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

健康和疾病中 TSEN 复合物 tRNA 剪接的分子基础

• 批准号: MR/T011025/1
• 负责人: Alessandro Vannini
• 金额: $ 56.43万
• 依托单位: Institute of Cancer Research
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FT011025%2F1
• 关键词: Molecular; basis; tRNA; splicing; TSEN

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.

细胞的 DNA 包含产生执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，细胞必须首先解释该遗传密码。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，其中大型细胞机器与 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重要的细胞过程至关重要。然而，为了达到这个功能阶段，tRNA 需要通过多种加工复合物的作用进行修饰。最初，RNA 聚合酶 III 产生前体，即前 tRNA，其中包含侧翼序列，在 tRNA 发挥作用之前必须先将其去除。此外，在所有 tRNA 的 6% 中，存在一个干扰序列或“内含子”，它会破坏 tRNA 序列，也必须将其切除。这种内含子切除是由 TSEN/hClp1 复合物以高度特异性和受调控的方式进行的。该复合物失去调节会导致非特异性裂解和有毒 tRNA 片段的产生，从而引发细胞死亡，并在人类患者中表现为严重的神经退行性疾病。事实上，TSEN/hClp1 复合物的突变与前小脑发育不全的发生有关，前小脑发育不全是一种早发性神经变性，会导致严重的智力低下和过早死亡。尽管这一基本过程对正常大脑发育很重要，但该过程的确切机制和调节仍然未知。因此，我们的目标是解析 TSEN/hClp1 复合物及其同源前 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质样品的其他生化研究结合起来，以了解这种作用是如何调节的。最近，使用纯化的 TSEN/hClp1，我们发现了一种新的 TSEN/hClp1 调节模式，其中小分子（ATP，在细胞代谢中至关重要）结合并调节复合物的特异性。这表明了一种潜在的调控策略，即 TSEN 利用细胞内该分子的水平来调节其活性和细胞在应激条件下的存活。此外，了解这种调节可能会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。