Molecular mechanism of mTORC1-dependent translation and ribosome biogenesis

 描述（由申请人提供）：雷帕霉素复合物1（mTORC 1）的哺乳动物靶标是一种进化上保守的多蛋白激酶复合物，其耦合营养和生长因子传感以调节广泛的基本细胞过程。mTORC 1刺激蛋白质合成和核糖体生物合成，这是调节细胞生长和增殖的重要细胞过程。mTORC 1信号转导的过度活化在许多类型的癌细胞中发现，并导致蛋白质合成的异常增加和不受控制的细胞增殖。尽管mT 0 RC 1在通过eIF 4 E活化增强一般帽依赖性翻译中具有积极作用，但最近的研究揭示mT 0 RC 1优先刺激mRNA的子集，包括编码翻译以及细胞生长、增殖和侵袭所需的蛋白质的那些。有趣的是，这些mRNA在其5 'UTR中具有嘧啶富集序列（PES），例如5' TOP，其被认为在其表达中起重要作用。然而，mTORC 1主要促进这些mRNA的一个子集的翻译的分子机制仍然难以捉摸。我们发现了一种新的mTORC 1底物LARP 1，它直接与5' TOP mRNA的5' UTR中的PES相互作用。我们的初步数据表明，LARP 1不仅作为分子开关来调节含有PES的mRNA翻译以响应mTOR活性，而且还作为mTORC 1的调节剂来磷酸化具有免疫活性的mRNA上的4 EBP 1和S6 K1。LARP 1的敲低显著减少了与LARP 1相互作用的mRNA的翻译，并显著抑制各种癌细胞中的核糖体生物合成和细胞增殖。我们认为LARP 1是一种非典型的mTORC 1底物，调节特定PES mRNA的翻译并增强其mTORC 1依赖性翻译。我们将探讨mTORC 1依赖的LARP 1磷酸化有助于其相互作用的mRNA的翻译的分子机制。其次，我们将确定在LARP 1结合的mRNA中富集的5 'UTR PES基序的功能重要性。最后，我们将探索LARP 1的体内功能以及LARP 1在PI 3 K-Akt-mTORC 1介导的癌症发展的动物模型中的病理生理作用。我们将通过利用生物化学，分子生物学，蛋白质组学，高通量RNA测序和小鼠模型相结合的多方面但综合的方法来实现我们的目标。通过阐明mTORC 1-LARP 1依赖性翻译的分子机制，我们提出的研究将为mTORC 1介导的翻译调控提供新的见解，并导致针对mTORC 1活性下游效应物的癌症治疗新方法。

 DESCRIPTION (provided by applicant): The mammalian target of rapamycin complex 1 (mTORC1) is an evolutionarily conserved multi- protein kinase complex that couples nutrient and growth factor sensing to regulate a wide array of essential cellular processes. mTORC1 stimulates protein synthesis and ribosome biogenesis, which are vital cellular processes that regulate cell growth and proliferation. Hyperactivation of mTORC1 signaling is found in many types of cancer cells and leads to aberrant increases in protein synthesis and uncontrolled cell proliferation. Despite the positive roles of mTORC1 in enhancing general cap- dependent translation through eIF4E activation, recent studies reveal mTORC1 preferentially stimulates a subset of mRNAs, including those that encode proteins required for translation, and cell growth, proliferation, and invasion. Interestingly, these mRNAs possess pyrimidine-enriched sequences (PES) such as 5'TOP in their 5'UTRs that are thought to play important roles in their expression. However, the molecular mechanisms by which mTORC1 primarily facilitates the translation of a subset of those mRNAs remain elusive. We have discovered a novel mTORC1 substrate, LARP1 that directly interacts with the PES in the 5'UTRs of 5' TOP mRNAs. Our preliminary data suggest that LARP1 functions not only as a molecular switch to regulate PES- containing mRNA translation in response to mTOR activity, but also as a regulator for mTORC1 to phosphorylate 4EBP1 and S6K1 on translationally-competent mRNAs. Knockdown of LARP1 significantly reduces translation of mRNAs that interact with LARP1 and dramatically inhibits ribosome biogenesis and cell proliferation in various cancer cells. We propose that LARP1 is an atypical mTORC1 substrate that regulates the translation of specific PES mRNAs and enhances their mTORC1-dependent translation. We will explore the molecular mechanisms by which mTORC1- dependent phosphorylation of LARP1 contributes to the translation of its interacting mRNAs. Second, we will determine the functional importance of the 5'UTR PES motif enriched in LARP1-bound mRNAs. Finally, we will explore the in vivo functions of LARP1 as well as pathophysiological role of LARP1 in PI3K-Akt-mTORC1-mediated cancer development in animal models. We will accomplish our goals by exploiting a multifaceted but integrated approach combining biochemistry, molecular biology, proteomics, high-throughput RNA sequencing, and mouse models. By elucidating the molecular mechanisms of mTORC1-LARP1-depenent translation, our proposed studies will provide new insights into the regulations of mTORC1-mediated translation and lead to new therapeutic approaches for cancer that target downstream effectors of mTORC1 activity.