Structure and regulation of tRNA ligases involved in stress response

RNA连接酶密封RNA断裂，是响应细胞应激信号的RNA加工途径的关键组成部分，包括病毒感染、真核生物的未折叠蛋白反应和细菌的抗生素应激。含有内含子的前trna和前rnas的位点特异性切割产生2′，3′-环磷酸（cPRNA）和5′-OH末端。已经描述了三种连接这些末端的模式：(i)愈合封闭或5′-3′连接（由病毒T4 Pnkp-Rnl1和酵母和植物特异性Trl1-Tpt1酶介导），（ii）直接连接或3′-5′连接（由古细菌和后生动物的RtcB介导）和（iii）可能的2′-5′连接（由光t）。light是一种2H家族磷酸酶，通过2′-5′连锁不可逆地连接cPRNA/5′-OH末端。然而，大肠杆菌light被证明优先表现出环磷酸二酯酶（CPDase）活性，其生物学功能尚不清楚。越来越多的证据表明，RtcB在后生动物和细菌的应激反应中起直接作用。除了剪接含内含子的rna外，古细菌RtcB同源物的功能尚不清楚，古细菌的应激反应也没有得到详细的表征。在本提案中，我旨在利用euryarchaeal （Haloferax volcanii）和crenarchaeal （Sulfolobus acidocalarius）模式生物的成熟遗传系统来描述RtcB在古细菌应激反应中的功能。本研究通过比较RtcB耗竭菌株的转录组和基因组内标记RtcB的相互作用组，将确定其在氧化DNA损伤应激反应中的功能。我将定制一个转录组学管道来破译古细菌的应激反应成分，重点是RNA加工途径。此外，我将利用生物化学和结构的方法来研究RtcB通过翻译后修饰调控的机制，并定义光在调节RtcB功能中的作用。在这种情况下，我将分析光t的生物学相关功能，并将探索两种依赖于光t活性的假设途径。古生菌是真核生物最接近的原核亲戚，具有保守的tRNA加工酶，我们设想破译后生动物tRNA连接酶复合物的进化相关性。总之，我的目标是结合跨学科的方法来表征古菌的应激反应，并确定RtcB在这一过程中的影响。

RNA ligases seal RNA breaks and are key components of RNA processing pathways in response to cellular stress signals, including viral infections, the unfolded protein response in eukaryotes, and antibiotic stress in bacteria. Site-specific cleavage of intron-containing pre-tRNAs and pre-rRNAs generates 2ʹ,3ʹ-cyclic phosphate (cPRNA) and 5ʹ-OH termini. Three modes of ligating these termini have been described: (i) healing-sealing or 5ʹ-3ʹ ligation (mediated by viral T4 Pnkp-Rnl1 and yeast and plant-specific Trl1-Tpt1 enzymes), (ii) direct ligation or 3ʹ-5ʹ ligation (mediated by RtcB in archaea and metazoa) and (iii) possibly 2ʹ-5ʹ ligation (by LigT). LigT is a 2H family phosphoesterase that irreversibly ligates cPRNA/5ʹ-OH ends via 2ʹ-5ʹ linkage. However, Escherichia coli LigT was shown to preferentially exhibit cyclic phosphodiesterase (CPDase) activity and its biological function remains unclear. Incremental evidence suggests that RtcB plays a direct role during stress response in metazoan and bacteria. Functions of archaeal RtcB homologs beyond splicing of intron-containing-RNAs are not known and the archaeal stress response has not been characterized in detail. In this proposal, I aim to take advantage of the well-established genetic systems of euryarchaeal (Haloferax volcanii) and crenarchaeal (Sulfolobus acidocaldarius) model organisms to delineate the function of RtcB in the archaeal stress response. Here, comparative transcriptomes of the RtcB depletion strains and interactomes of the in-genome tagged RtcB will pinpoint its function in response to oxidative, DNA damage stress. I will customize a transcriptomics pipeline to decipher archaeal stress response components focusing on RNA processing pathways. Furthermore, I will use biochemical and structural approaches to investigate mechanisms of RtcB regulation by post-translational modifications and define the role of LigT in regulating of RtcB function. In this context, I will analyze the biologically relevant function of LigT and will explore two hypothetical pathways that would rely on LigT activity. Archaea being the closest prokaryotic relative of eukaryotes and having conserved tRNA processing enzymes, we envision deciphering the evolutionary relevance of the metazoan tRNA ligase complex. In conclusion, I aim to combine interdisciplinary approaches to characterize the archaeal stress response and to define the impact of RtcB in this process.