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

转运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，是解码信息的核心，从信使rna到翻译核糖体上的蛋白质。除了在蛋白质生物合成过程中的典型作用外，trna还作为信号分子在许多代谢和细胞过程的调节中发挥额外的功能，如应激传感器和trna依赖性生物合成途径。tRNA片段已被确定为一种新型的小非编码rna，在翻译控制、基因调控和沉默以及进行性运动神经元丢失中起着重要作用。trna被编码为前体分子，前体分子经历过多的修饰，包括内含子序列的去除。在人类中，内含子被与RNA激酶CLP1相关的异四聚体tRNA剪接内切酶（TSEN）切割。TSEN亚基或CLP1的突变可导致严重神经系统疾病的发展。这些突变是如何参与疾病发展的还完全不清楚。先前的研究揭示了人类前trna剪接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，对内切酶亚基的组装和底物识别的详细见解仍然难以捉摸。TSEN识别和切割前trna的一般原理仅从生化研究中推断出来。CLP1在pre-tRNA剪接过程中的作用仍然是一个谜。我将通过三个主要目标来解决有关trna前处理的基本问题。通过解决人类内切酶的高分辨率结构，我们将为理解前trna剪接的分子机制提供一个结构框架，解释古细菌和真核生物系统之间的差异，为CLP1的作用提供证据，揭示含有内含子的前trna分子的结构，并解释疾病突变的影响。我们将使用定制的预trna荧光团/淬灭探针在亚细胞分辨率下跟踪单细胞中的预trna剪接，这些探针可以报告定位和剪接状态。我们将在细胞疾病模型中使用这个tRNA报告系统来研究突变对TSEN成分定位、tRNA和剪接的影响。此外，预trna探针将使我们能够推断出野生型和突变型内切酶复合物在体外的内含子切除的实时动力学。最后，我们将通过UV交联免疫沉淀-高通量测序（CLIP-seq）鉴定人类前trna内切酶的新靶标rna。酵母tRNA内切酶在信使rna和核糖体rna上的兼职活性已经被确定。我们的数据将首次揭示人类前trna核酸内切酶直接参与细胞过程，不同于可能由致病突变强制的前trna剪接。

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.