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

该项目研究了参与神经细胞发育的基因的表达如何由一种名为TRIM 2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调控。神经元调节基因表达的一种方式是通过RNA结合蛋白，如TRIM 2，这是最近才发现的。 该项目中的实验将揭示TRIM 2用于调节基因表达的确切机制，利用基于诱导多能干细胞的新技术。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TRIM 2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，将进一步科学地理解神经细胞和大脑发育的方式，并确定更好地控制这些过程的潜在目标，以补救异常发育或疾病的影响。这项研究还将通过为本科生和研究生提供项目，为科学领域的少数族裔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，这是通过在大脑发育期间对基因表达进行严格的空间和时间调节来实现的。 PI先前的工作表明，TRIM 2，一种E3连接酶，也可能作为一种RNA结合蛋白，调节RNA表达和降解。 TRIM 2用于结合神经元RNA的机制将被详细表征，包括分析TRIM 2靶向的关键RNA结构，以及通过TRIM 2与Argonaut 2蛋白之间的相互作用调节RNA降解（使用免疫沉淀方法表征）。TRIM 2与RNA的结合将使用RIP-seq方法测量，数据将使用生物信息学方法分析。将在神经元发育期间测量RNA与TRIM 2的结合，并将确定TRIM 2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TRIM 2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TRIM 2对神经元功能的影响。 这项研究的结果将是TRIM 2蛋白如何调节神经元功能和发育的全面理解。这个奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

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.