Do chemical modifications of tRNAs contribute to the aggregation resilience of the highly Q-rich proteome in D. discoideum?

tRNA 的化学修饰是否有助于盘状 D. discoideum 中高度富含 Q 的蛋白质组的聚集弹性？

• 批准号: 405030915
• 负责人: Professor Dr. Christian Hammann
• 金额: --
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/405030915?language=en
• 关键词: Do; chemical; modifications; tRNAs; contribute

The wobble uridines at position 34 (U34) in transfer RNAs (tRNAs) are chemically modified in eukaryotes by the Elongator complex. In the absence of these modifications, several severe phenotypes are observed. In yeast, these phenotypes can be attributed to distorted translation, which results in protein aggregation. Elevated levels of hypomodified tRNAs, however, can rescue these phenotypes.The amoeba Dictyostelium discoideum, the model organism of the evolutionary supergroup of Amoebozoa, features a proteome with exceptionally long stretches of the amino acids glutamine (Q) that in other organisms cause protein aggregation. Concurrent, it harbors a significantly increased copy number of tRNA genes that are modified by the elongator complex. We have generated strains in which the elongator-dependent modification pathway is disrupted at all major steps. Additionally, we have generated the first unconditional knockout of the tRNA gene of tQCUG in a eukaryotic species. Our analyses indicate that the chemical modifications introduced by the Elongator complex are conserved in the amoeba, but the structural composition deviates from that seen in yeast. Within this project, we will determine the structure of the Elongator complex of the amoeba at atomic resolution. D. dictyostelium can live in a unicellular or multicellular lifestyle. This offers the unique opportunity to compare the function of the Elongator complex in the unicellular and multicellular lifestyles. Using the generated mutant strains, we will quantitatively determine the exact chemical modifications that are present in either form of its life, and correlate this to phenotypic changes in the timing and progress of development.The unique tQCUG null strain, which we have generated, appears to depend on the presence of a functional Elongator complex, likely because the chemical modifications introduced in the isoacceptor tQUUG are important for decoding. We will generate a conditional knockout system, which will allow us to investigate whether the elongator-dependent modifications indeed are essential for the viability of the tQCUG null strain. Using reporter constructs, we will analyze, whether elongator mutants in the amoeba become susceptible to protein aggregation, in both the unicellular and the multicellular lifestyle. These investigations will be complemented by global studies on translation in these mutants, for which we will employ ribosome profiling. By these investigations, we want to unravel the contribution of chemical modifications on the resilience of the amoeba to protein aggregation. Understanding the mechanisms of how protein aggregates with their often devastating effects can be prevented should be important also for other species.

转移RNA（tRNA）中34位（U34）的摆动尿苷在真核生物中被Elongator复合物化学修饰。在没有这些修饰的情况下，观察到几种严重的表型。在酵母中，这些表型可以归因于扭曲的翻译，这导致蛋白质聚集。然而，低修饰的tRNA水平的升高可以挽救这些表型。盘状网囊变形虫是变形虫进化超类群的模式生物，其特征是蛋白质组中含有特别长的氨基酸谷氨酰胺（Q），在其他生物中这种氨基酸会引起蛋白质聚集。同时，它含有显著增加的由延长子复合物修饰的tRNA基因的拷贝数。我们已经产生了其中延伸子依赖性修饰途径在所有主要步骤都被破坏的菌株。此外，我们已经在真核生物中产生了tQCUG的tRNA基因的第一个无条件敲除。我们的分析表明，引入的延伸复合物的化学修饰是保守的变形虫，但结构组成偏离酵母中看到的。在这个项目中，我们将以原子分辨率确定变形虫的拉长子复合体的结构。D.网骨藻可以以单细胞或多细胞的方式生活。这提供了一个独特的机会来比较单细胞和多细胞生活方式中延长子复合体的功能。使用产生的突变株，我们将定量确定确切的化学修饰，存在于任何形式的生活，并将其与表型变化的时间和进展的发展。独特的tQCUG无效株，我们已经产生，似乎取决于存在的功能性延长复合物，可能是因为化学修饰引入的isoacceptor tQUUG是重要的解码。我们将产生一个条件性敲除系统，这将使我们能够调查是否延伸依赖性修饰确实是必不可少的tQCUG空菌株的生存能力。使用报告结构，我们将分析，无论是在单细胞和多细胞的生活方式，在阿米巴的延长突变体变得容易蛋白质聚集。这些调查将通过对这些突变体翻译的全球研究来补充，我们将采用核糖体分析。通过这些研究，我们希望揭示化学修饰对变形虫蛋白质聚集的弹性的贡献。了解蛋白质聚集体的机制及其通常的破坏性影响是如何预防的，对其他物种也很重要。