Prospective Identification of Translational Regulators in EMT

 描述（由申请人提供）：mRNA定位、稳定性和翻译的转录后控制是一种非常重要的基因调控机制，但在很大程度上尚未探索，该机制有助于细胞生理学的几乎每个方面。在许多情况下，给定mRNA转录物的3'非翻译区（UTR）内的元件赋予对微RNA和/或结合蛋白的响应性，这可能决定转录物的命运。其中基因表达的转录后调节已被证明发挥重要作用的一个生理过程是上皮向间充质转化（EMT）。EMT被认为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。乳腺上皮细胞系MCF 10a响应于用细胞因子TGF-β处理而经历EMT。使用密度梯度分离和下一代测序，我们已经研究了MCF 10a细胞在上皮和间充质状态下的多聚体富集和耗尽。在我们的模型系统中，我们已经确定了54种基因产物，它们在间充质状态下优先翻译（比总mRNA富集4倍或更多）。在这些基因产物中，SNAI 1是几个实验系统中已知的EMT驱动因子。驱动这种多聚体富集的机制的性质，以及所观察到的基因产物的其余部分是否有助于间充质状态的启动或维持，都不清楚。该研究旨在评估在我们的系统中前瞻性识别新型翻译调节因子的模板的可行性。我们的假设是双重的：首先，这些基因产物在功能上有助于间充质状态的起始或维持，其次，它们的优先翻译受共同因子与这些转录物的3 ′ UTR的相互作用的调节。为了验证这些假设，我们将采用配对的功能基因组方法，这些方法相互提供信息，但不相互依赖。通过过表达和RNAi敲低，我们将确定所鉴定的基因产物是否是EMT在该过程的三种不同模型中所必需和足够的。与此同时，我们将采用一种新的、可扩展的、基于转座子的报告平台来定义3' UTR赋予这些mRNA转录本中的哪一个翻译控制。是否顺式调控元件共同的许多这些成绩单的基础，这种控制也将直接评估。最后，我们将使用生物化学和分子生物学的方法来前瞻性地识别和表征假定的反式调节结合这些假定的顺式元件，根据这些反式调节可能整合在EMT程序中的转录后基因调控的基本原理。在我们的模型系统中鉴定的EMT的新型调节剂，包括直接效应子和调节它们的反式因子，是旨在预防早期癌症转移的治疗干预的潜在靶点。此外，我们的方法的成功实施将产生一个通用的模板，可以在其他基于细胞的肿瘤发生模型中利用。

 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.