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

这个项目调查了参与神经细胞发育的基因的表达如何由一种名为TRIM2的蛋白质控制。为了使脑细胞能够正常发育，神经元中的发育基因表达随着神经元的生长而受到严格的调控。神经元调节哪些基因表达的一种方式是通过RNA结合蛋白，如最近才发现的TRIM2。该项目中的实验将揭示TRIM2利用基于诱导多能干细胞的新技术来调节基因表达的确切机制(S)。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，并促进操纵性实验，这些实验将阐明TRIM2用来调节基因表达的详细分子机制，以及这些机制对神经功能的影响。通过解决这些“如何”的问题，将进一步促进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补救异常发育或疾病的影响的潜在目标。这项研究还将通过为本科生和研究生提供项目，为少数民族理科学生提供招生和培训机会。神经元具有复杂的结构-功能关系，通过在大脑发育过程中对基因表达进行严格的空间和时间调节而实现。PI以前的工作表明，TRIM2，一种E3连接酶，也可能作为一种RNA结合蛋白，调节RNA的表达和降解。将详细描述TRIM2与神经元核糖核酸结合的机制(S)，包括对TRIM2靶向的关键核糖核酸结构的分析，以及通过TRIM2与ArgAert2蛋白的相互作用调节核糖核酸降解(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中不同发育时期的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被用来证明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了解。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.