We use in vivo and in vitro models of stroke and apply a variety of methodologies such as in situ hybridization, immunoprecipitation, immunochemistry, high-throughput sequencing, microarrays, reporter assays, gain- or loss-of-function strategies, imaging, and bioinformatics to study RNA biology and function in brain damage.
LncRNAs and Regulation of Transcription
LncRNAs directly interact with regulatory proteins such as chromatin modifiers and transcription factors at genomic sites to alter the local transcriptional landscape. This influences gene expression and phenotypic outcomes. The key lncRNA-protein complexes that are differentially active in the normal versus post-stroke cortex are not yet known. What are their genomic targets and what specific actions do they perform at these sites? How do these activities influence the post-stroke pathophysiology? Our lab is currently addressing these questions. Using data from this work, we seek to map the transcriptomic networks that are modulated by specific stroke-relevant lncRNA-protein modules in response to the ischemic injury.
The roles of enhancer RNAs in regulating post-stroke gene expression and brain damage
Enhancers undergo activity-dependent transcription to produce noncoding enhancer RNAs (eRNAs). Some of these eRNAs have been shown to play central roles in organizing functional interactions between the enhancers and their downstream gene targets and influence cellular outcomes. We recently identified several novel stroke-responsive eRNAs in the post-stroke cerebral cortex. Loss-of-function experiments resulted in pronounced molecular and phenotypic outcomes, which suggest important roles for the eRNAs in the post-stroke brain. Our current work in this area is focused on identifying the molecular targets of these eRNAs, the cellular and physiological processes that they influence, and their sex-based expression and functional characteristics using in vitro and in vivo models of stroke.
Discovery of novel protein isoforms in the post-stroke brain
During gene expression, one of the most important processes is alternative splicing of the pre-RNA to produce multiple, distinct mRNAs. Alternative splicing enables the cell to generate a diverse complement of transcripts from a limited number of genes to facilitate diversification of function, and such molecular and functional diversity is bound to have implications for development and disease. In mammals, alternative splicing is most prevalent in the brain. Recently, we identified a number of novel alternatively spliced mRNA isoforms that were significantly altered in the post-stroke cortex as compared to controls. Sequence analysis suggested that they have the potential to yield novel proteins. Altered spatiotemporal expression of such novel proteins may have important implications for the pathophysiological outcomes in stroke. Our current work is focused on investigating the putative translational products of the novel mRNAs, evaluating their cellular and subcellular distribution in the mouse brain during stroke, identifying their functions and functional partners, and determining their expression patterns as a function of age and sex. Our overarching goal is to obtain a clearer understanding of the post-stroke pathophysiology by elucidating the significance of the alternatively spliced transcriptome in stroke.