This project focuses on the gene expression profile of a single nucleus to identify pre-mRNA transcript signatures in vivo. Pre-mRNA is a precursor of cytosolic translation-ready mRNA in the cytosol, which is translated into protein, a fundamental biological element. The existence of pre-mRNA in the nucleus has long been known and recent progress in single cell biology raises the importance of profiling it for comprehensive research in genetic information flow from DNA to protein. However, due to the lack of proper technology, investigation of nucleic pre-mRNA profile in in vivo has not been possible. Although much different pathology have been linked to genetic information flow, the correlation between nucleic and cytosolic mRNA has never been studied. One of the main reasons is a lack of methods to capture the total pre-mRNA in a single nucleus in vivo. We suggest that pre-mRNA content reflects a cell's natural microenvironment in tissue. In this proposal, we will first perform pre-mRNA profile analysis of single nuclei of different cell types from multiple brain locations in order to build a database to correlate pre-mRNA signatures with their resultant cellular phenotype in vivo. A cell-type specific genetically modified mouse model will be used to identify specific cell types and used to snap capture the pre-mRNA within a single nuclei of each cell type. High resolution microscopy and optical manipulation of a novel mRNA capturing compound (TIVA-Nuc) will be employed to capture the pre-mRNA transcript profile with and without physiological stimulation. The main goal of this project is to establish the heterogeneous nature of pre-mRNA profiles based on their cell types and anatomical location in brain as well as their functional connections, such as interacting with other neurons in tissue. These results will generate a novel database for assessing the role of pre-mRNA, which will greatly facilitate understanding their specific roles in regulating and maintaining brain cellular function.

Public Health Relevance

This research focuses on analysis of single nuclei to identify the pre-mRNA contents in vivo. The success of this project will generate an in vivo single nucleus pre-mRNA database, which will open new avenues not only for single cell research but also for clinical interventions and pharmaceutical drug development for brain diseases.

National Institute of Health (NIH)
National Institute of Mental Health (NIMH)
Exploratory/Developmental Grants Phase II (R33)
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Special Emphasis Panel (ZRG1-BST-A (50))
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Beckel-Mitchener, Andrea C
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University of Pennsylvania
Schools of Medicine
United States
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Spaethling, Jennifer M; Na, Young-Ji; Lee, Jaehee et al. (2017) Primary Cell Culture of Live Neurosurgically Resected Aged Adult Human Brain Cells and Single Cell Transcriptomics. Cell Rep 18:791-803
Morris, Jacqueline; Na, Young-Ji; Zhu, Hua et al. (2017) Pervasive within-Mitochondrion Single-Nucleotide Variant Heteroplasmy as Revealed by Single-Mitochondrion Sequencing. Cell Rep 21:2706-2713