Molecular basis of tRNA splicing by the TSEN complex in health and disease

细胞的DNA包含了产生进行所有细胞过程所需的蛋白质所需的所有遗传信息。为了产生所需的蛋白质，细胞必须首先解释这种遗传密码。这种解释或翻译是通过信使（mRNA）和转移（tRNA）的共同努力来完成的。mRNA（由RNA聚合酶II产生）将信息携带到细胞质中，在细胞质中，大型细胞机器与tRNA（由RNA聚合酶III产生）一起将代码翻译成蛋白质序列。因此，tRNA的功能对于表达所需基因和维持必要的细胞过程至关重要。然而，为了达到这个功能阶段，tRNA被几个加工复合物的作用所修饰。最初，RNA聚合酶III产生一个前体，前tRNA，它包含在tRNA起作用之前必须先去除的侧翼序列。此外，在6%的tRNA中有一个干扰序列或“内含子”，它会破坏tRNA序列，也必须被切除。这种内含子切除是由TSEN/hClp1复合体以高度特异性和受调节的方式进行的。这种复合物的调控缺失导致非特异性切割和有毒tRNA片段的产生，从而引发细胞死亡，在人类患者中表现为严重的神经退行性变。事实上，TSEN/hClp1复合体的突变与前脑小脑发育不全的发展有关，这是一种早发性神经变性，可导致严重的智力迟钝和过早死亡。尽管这一基本过程对正常的大脑发育很重要，但这一过程的确切机制和调控尚不清楚。因此，我们的目标是解决TSEN/hClp1复合物及其同源的前trna底物的结构，以了解该酶如何特异性识别和切除内含子。此外，我们的目标是将这种方法与重组纯化蛋白样品的其他生化研究相结合，以了解这种作用是如何被调节的。最近，利用纯化的TSEN/hClp1，我们发现了一种新的TSEN/hClp1调节模式，其中一个小分子（ATP，在细胞代谢中起关键作用）结合并调节复合物的特异性。这表明了一种潜在的调节策略，即TSEN利用细胞内这种分子的水平来调节其活性和应激条件下的细胞存活。此外，了解这种调节可能为这些神经退行性疾病提供基于修改TSEN/hClp1活性的治疗策略。

The DNA of the cell contains all the genetic information required to produce the proteins required to carry out all cellular processes. In order to produce the desired protein, the cell must first interpret this genetic code. This interpretation, or translation, is carried out by the combined efforts of messenger (mRNA) and transfer (tRNA). The mRNA (produced by RNA polymerase II) carries the message to the cytoplasm, where large cellular machinery together with tRNA (produced by RNA polymerase III) translates the code into a protein sequence. Therefore, the function of the tRNA is paramount to expressing the desired gene and maintaining essential cellular processes. However, to reach this functional stage, the tRNA is modified by the action of several processing complexes. Initially, RNA polymerase III produces a precursor, pre-tRNA, which contains flanking sequences that must first be removed before the tRNA can function. Additionally, in 6% of all tRNAs there is an intervening sequence or 'intron' which disrupts the tRNA sequence and must also be excised out. This intron excision is carried out by the TSEN/hClp1 complex in a highly specific and regulated manner. Loss of regulation of this complex leads non-specific cleavage and the generation of toxic tRNA fragments which trigger cell death and manifests in human patients as severe neurodegeneration. Indeed, mutations in the TSEN/hClp1 complex have been associated with the development of prontocerebellar hypoplasia, an early-onset neurodegeneration which causes severe mental retardation and premature death. Despite the importance of this fundamental process to normal brain development the exact mechanism and regulation of this process remains unknown. Therefore we aim to solve the structure of the TSEN/hClp1 complex together with its cognate pre-tRNA substrate to understand how the enzyme specifically recognises and excises the intron. Furthermore, we aim to combine this approach with other biochemical studies with recombinant purified protein samples in order to understand how this action is regulated. Recently, using purified TSEN/hClp1, we have discovered a novel mode of TSEN/hClp1 regulation where a small molecule (ATP, which is pivotal in the cells metabolism) binds to and regulates the specificity of the complex. This suggests a potential regulatory strategy where TSEN uses the levels of this molecule inside the cell to regulate its activity and cell survival under stress conditions. Additionally, understanding this regulation may provide a therapeutic strategy based on modifying TSEN/hClp1 activity for these neurodegenerative conditions.