Excellence In Research: Spatial and temporal mechanisms of gene expression regulation

这个项目研究了参与神经细胞发育的基因的表达是如何被一种叫做TRIM2的蛋白质控制的。为了使脑细胞正常发育，神经元中的发育基因表达在神经元生长过程中受到严格调控。神经元调节基因表达的一种方式是通过像TRIM2这样的rna结合蛋白，这是最近才发现的。该项目的实验将利用基于诱导多能干细胞的新技术，揭示TRIM2调节基因表达的确切机制。这些技术允许在细胞培养皿中跟踪特定类型的未成熟神经元的发育，并促进操纵实验，这些实验将阐明TRIM2用于调节基因表达的详细分子机制，以及这些对神经功能的影响。通过解决这些“如何”的问题，对神经细胞和大脑发育方式的科学理解，以及确定更好地控制这些过程以纠正异常发育或疾病的影响的潜在目标将得到进一步发展。这项研究还将通过为本科生和研究生提供项目，为科学领域的少数民族学生提供招聘和培训机会。神经元具有复杂的结构-功能关系，这是通过大脑发育过程中基因表达的时空调控来实现的。PI先前的工作表明，TRIM2，一种e3连接酶，也可能作为RNA结合蛋白调节RNA的表达和降解。我们将详细描述TRIM2与神经元RNA结合的机制，包括分析TRIM2靶向的关键RNA结构，以及通过TRIM2与Argonaut2蛋白的相互作用调节RNA降解（使用免疫沉淀方法表征）。TRIM2与RNA的结合将使用RIP-seq方法进行测量，数据将使用生物信息学方法进行分析。将在神经元发育过程中测量rna与TRIM2的结合，并确定TRIM2耗尽后基因表达的变化。这些研究将同时测量神经元不同发育时期的RNA水平和TRIM2与RNA的结合。最后，使用显微成像和电生理记录的蛋白质测量将用于记录TRIM2对神经元功能的影响。这项研究的结果将是对TRIM2蛋白如何调节神经元功能和发育的全面理解。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

This project investigates how the expression of genes that are involved in nerve cell development are controlled by a protein called TRIM2. To enable the brain cells to develop properly, developmental gene expression in neurons is tightly regulated as neurons grow. One way that neurons regulate which genes get expressed is via RNA-binding proteins like TRIM2, which has been discovered only recently. The experiments in this project will reveal the exact mechanism(s) that TRIM2 uses to regulate gene expression, taking advantage of novel technologies based on induced pluripotent stem cells. These techniques allow the development of specific types of immature neurons to be followed in cell culture dishes, and facilitate manipulative experiments that will elucidate both the detailed molecular mechanisms that TRIM2 uses to regulate gene expression, and what consequences these have for neural function. By addressing these “how” questions, the scientific understanding of the ways in which nerve cells and brains develop, as well as identifying potential targets for better controlling these processes to remediate the effects abnormal development or disease will be furthered. This research will also offer recruitment and training opportunities for minority students in the sciences, by providing projects for both undergraduate and graduate students. Neurons have a complex structure-function relationship, achieved through a tight spatial and temporal regulation of gene expression during brain development. Previous work by the PI suggested that TRIM2, an E3ligase, may also function as an RNA binding protein that regulates RNA expression and degradation. The mechanism(s) that TRIM2 uses to bind to neuronal RNA will be characterized in detail, including an analysis of the key RNA structures targeted by TRIM2, and the regulation of RNA degradation via the interaction between TRIM2 with Argonaut2 protein (characterized using an immunoprecipitation approach). TRIM2 binding to RNA will be measured using RIP-seq methodology, and data will be analyzed using bioinformatics approaches. The binding of RNAs to TRIM2 will be measured during neuronal development, and changes to gene expression following TRIM2 depletion will be determined. These studies will simultaneously measure RNA levels and TRIM2 binding to RNA at different developmental periods in neurons. Finally, protein measurements using microscopic imaging and electrophysiological recordings will be used to document the effects of TRIM2 on neuronal function. The result of this study will be a comprehensive understanding of how TRIM2 proteins regulate neuronal function and development.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.