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

保守的真核蛋白复合体Elongator(Elp1-Elp6)主要作用于转移RNA(TRNAs)的修饰。因此，它确保了在mRNA翻译和蛋白质生物合成过程中解码信使RNA(MRNAs)遗传信息所需的适当tRNA功能。因此，人们可能会认为Elongator在任何时候都是永久有效的，以进行准确的tRNA解码。然而，数据显示tRNA修饰对各种刺激的反应是振荡的，这表明Elongator的活性是可调节的。为了支持这一点，我们已经证明Elongator经历了依赖于激酶Hrr25和磷酸酶Sit4的动态磷酸化，并且重要的是，可以被Kti12调节，Kti12是一种Elongator伙伴蛋白，与tRNA依赖的激酶(PSTK)和其他核苷酸(NTP)结合蛋白有关。Kti12如何准确地参与Elongator功能，以及它是否作为潜在的Elongator调节器运行，是需要回答的关键问题。因此，我们使用酵母作为模型系统来研究Kti12对Elongator的调控，我们的目标是实现以下目标：-(与Stark，Dundee教授合作)通过修改位点的质谱图和随后的定点突变来研究翻译后的Kti12修饰，以验证它们的意义。-使用我们的合作者(Stark，Dundee教授)先前分离的Kti12突变体库，并通过KTI12基因的定点突变来补充，以确定Kti12上潜在参与Elongator功能和调节的蛋白质结构域。-利用Kti12的真正性质(Elongator相互作用，Hrr25激酶招募到Elongator，Elp1磷酸化调节，Sit4拮抗，Sit4，等)，以研究上述Kti12结构域对Elongator功能和调节的需求。-开发体外分析Kti12的tRNA结合能力及其与NTP辅因子结合的潜在能力(见上文)。这些研究将涉及上面确定的重组Kti12和突变体，用于tRNA依赖的凝胶位移分析和辅因子结合研究。总之，该提案围绕体内和体外方法展开，旨在确定Kti12与Elongator合作的结构功能要求，并允许Elongator依赖的tRNA修饰受到差异化调控。此外，该项目在生物医学上具有长期改善健康和生活质量的潜力。这是由于大量最近的证据表明tRNA修饰与人类神经病变和肿瘤形成有关，因此，该提议有助于更好地理解协调形成Elongator连接的tRNA修饰在包括我们自己的细胞在内的高等真核细胞模型中的翻译控制和神经生成中所起的作用。

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.