Regulation and function of the DENR/MCT-1 complex

对mRNA翻译的调控代表了一层与转录基因表达垂直的基因表达。影响其翻译的一个因素是短上游开放阅读框架(UORF)。当核糖体在真核生物中翻译uORF时，它们‘消耗’含有启动子tRNA的三元复合体，这引发了一个问题，即进一步下游的主要ORF如何被翻译。通过邓肯和特尔曼实验室之间的长期合作，我们最近发现，由两个非规范翻译因子组成的DENR/MCT-1复合体，促进了uORF下游主要ORF的翻译重新启动。我们发现，该复合体在增殖细胞中有效，但在静止细胞中不活跃，这可能是由于差异磷酸化所致。这项工作提出了两个重要的问题，我们打算在这项拨款申请中解决：1)DENR/MCT-1复合体是如何受磷酸化调控的？在目标1中，我们建议确定DENR/MCT-1复合体上的磷酸化位点，以及负责这些磷酸化的激酶(Teleman Lab)。2)在分子水平上，DENR/MCT-1复合体如何促进翻译的重新启动？在目标2中，我们建议使用翻译提取物和半重组翻译系统在分子水平上研究DENR/MCT-1的功能(Duncan Lab)。最后，我们将在目标3中结合这两个工作流程来研究目标1中确定的磷酸化如何影响目标2中确定的DENR/MCT-1的分子功能。这项工作将对基础生物学和医学都有意义。这将有助于揭示翻译再启动的基本和基本过程及其调控。由于DENR和MCT-1是癌基因，而且许多癌症相关基因都含有uORF，因此这项工作也将与癌症相关。

Regulation of mRNA translation represents a layer of gene expression that is orthogonal to the transcriptional one. One element on mRNAs that influences their translation are short upstream Open Reading Frames (uORFs). When ribosomes translate uORFs in eukaryotes, they 'consume' the ternary complex containing the initiator tRNA, raising the question of how the main ORF further downstream can be translated. Through a long-standing collaboration between the Duncan and Teleman labs, we recently showed that the DENR/MCT-1 complex, consisting of two non-canonical translation factors, promotes translation re-initiation on main ORFs downstream of uORFs. We showed that this complex is active in proliferating cells but not in quiescent cells, likely due to differential phosphorylation. This work raised two important questions which we propose to address in this grant application: 1) How is the DENR/MCT-1 complex regulated by phosphorylation? In Aim 1 we propose to identify phosphorylation sites on the DENR/MCT-1 complex, and the kinases responsible for these phosphorylations (Teleman Lab). 2) How, at the molecular level, does the DENR/MCT-1 complex promote translation reinitiation? In Aim 2 we propose to use translation extracts and semi-reconstituted translation systems to study DENR/MCT-1 function at the molecular level (Duncan Lab). Finally, we will combine these two workstreams in Aim 3 to study how the phosphorylations identified in Aim 1 affect the molecular functions of the DENR/MCT-1 identified in Aim 2. This work will have implications for both basic biology and medicine. It will shed light on the basic and fundamental process of translation re-initiation as well as its regulation. Since DENR and MCT-1 are oncogenes, and many cancer-related genes contain uORFs, this work will also be of cancer relevance.