Regulation of the Elongator and the DPH complex by the Kti11/Kti13 heterodimer

RNA和蛋白质的转录后和翻译后修饰在核糖体蛋白质合成中起重要作用。引人注目的是，两个非常不同的修饰反应，tRNA反密码子的尿苷修饰和真核延伸因子EF-2的邻苯二甲酰胺修饰，分别由Elongator或DPH复合物催化，共享一个共同的组分，Kti11/Kti13异二聚体。这些修饰特别重要，因为它们影响核糖体上tRNA的结合和易位，从而可以同时影响大量基因的翻译效率和准确性。Elongator和DPH复合物以及Kti11/Kti13的结构在真核生物中是保守的，但这些复合物的分子功能及其合作方式仍然知之甚少。Elongator是由六种不同的蛋白质组成的大型且高度保守的大分子组装体，其最初在酵母中被鉴定为RNA聚合酶II（Pol II）相关的转录延伸因子，其名称来源于此。已经描述了由伸长肌功能障碍引起的不同表型，表明其涉及各种细胞功能。在人类中，Elongator与神经系统疾病（例如家族性自主神经功能障碍）和致癌作用有关。在分子水平上，蛋白质复合物已被证明是必不可少的尿苷修饰的tRNA。不能排除Elongator具有额外的细胞和分子功能，其确切活性仍有争议。然而，越来越多的证据强烈支持尿苷的氨基甲酰基和甲氧羰基侧链在生物合成中的催化作用。该功能需要与Kti11/Kti13异二聚体复合物相互作用，可能是短暂的。DPH复合物与Dph 4一起催化二苯二甲酰胺生物合成的第一步（组氨酸残基的修饰），由Dph 1、Dph 2和Dph 3（别名Kti 11）组成。DPH1别名OVCA1是卵巢癌中的候选肿瘤抑制因子，小鼠纯合缺失突变体是胚胎致死的，而酵母可以在没有联苯二甲酰胺的情况下生长。我们计划表征Kti11/Kti13与Elongator和双苯二甲酰胺生物合成（DPH）复合物之间的功能相互作用。来自最近的结构见解的假设将指导功能在体内和体外的研究，以获得全面的机械理解的三种蛋白质复合物，并获得进一步了解其生物学作用。在这项拟议的研究计划中解决的科学问题不仅将增强对这些中央翻译控制网络的理解，而且还将促进对几种严重人类疾病的新型治疗策略的设计。

Post-transcriptional and post-translational modifications of RNA and proteins play an important role in ribosomal protein synthesis. Strikingly, two very different modification reactions, uridine modifications of tRNA anticodons and diphthamide modification of eukaryotic elongation factor EF-2, catalyzed by the Elongator or DPH complex, respectively, share a common component, the Kti11/Kti13 heterodimer. These modifications are of particular importance, as they affect the binding and translocation of tRNAs on the ribosome and thus can influence the translation efficiency and accuracy of a multitude of genes simultaneously. The structure of Elongator and the DPH complex as well as that of Kti11/Kti13 is conserved among eukaryotes, but the molecular functions of these complexes and the way they cooperate are still poorly understood. Elongator is a large and highly conserved macromolecular assembly built up by six distinct proteins that was initially identified in yeast as a RNA polymerase II (Pol II) associated transcription elongation factor from where its name originates. Diverse phenotypes arising from Elongator dysfunction have been described suggesting that it is involved in a whole variety of cellular functions. In humans, Elongator has been associated with neurological disorders (e.g. familial dysautonomia) and carcinogenesis. At the molecular level the protein complex has been shown to be essential for the uridine modification in tRNAs. It cannot be excluded that Elongator fulfills additional cellular and molecular functions and its precise activities are still controversially discussed. However, evidence is accumulating that strongly supports a catalytic role in biosynthesis of the carbamoyl and methoxycarbonyl side chains of uridine. This function requires interaction, perhaps transiently, with the Kti11/Kti13 heterodimeric complex. The DPH complex catalyzes the first step in diphthamide biosynthesis (a modification of a histidine residue) together with Dph4 and is composed of Dph1, Dph2 and Dph3 alias Kti11. DPH1 alias OVCA1 is a candidate tumor suppressor in ovarian cancer and mouse homozygous deletion mutants are embryonic lethal whereas yeast can grow in the absence of diphthamide. We plan to characterize the functional interplay between Kti11/Kti13 with Elongator and the diphthamide biosynthetic (DPH) complex. Hypotheses derived from recent structural insights will guide functional in vivo and in vitro studies to obtain a comprehensive mechanistic understanding of the three protein complexes and to gain further insight in their biological roles. The scientific questions addressed in this proposed research plan will not only enhance the understanding of these central translational control networks, but also facilitate the design of novel therapeutic strategies for several severe human diseases.