Structural and mechanistic basis of pre-tRNA processing by the human tRNA splicing endonuclease in health and neurodegenerative disease

健康和神经退行性疾病中人 tRNA 剪接核酸内切酶前 tRNA 加工的结构和机制基础

• 批准号: 428858078
• 负责人: Dr. Simon Trowitzsch
• 金额: --
• 依托单位: Institut für Biochemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/428858078?language=en
• 关键词: Structural; mechanistic; basis; pre; tRNA

Transfer RNAs (tRNAs) are among the most ubiquitous molecules in cells from all three domains of life and central to decoding information from messenger RNAs to proteins on translating ribosomes. Beyond their canonical role during protein biosynthesis, tRNAs also perform additional functions as signaling molecules in the regulation of numerous metabolic and cellular processes, as stress sensors and in tRNA-dependent biosynthetic pathways. tRNA fragments have been identified as novel species of small non-coding RNAs contributing to translational control, gene regulation and silencing, and progressive motor neuron loss. tRNAs are encoded as precursor molecules, which undergo a plethora of modifications, including removal of intronic sequences. In humans, introns are cleaved by the heterotetrameric tRNA splicing endonuclease (TSEN), which associates with the RNA kinase CLP1. Mutations in TSEN subunits or CLP1 lead to the development of severe neurological disorders. How these mutations engage in the development of the disease is totally unclear. Previous studies revealed first functional and structural aspects of the human pre-tRNA splicing endonuclease, but detailed insights into the assembly of the endonuclease subunits and substrate recognition remain elusive due to the lack of high-resolution structures. General principles of pre-tRNA recognition and cleavage by TSEN were only deduced from biochemical studies. The role of CLP1 in the process of pre-tRNA splicing remains enigmatic. I will address fundamental questions on pre-tRNA processing by three major objectives. By solving high-resolution structures of the human endonuclease, we will provide a structural framework for understanding the molecular mechanism of pre-tRNA splicing, explain differences between archaeal and eukaryotic systems, provide evidence for the role of CLP1, reveal the structure of an intron-containing pre-tRNA molecule, and explain the impact of disease mutations. We will track pre-tRNA splicing in single cells at sub-cellular resolution using tailored pre-tRNA fluorophore/quencher probes that report on localization and splice status. We will use this tRNA reporter system in a cellular disease model to study consequences of mutations on localization of TSEN components, tRNA and splicing. Furthermore, the pre-tRNA probes will allow us to deduce real-time kinetics of intron excision for wild type and mutant endonuclease complexes in vitro. Ultimately, we will identify novel target RNAs of the human pre-tRNA endonuclease by UV cross-linking immunoprecipitation-high-throughput sequencing (CLIP-seq). Moonlighting activities on messenger and ribosomal RNAs have been identified for the yeast tRNA endonuclease. Our data will for the first time reveal direct involvement of the human pre-tRNA endonuclease in cellular processes distinct from pre-tRNA splicing possibly enforced by disease-causing mutations.

转运RNA（transfer RNA，tRNA）是细胞中最普遍存在的分子之一，来自生命的所有三个领域，是将信使RNA的信息解码为翻译核糖体上的蛋白质的核心。除了在蛋白质生物合成过程中的典型作用外，tRNA还在许多代谢和细胞过程的调节中作为信号分子，作为压力传感器和tRNA依赖的生物合成途径中发挥额外的功能。tRNA片段已被鉴定为有助于翻译控制、基因调控和沉默以及进行性运动神经元丧失的小的非编码RNA的新种类。tRNA被编码为前体分子，其经历过多的修饰，包括去除内含子序列。在人类中，内含子被异源四聚体tRNA剪接核酸内切酶（TSEN）切割，该酶与RNA激酶CLP 1相关。TSEN亚基或CLP 1的突变导致严重神经系统疾病的发展。这些突变如何参与疾病的发展尚不清楚。先前的研究揭示了人类前tRNA剪接核酸内切酶的第一个功能和结构方面，但由于缺乏高分辨率结构，对核酸内切酶亚基组装和底物识别的详细了解仍然难以捉摸。TSEN识别和切割前tRNA的一般原理仅从生物化学研究中推导出来。CLP 1在前体tRNA剪接过程中的作用仍然是个谜。我将通过三个主要目标来解决前tRNA加工的基本问题。通过解决人类内切核酸酶的高分辨率结构，我们将为理解前tRNA剪接的分子机制提供结构框架，解释古细菌和真核系统之间的差异，为CLP 1的作用提供证据，揭示含内含子的前tRNA分子的结构，并解释疾病突变的影响。我们将使用定制的前tRNA荧光团/淬灭剂探针以亚细胞分辨率跟踪单细胞中的前tRNA剪接，该探针报告定位和剪接状态。我们将在细胞疾病模型中使用这种tRNA报告系统来研究突变对TSEN组分、tRNA和剪接定位的影响。此外，前tRNA探针将使我们能够推导出实时动力学的内含子切除野生型和突变体核酸内切酶复合物在体外。最终，我们将通过UV交联免疫沉淀-高通量测序（CLIP-seq）鉴定人前tRNA内切酶的新靶RNA。酵母tRNA内切酶对信使和核糖体RNA的兼职活动已经被鉴定。我们的数据将首次揭示人类前tRNA内切酶直接参与细胞过程，而不是可能由致病突变引起的前tRNA剪接。