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

在真核生物中，转移RNA(TRNAs)中34位(U34)的摆动尿苷被Elongator复合体化学修饰。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，从而导致蛋白质聚集。然而，低修饰tRNA水平的升高可以挽救这些表型。盘状阿米巴是变形虫进化超群的模式生物，其特点是蛋白质组具有异常长的氨基酸谷氨酰胺(Q)，在其他生物体中会引起蛋白质聚集。同时，它含有显著增加的tRNA基因的拷贝数，这些tRNA基因被延伸器复合体修饰。我们已经产生了在所有主要步骤中依赖于延伸器的修饰途径都被破坏的菌株。此外，我们还在一个真核物种中首次无条件地敲除了tQCUG的tRNA基因。我们的分析表明，Elongator复合体引入的化学修饰在阿米巴中是保守的，但结构组成与酵母中的结构组成不同。在这个项目中，我们将在原子分辨率下确定阿米巴的Elongator复合体的结构。网柄网柄菌可以以单细胞或多细胞的生活方式生活。这为比较Elongator复合体在单细胞和多细胞生活方式中的功能提供了独特的机会。使用产生的突变菌株，我们将定量确定其生命中任何一种形式存在的确切化学修饰，并将其与发育时间和进展中的表型变化相关联。我们产生的唯一tQCUG零菌株似乎依赖于功能Elongator复合体的存在，这可能是因为等受体tQUG中引入的化学修饰对解码很重要。我们将产生一个有条件的基因敲除系统，它将允许我们调查依赖于延伸子的修饰是否确实对tQCUG零菌株的生存是必不可少的。使用报告构建，我们将分析，在单细胞和多细胞生活方式中，阿米巴中的伸长突变体是否变得对蛋白质聚集敏感。这些研究将得到这些突变体翻译的全球研究的补充，我们将使用核糖体图谱。通过这些研究，我们想要揭示化学修饰对阿米巴对蛋白质聚集的弹性的贡献。理解如何防止蛋白质聚集及其往往具有破坏性的影响的机制，对其他物种也应该是重要的。

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.