Tracking Gene Expression Dynamics from Transcription to Degradation

 描述(由申请人提供)： 基因表达的调节决定了蛋白质异构体产生的时间、地点和程度，从而满足了构成器官和生物的各种细胞类型的新陈代谢需求。考虑到不断变化的微环境，这个过程是高度动态的，能够立即调整到多个外部线索，或在较长时间内积累基因表达反应。为了实现不同的基因表达反应，细胞依赖于多个调节点，包括调节转录的来源和速度， 改变信使核糖核酸的前处理以诱导选择性剪接信使核糖核酸异构体的产生，通过选择性多聚腺苷基化改变3‘非编码区的长度或位置，选择性信使核糖核酸异构体翻译，以及改变信使核糖核酸库的稳定性。所有这些基因表达步骤都是整合的，相互依赖。然而，我们对基因表达的动态性质的理解是有限的，因为大多数基于细胞的研究只分析基因表达的稳态水平，而且因为详细的分析只集中在表示基因表达谱所涉及的多个步骤中的一个。这项研究的应用重点是评估基因表达的动态，从前mRNA的产生到核内的RNA加工，再到细胞质中的翻译和mRNA的降解，长期目标是绘制所有表达的mRNAs在细胞转化时的寿命。我们将测试这一假设，即基因表达步骤动力学的变化设定在石头独特的基因表达程序，决定细胞的命运。使用新的代谢标记技术，我们将跟踪细胞核中的前信使核糖核酸动态(目标1)和细胞质中的信使核糖核酸动态(目标2)，以全面了解建立稳态基因表达模式的动力学相互作用。Aim 3将利用这种理解和工具来确定基因表达步骤的动态变化在多大程度上有助于伴随着Src诱导的乳腺上皮细胞转化的基因表达谱的整体变化。所描述的实验将突出转录、RNA加工和信使核糖核酸翻译之间的相互作用对于改变基因表达程序的发展是多么关键。此外，了解建立新的基因表达谱所涉及的动力学可能为人类乳腺癌的新疗法铺平道路。在整个项目中，我们将依靠我们在动力学、RNA生物学和生物信息学方面的专业知识，获得Src诱导的乳腺上皮细胞转化所特有的基因表达的完整动力学图谱，从而产生对基因表达和乳腺癌研究社区有用的丰富数据。乳腺癌特异性基因表达事件的识别可能对开发新的早期检测或预测工具有价值。

 DESCRIPTION (provided by applicant): The regulation of gene expression dictates when, where, and to what degree protein isoforms are generated, thereby meeting the metabolic needs of various cell types that make up organs and living beings. Given the constantly changing microenvironment, this process is highly dynamic, capable of adjusting instantly to multiple external cues, or mounting gene expression responses within extended periods of time. To achieve diverse gene expression responses cells rely on multiple points of regulation that include modulating the origin and rate of transcription, altering pre-mRNA processing to induce the generation of alternatively spliced mRNA isoforms, changing the length or location of the 3' UTR through alternative polyadenylation, selective mRNA isoform translation, and altering the stability of mRNA pools. All of these gene expression steps are integrated and co-dependent. However, our understanding of the dynamic nature of gene expression is limited because most cell-based investigations analyze only steady-state levels of gene expression and because detailed analyses focus only on one of the multiple steps involved in representing a gene expression profile. This research application focuses on evaluating the dynamics of gene expression from pre-mRNA generation through RNA processing in the nucleus to translation and mRNA degradation in the cytoplasm with the long-term goal to chart the life span of all expressed mRNAs upon cellular transformation. We will test the hypothesis that alterations in the kinetics of gene expression steps set in stone unique gene expression programs that dictate cellular fate. Using novel metabolic labeling techniques we will track pre-mRNA dynamics in the nucleus (Aim 1) and mRNA dynamics in the cytoplasm (Aim 2) to develop a comprehensive understanding on the kinetic interplay that establishes steady-state gene expression patterns. Aim 3 will use this understanding and tools to determine to what degree alterations in the dynamics of gene expression steps contribute to the overall change in gene expression profiles that accompany Src-induced breast epithelial cell transformation. The experiments described will highlight how crucial the interplay between transcription, RNA processing and mRNA translation is for the development of altered gene expression programs. Furthermore, understanding the kinetics involved in establishing novel gene expression profiles may pave the way for novel therapies of human breast cancer. Throughout this project we will rely on our expertise in kinetics, RNA biology and bioinformatics to obtain complete kinetic profiles of gene expression unique to Src-induced breast epithelial cell transformation, thus producing a wealth of data useful to the gene expression and breast cancer research communities. The identification of breast cancer-specific gene expression events may be valuable for the development of novel early detection or prediction tools.