RNA Ligation Pathways in Mammalian Unfolded Protein Response

项目摘要 未折叠蛋白反应（UPR）是一种众所周知的细胞适应性机制， ER稳态与多种人类疾病的发病机制有关。 UPR信号传导元件的基因缺失可以显著影响疾病 在糖尿病、肝脂肪变性、IBD、ALS和其他疾病的小鼠模型中的进展。我们 最近进行了合成生物学筛选，以询问哺乳动物的UPR途径。 从这个筛选中，我们已经鉴定出RtcB是一种长期寻找的RNA连接酶， 在ER应激期间的非常规XBP-1剪接。RtcB是一种多功能RNA连接酶， 参与tRNA剪接。RtcB敲除细胞的遗传拯救表明RtcB介导的 XBP 1和tRNA的连接可以发生在不同的亚细胞区室。因此我们将 确定亚细胞区室特异性RtcB功能。通过生成Trpt 1和RtcB双 敲除细胞和进行基因拯救，我们进一步证明，第二个 生物化学上不同的RNA连接途径与哺乳动物细胞中的RtcB合作， 调节XBP 1和tRNA剪接。因此，我们将研究其他假定的 RNA连接途径中的组分。最后，我们将确定未知的RNA连接酶的 通过使用RtcB敲除细胞的遗传抑制筛选新的连接途径。通过揭示 完整的RNA连接途径的分子库，我们将有希望获得新的见解 并发现调节哺乳动物UPR的新治疗靶点。

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.