胞特异性敲除L13a。从这些小鼠中分离的巨噬细胞将进行离体翻译沉默测试。另一方面，这些小鼠将受到炎症诱导剂的刺激，反应将在多个层面上进行检查。(3) L13a在核糖体功能和IRES活性中的作用。在这个目的中，我们将使用核糖体分离、免疫印迹分析和体外核糖体结合的联合方法来确定L13a的核糖体结合位点。我们将使用L13a枯竭细胞的翻译胜任提取物来确定L13a依赖IRES活性的机制。我们相信这些研究将发现新一代抗炎小分子对抗动脉粥样硬化和其他炎症性疾病的新的细胞靶点。

DESCRIPTION (provided by applicant): Atherosclerosis is a pathogenic consequence of uncontrolled inflammation of the resident cells of vessel wall. Using cell-based model we showed that ribosomal protein L13a-dependent translational silencing pathway could terminate the expression of a group of inflammatory genes. In this proposal we will test whether this mechanism can resolve inflammation. We have demonstrated that IFN-3 induced synthesis of Ceruloplasmin (Cp) and a group of other inflammatory proteins in monocytic cells is under translational control. Further, we discovered a crucial role of ribosomal protein L13a and its release from 60S ribosome in the formation of IFN- Gamma-Activated Inhibitor of Translation (GAIT) complex. The translational silencing mechanism relies on the recognition of L13a-dependent GAIT complex by the GAIT element present in the 3< untranslated region (UTR) of the target mRNAs. This is based on our observation that shows the depletion of L13a by RNA silencing can overcome the translation inhibition of these target mRNAs. To our surprise, the depletion of L13a had no effect on overall protein synthesis, however several Internal Ribosome Entry Site (IRES) dependent translation were inhibited. This proposal wants to test the hypothesis that L13a-dependent translational silencing of these target mRNAs coding a cluster of inflammatory proteins may serve as an endogenous defense mechanism against uncontrolled inflammation and atherosclerosis. On the other hand, the role of L13a may be confined to specialized rather than overall function of the ribosome. We will test our hypothesis by pursuing the following three specific aims: (1) Analysis of the L13a regulated and inflammation responsive post-transcriptional operon in IFN-3 activated monocytes. In these aim we will undertake a comprehensive analysis of the new target mRNAs the member of the post-transcriptional operon by assessing the de novo translation, identifying the cis-active elements and the ribonucleoprotein complex recognized by these elements of the new target mRNAs. (2) Studies on the physiological role of the GAIT complex mediated translational silencing. We have generated mice harboring the conditional null allele (floxed allele) of L13a. Crossing LysMCre mouse with the floxed mouse will generate the macrophage specific knockout of L13a. Macrophages isolated from these mice will be tested ex-vivo for translational silencing. On the other hand these mice will be challenged with the inducer of inflammation and the response will be examined on multiple levels. (3) Role of L13a in ribosome function and IRES activities. In this aim using a combined approach involving ribosome fractionation, immunoblot analysis and in vitro ribosome binding we will determine the ribosome-binding site of L13a. We will use the translation competent extract of L13a depleted cell to determine the mechanism of L13a dependence of the IRES activities. We believe these studies will discover novel cellular targets for new generation of anti-inflammatory small molecules against atherosclerosis and other inflammatory diseases. PUBLIC HEALTH RELEVANCE: Uncontrolled inflammation is the cause of many diseases such as atherosclerosis or cardiovascular disease. This research will uncover the insight about the endogenous cellular mechanisms to control the expression of inflammatory molecules and help to generate novel therapeutic agents against inflammatory diseases.