Structure and regulation of tRNA ligases involved in stress response

RNA连接酶密封RNA断裂并且是响应细胞应激信号的RNA加工途径的关键组分，所述细胞应激信号包括病毒感染、真核生物中的未折叠蛋白质反应和细菌中的抗生素应激。含有内含子的前体tRNA和前体rRNA的位点特异性切割产生2 β，3 β-环磷酸（cPRNA）和5 β-OH末端。已经描述了连接这些末端的三种模式：（i）愈合-密封或5 ′-3 ′连接（由病毒T4 Pnkp-Rnl 1和酵母和植物特异性Trl 1-Tpt 1酶介导），（ii）直接连接或3 ′-5 ′连接（由古生菌和后生动物中的RtcB介导）和（iii）可能的2 ′-5 ′连接（由LigT介导）。LigT是一种2 H家族磷酸酯酶，其通过2 ′-5 ′键不可逆地连接cPRNA/5 ′-OH末端。然而，大肠杆菌LigT被证明优先表现出环磷酸二酯酶（CPD酶）的活性，其生物学功能仍不清楚。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着直接的作用。古细菌RtcB同源物的功能超出剪接的内含子含有RNA是未知的，古细菌的应激反应还没有详细的特点。在这个建议中，我的目标是利用euryarchaeal（Haloferax volcanii）和crenarchaeal（Sulfolobus acidocaldarius）模式生物的成熟的遗传系统来描绘RtcB在古细菌应激反应中的功能。在这里，RtcB耗竭菌株的比较转录组和基因组内标记的RtcB的相互作用组将查明其响应氧化、DNA损伤应激的功能。我将定制一个转录组学管道来破译古细菌的应激反应组件，重点是RNA加工途径。此外，我将使用生物化学和结构的方法来研究RtcB的翻译后修饰的调节机制，并确定LigT在调节RtcB功能中的作用。在这种情况下，我将分析的生物相关功能的LigT，并将探讨两个假设的途径，将依赖于LigT的活动。作为真核生物的最接近的原核亲属，并具有保守的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，我的目标是联合收割机跨学科的方法来表征古细菌的压力反应，并确定在这个过程中的影响RtcB。

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.