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着重要作用。值得注意的是，两种截然不同的修饰反应，tRNA反密码子的尿苷修饰和真核细胞延伸因子EF-2的二苯胺修饰，分别由Elongator或DPH络合物催化，它们共享一个共同的组分，即Kti11/Kti13异源二聚体。这些修饰特别重要，因为它们影响核糖体上tRNAs的结合和转位，从而可以同时影响多个基因的翻译效率和准确性。Elongator和DPH复合体以及Kti11/Kti13的结构在真核生物中是保守的，但这些复合体的分子功能及其相互作用的方式仍然知之甚少。Elongator是一个大的、高度保守的大分子组件，由六种不同的蛋白质组成，最初在酵母中被鉴定为与RNA聚合酶II(POL II)相关的转录延伸因子，它的名字就是从那里来的。由Elongator功能障碍引起的不同表型已被描述，这表明它参与了一系列细胞功能。在人类中，Elongator与神经疾病(如家族性自主神经障碍)和癌症的发生有关。在分子水平上，蛋白质复合体对于tRNAs中的尿苷修饰是必不可少的。不能排除Elongator具有额外的细胞和分子功能，其确切的活性仍存在争议。然而，越来越多的证据强烈支持在尿苷的氨甲酰基和甲氧基羰基侧链的生物合成中起催化作用。这一功能可能需要与Kti11/Kti13异二聚体复合体的相互作用，也许是暂时的。DPH络合物与Dph4一起催化双苯二甲胺生物合成的第一步(组氨酸残基的修饰)，由Dph1、Dph2和Dph3别名Kti11组成。DPH1别名OVCA1是卵巢癌的候选抑癌基因，小鼠纯合子缺失突变体对胚胎是致命的，而酵母可以在没有敌草胺的情况下生长。我们计划表征Kti11/Kti13与Elongator和联苯二甲胺生物合成(DPH)复合体之间的功能相互作用。根据最近的结构洞察力得出的假说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体的全面机制理解，并进一步了解它们的生物学作用。这项拟议的研究计划中涉及的科学问题不仅将加强对这些中央翻译控制网络的理解，还将促进针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.