RNA Ligation Pathways in Mammalian Unfolded Protein Response

项目摘要 未折叠蛋白反应(UPR)，一种众所周知的细胞维持适应机制 内质网稳态参与了多种人类疾病的发病机制。 UPR信号成分的基因缺失会显著影响疾病 糖尿病、肝脏脂肪变性、炎症性肠病、肌萎缩侧索硬化症等疾病小鼠模型的研究进展。我们 最近进行了一项合成生物学筛选，以询问哺乳动物的UPR途径。 从这个筛选中，我们已经确定RtcB是一种长期寻找的RNA连接酶，它催化 内质网应激过程中的非常规XBP-1剪接。RtcB是一种多功能RNA连接酶，它还 在tRNA剪接中起作用。RtcB基因敲除细胞的遗传挽救表明RtcB介导的 XBP1和tRNA的连接可能发生在不同的亚细胞室。因此，我们将 确定亚细胞室特异的RtcB功能。通过生成Trpt1和RtcB Double 敲除细胞并进行遗传拯救，我们进一步证明了一秒钟 在哺乳动物细胞中，生物化学上不同的RNA连接途径与RtcB合作，以 调节XBP1和tRNA剪接。因此，我们将研究其他推定的 RNA连接途径中的组件。最后，我们将鉴定未知的RNA连接酶 利用RtcB基因敲除细胞筛选遗传抑制基因的新连接途径。通过揭示 完整的RNA连接途径的分子谱系，我们将有望获得新的见解 并发现新的治疗靶点来调节哺乳动物的UPR。

Project Summary The unfolded protein response (UPR), a well-known adaptive mechanism for cells to maintain ER homeostasis, has been implicated in the pathogenesis of a variety of human diseases. Genetic deletion of the UPR signaling components can significantly influence the disease progression in mouse models for diabetes, hepatic steatosis, IBD, ALS and other diseases. We have recently performed a synthetic biology screen to interrogate the mammalian UPR pathway. From this screen, we have identified RtcB as a long sought RNA ligase that catalyzes unconventional XBP-1 splicing during ER stress. RtcB is a multifunctional RNA ligase that also acts in tRNA splicing. Genetic rescue of RtcB knockout cells suggests that RtcB-mediated ligation of XBP1 and tRNA may occur at different subcellular compartments. We will therefore determine subcellular compartment-specific RtcB function. By generating Trpt1 and RtcB double knockout cells and performing genetic rescue, we further demonstrate that a second biochemically distinct RNA ligation pathway cooperates with RtcB in mammalian cells to regulate XBP1 and tRNA splicing. Therefore, we will examine the role of other putative components in the RNA ligation pathway. Lastly we will identify unknown RNA ligases of the new ligation pathway by a genetic suppressor screen using RtcB knockout cells. By revealing the complete molecular repertoire of RNA ligation pathways, we will hopefully gain new insights and uncover new therapeutic targets to modulate the mammalian UPR.