Structure and regulation of tRNA ligases involved in stress response

参与应激反应的 tRNA 连接酶的结构和调控

• 批准号: 503940636
• 负责人: Dr. Ankan Banerjee
• 金额: --
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/503940636?language=en
• 关键词: Structure; regulation; tRNA; ligases; involved

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.

RNA连接酶封闭RNA断裂，是响应细胞应激信号的RNA处理途径的关键组成部分，包括病毒感染、真核生物中的未折叠蛋白反应和细菌中的抗生素应激。含有内含子的前tRNA和前rRNA的定点切割产生2个ʹ，3个ʹ-环磷酸(CPRNA)和5个ʹ-OH末端。已描述了三种连接这些末端的方式：(I)愈合封闭或5ʹ-3ʹ连接(由病毒T4Pnkp-Rnl1、酵母和植物特有的Trl1-Tpt1酶介导)，(Ii)直接连接或3ʹ-5ʹ连接(由古生物和后生动物的rtcB介导)和(Iii)可能的2ʹ-5ʹ连接(通过LIGT)。LIGT是一个2H家族的磷酸酯酶，它通过2个ʹ-5ʹ链不可逆地连接cPRNA/5ʹ-OH末端。然而，大肠杆菌LIGT具有较强的环磷酸二酯酶(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发挥着直接作用。古生菌RtcB同系物除了剪接含内含子的RNA外，其功能尚不清楚，古生菌的应激反应也没有详细的描述。在这项提议中，我的目标是利用已建立的古细菌(Haloferax Volcanus II)和古细菌(Sulfolobus Acidocaldarius)模式生物的遗传系统来描述RtcB在古细菌应激反应中的功能。在这里，RtcB缺失菌株的比较转录本和基因组内标记的RtcB的相互作用将准确地确定其在氧化和DNA损伤压力下的功能。我将定制一条转录组分管道来破译古生菌应激反应成分，重点是RNA加工途径。此外，我将使用生化和结构方法来研究翻译后修饰对RtcB的调控机制，并确定Ligt在RtcB功能调节中的作用。在此背景下，我将分析LIGT的生物学相关功能，并将探索依赖于LIGT活性的两条假想途径。古生菌是真核生物最接近的原核近亲，具有保守的tRNA加工酶，我们设想破译后生动物tRNA连接酶复合体的进化相关性。总之，我的目标是结合跨学科的方法来表征古生菌的应激反应，并确定RtcB在这一过程中的影响。