Regulation and function of the DENR/MCT-1 complex

mRNA翻译的调节代表了与转录水平正交的基因表达水平。mRNA上影响其翻译的一个元件是短的上游开放阅读框（uORF）。当核糖体在真核生物中翻译uORF时，它们“消耗”含有起始tRNA的三元复合物，这就提出了下游主要ORF如何翻译的问题。通过邓肯和Teleman实验室之间的长期合作，我们最近发现，由两个非经典翻译因子组成的DENR/MCT-1复合物促进uORF下游主要ORF的翻译重新启动。我们发现，这种复合物在增殖细胞中是活跃的，但在静止细胞中不活跃，可能是由于差异磷酸化。这项工作提出了两个重要的问题，我们建议在本基金申请中解决：1）DENR/MCT-1复合物如何通过磷酸化调节？在目标1中，我们提出鉴定DENR/MCT-1复合物上的磷酸化位点，以及负责这些磷酸化的激酶（Teleman Lab）。2)在分子水平上，DENR/MCT-1复合物是如何促进翻译再起始的？在目标2中，我们建议使用翻译提取物和半重构翻译系统在分子水平上研究DENR/MCT-1功能（邓肯实验室）。最后，我们将联合收割机在目标3中结合这两个工作流程，研究目标1中鉴定的磷酸化如何影响目标2中鉴定的DENR/MCT-1的分子功能。这项工作将对基础生物学和医学产生影响。本文将揭示翻译再启动的基本过程及其调控。由于DENR和MCT-1是癌基因，并且许多癌症相关基因含有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.