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合成中起着重要作用。引人注目的是，两种截然不同的修饰反应——tRNA反密码子的尿苷修饰和真核延伸因子EF-2的双苯二胺修饰，分别由拉长子或DPH络合物催化，它们有一个共同的成分，即Kti11/Kti13异源二聚体。这些修饰是特别重要的，因为它们影响核糖体上trna的结合和易位，从而可以同时影响多种基因的翻译效率和准确性。细长体和DPH复合物以及Kti11/Kti13复合物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。伸长子是由六种不同的蛋白质组成的大型高度保守的大分子组装体，最初在酵母中被鉴定为RNA聚合酶II （Pol II）相关的转录伸长因子，其名称来源于此。细长体功能障碍引起的多种表型已被描述，表明它涉及多种细胞功能。在人类中，细长子与神经系统疾病（如家族性自主神经障碍）和癌变有关。在分子水平上，蛋白质复合物已被证明是trna中尿苷修饰所必需的。不能排除伸长器还具有额外的细胞和分子功能，其确切的活性仍有争议。然而，越来越多的证据强烈支持尿苷在氨基甲酰和甲氧羰基侧链的生物合成中的催化作用。这种功能需要与Kti11/Kti13异二聚物相互作用，可能是短暂的。DPH配合物与Dph4一起催化双酞胺生物合成（组氨酸残基的修饰）的第一步，由Dph1、Dph2和Dph3别名Kti11组成。DPH1别名OVCA1是卵巢癌的候选肿瘤抑制因子，小鼠纯合缺失突变体是胚胎致死的，而酵母可以在没有双苯二胺的情况下生长。我们计划表征Kti11/Kti13与伸长子和双苯二胺生物合成（DPH）复合物之间的功能相互作用。从最近的结构见解中得出的假设将指导体内和体外的功能研究，以获得对这三种蛋白质复合物的全面机制理解，并进一步了解它们的生物学作用。在本研究计划中提出的科学问题不仅将增强对这些中央转译控制网络的理解，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.