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

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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.