Prospective Identification of Translational Regulators in EMT

 描述(由申请人提供)：转录后控制信使核糖核酸的定位、稳定性和翻译是一种基本的重要的，但在很大程度上未被探索的基因调控机制，几乎对细胞生理学的每一个方面都有贡献。在许多情况下，给定的mRNA转录本的3‘非翻译区(UTR)中的元件对microRNAs和/或结合蛋白具有反应性，这可能决定转录本的命运。转录后调控基因表达的一个生理过程是上皮向间充质转化(EMT)。EMT被认为是通过改变原发肿瘤内癌细胞的形状、侵袭性和运动性来启动肿瘤转移的。乳腺上皮细胞系MCF10A在细胞因子转化生长因子的作用下进行EMT。利用密度梯度分离和下一代测序，我们检测了MCF10A细胞在上皮和间充质状态下的多体浓缩和缺失。在我们的模型系统中，我们已经确定了54个基因产物在间充质状态下优先翻译(比总mRNA丰度高4倍或更多)。在这些基因产物中有SNAI1，它是几个实验系统中已知的EMT的驱动因素。驱动这种多体浓缩的机制的本质，以及所观察到的其余基因产物是否有助于启动或维持间充质状态，都不清楚。这项拟议的研究旨在评估模板的可行性，以期在我们的系统中识别新的翻译调控因子。我们的假设有两个方面：第一，这些基因产物在功能上有助于启动或维持间充质状态，第二，它们的优先翻译受到共同因子与这些转录本的3‘UTRs相互作用的调节。为了检验这些假设，我们将使用相互提供信息但不相互依赖的配对功能基因组方法。通过过表达和RNAi敲除，我们将确定在这一过程的三个不同模型中，所识别的基因产物是否对于EMT是必要的和充分的。同时，我们将使用一个新颖的、可扩展的、基于转座子的报告平台来定义3‘UTR对这些mRNA转录产物中的哪些给予翻译控制。还将直接评估这些转录本中许多共同的顺式调控元件是否构成这种控制的基础。最后，我们将使用生化和分子方法来前瞻性地鉴定和表征与这些假定的顺式元件结合的假定的反式调节因子，理由是这些反式调节因子可能会将转录后基因调节整合到EMT计划中。在我们的模型系统中发现的新的EMT调节因子，包括直接效应因子和调节它们的反式因子，都是旨在防止早期癌症转移的治疗干预的潜在靶点。此外，我们方法的成功实施将产生一个通用模板，可以在其他基于细胞的肿瘤发生模型中加以利用。

 DESCRIPTION (provided by applicant): Post-transcriptional control of mRNA localization, stability, and translation is a fundamentally important, yet largely underexplored, mechanism of gene regulation that contributes to virtually every aspect of cellular physiology. In many cases, elements within the 3' untranslated region (UTR) of a given mRNA transcript confer responsiveness to microRNAs and/or binding proteins, which may dictate the fate of the transcript. One physiological process in which post-transcriptional regulation of gene expression has been demonstrated to play an important role is the epithelial to mesenchymal transition (EMT). EMT is thought to initiate tumor metastasis by altering the shape, invasiveness, and motility of carcinoma cells within the primary tumor. The breast epithelial cell line MCF10a undergoes EMT in response to treatment with the cytokine TGF-ß. Using density gradient fractionation and Next Generation Sequencing, we have examined polysomal enrichment and depletion in MCF10a cells in both the epithelial and mesenchymal states. Within our model system, we have identified 54 gene products that are preferentially translated (4-fold or more enrichment over total mRNA) in the mesenchymal state. Among these gene products is SNAI1, a known driver of EMT in several experimental systems. The nature of the mechanism driving this polysomal enrichment, and whether the remainder of the observed gene products contribute to the initiation or maintenance of the mesenchymal state, are both unclear. The proposed research is designed to assess the feasibility of a template for prospective identification of nove translational regulators within our system. Our hypothesis is two-fold: firstly, that these gene products functionally contribute to the initiation or maintenance of the mesenchymal state, and secondly, that their preferential translation is modulated by the interaction of a common factor with the 3' UTRs of these transcripts. To test these hypotheses, we will employ paired functional genomic approaches that are mutually informative but non-interdependent. Via overexpression and RNAi knockdown, we will determine whether the identified gene products are necessary and sufficient for EMT in three distinct models of this process. In parallel, we will employ a novel, scalable, transposon-based reporter platform to define for which of these mRNA transcripts the 3' UTR confers the translational control. Whether a cis-regulatory element common to many of these transcripts underlies this control will also be directly assessed. Finally, we will use biochemical and molecular methods to prospectively identify and characterize putative trans-regulators binding these putative cis-elements, under the rationale that these trans-regulators may integrate post-transcriptional gene regulation in the EMT programs. Novel regulators of EMT identified in our model system, both direct effectors and the trans-factors that regulate them, are potential targets for therapeutic intervention aimed at preventing metastasis of early-stage cancers. Further, successful implementation of our approach will result in a generalized template that can be leveraged in other cell-based models of tumorigenesis.