Kti12 - a regulator of the tRNA modification function of Elongator in yeast?

Kti12 - 酵母中 Elongator tRNA 修饰功能的调节因子？

• 批准号: 264621823
• 负责人: Professor Dr. Raffael Schaffrath
• 金额: --
• 依托单位: Fachgebiet Mikrobiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/264621823?language=en
• 关键词: Kti12; regulator; tRNA; modification; function

The conserved eukaryotic protein complex Elongator (Elp1-Elp6) primarily operates in the modification of transfer RNAs (tRNAs). Thus, it ensures proper tRNA functioning required for decoding the genetic information of messenger RNAs (mRNAs) during the processes of mRNA translation and protein biosynthesis. Hence, one might expect that Elongator is permanently active for accurate tRNA decoding at all times. However, data showing that the tRNA modifications oscillate in response to various stimuli suggest Elongator activity is regulatable. In support of this, we have shown Elongator undergoes dynamic phosphorylation dependent on kinase Hrr25 and phosphatase Sit4 and importantly, modulatable by Kti12, an Elongator partner protein related to a tRNA-dependent kinase (PSTK) and other nucleotide (NTP) binding proteins. How Kti12 is precisely involved in Elongator function and whether it operates as a potential Elongator regulator are key questions in need for answers. Therefore, and using yeast as model system to study Elongator regulation by Kti12, we aim to achieve the following goals:- to study (in collaboration with Prof. Stark, Dundee) posttranslational Kti12 modification by mass spectrometric mapping of modification sites followed by site-specific mutagenesis for validation of their significance.- to use a pool of kti12 mutants previously isolated by our collaborator (Prof. Stark, Dundee) and to be complemented by site-specific mutagenesis of the KTI12 gene for identifying protein domains on Kti12 hat are potentially involved in Elongator function and regulation.- to take advantage of bona fide Kti12 properties (Elongator interaction, Hrr25 kinase recruitment to Elongator, Elp1 phosphomodulation, Sit4 antagonism, etc) for examining the requirement of the above identified Kti12 domains for Elongator function and regulation.- to exploit in vitro assays for analysing the proposed tRNA binding capacity of Kti12 and its potential ability to bind NTP cofactors (see above). These studies will involve recombinant Kti12 and mutants identified above for use in tRNA-dependent gel-shift assays and cofactor binding studies. In summary, the proposal centers around in vivo & in vitro methods that aim at identifying structure-function requirements for Kti12 to partner with Elongator and allow Elongator-dependent tRNA modifications to be differentially regulated. Moreover, the project is biomedically relevant with potentials to improve the quality of health and life in the longer term. This lies with a strong body of recent evidence showing that tRNA modifications are linked to human neuropathies and tumour formation Hence, the proposal can contribute to a better understanding of the roles that coordinated formation of Elongator-linked tRNA modifications play in translational control and neurogeneration in higher eukaryal models including our own cells.

保守的真核蛋白复合物Elongator（Elp 1-Elp 6）主要在转运RNA（tRNA）的修饰中起作用。因此，它确保在mRNA翻译和蛋白质生物合成过程中解码信使RNA（mRNA）的遗传信息所需的适当tRNA功能。因此，人们可能会期望Elongator在任何时候都能永久有效地进行准确的tRNA解码。然而，数据显示tRNA修饰会响应各种刺激而振荡，这表明Elongator活性是可调节的。为了支持这一点，我们已经证明了Elongator经历了依赖于激酶Hrr 25和磷酸酶Sit 4的动态磷酸化，并且重要的是，可以通过Kti 12调节，Kti 12是一种与tRNA依赖性激酶（PSTK）和其他核苷酸（NTP）结合蛋白相关的Elongator伴侣蛋白。Kti 12如何精确地参与伸长器功能以及它是否作为潜在的伸长器调节器运行是需要回答的关键问题。因此，使用酵母作为模型系统研究Kti 12对Elongator的调节，我们旨在实现以下目标：-通过修饰位点的质谱图谱研究（与Stark教授、邓迪合作）翻译后Kti 12修饰，然后进行位点特异性突变以验证其重要性。-使用我们的合作者（Stark教授，邓迪）先前分离的kti 12突变体库，并通过Kti 12基因的位点特异性诱变进行补充，以鉴定Kti 12上可能参与Elongator功能和调节的蛋白质结构域。利用真正的Kti 12特性（延伸子相互作用、Hrr 25激酶向延伸子的募集、Elp 1磷酸调节、Sit 4拮抗等）来检查上述鉴定的Kti 12结构域对延伸子功能和调节的需求。利用体外测定法分析Kti 12的拟议tRNA结合能力及其结合NTP辅因子的潜在能力（见上文）。这些研究将涉及重组Kti 12和上述鉴定的突变体，用于tRNA依赖性凝胶迁移试验和辅因子结合研究。总之，该提案围绕体内和体外方法，旨在确定Kti 12与Elongator合作的结构-功能要求，并允许Elongator依赖性tRNA修饰受到差异调节。此外，该项目与生物医学有关，有可能长期改善健康和生活质量。这与最近的证据表明tRNA修饰与人类神经病变和肿瘤形成有关的强有力的身体有关。因此，该提议可以有助于更好地理解协调形成的延伸子连接的tRNA修饰在包括我们自己的细胞在内的更高真核模型中的翻译控制和神经生成中所起的作用。