Our lab utilizes in vivo and in vitro models of stroke to study lncRNA activity. We employ a variety of methodologies such as immunoprecipitation, high-throughput sequencing, microarrays, reporter assays, gain- or loss-of-function strategies and bioinformatics to assess lncRNA expression and functions; and histological approaches to assess tissue structure. The broad interests of our lab are as follows:
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.
LncRNAs and Post-Transcriptional Regulation of Gene Expression
LncRNAs localized to the cytoplasm participate in post-transcriptional processing of mRNAs via multiple mechanisms. One mechanism of particular interest to us is the functional interaction between cytoplasmic lncRNAs and miRNAs wherein miRNAs are bound by lncRNAs and sequestered away from the free miRNA pool in a process known as competing endogenous activity (or sponging). This leads to reduced availability of miRNAs to target mRNAs resulting in translational de-repression of the mRNAs and increased protein expression. We study lncRNA-miRNA interactions in vivo and in vitro to identify ischemia-responsive competing endogenous lncRNAs and understand how they contribute to post-ischemic gene expression alterations and cellular outcomes. We utilize a combined approach to estimate differential mRNA translation rates, protein expression changes, and pathways analyses to identify novel post-transcriptional regulatory networks underlying critical post-ischemic neural cell fate decisions.
Biochemistry Underlying LncRNA--Protein Interactions
Despite their low evolutionary sequence conservation, lncRNAs can bind highly conserved regulatory proteins. The molecular correlates driving these interactions are not clearly known. In general, RNAs contain sequence motifs or secondary structures that are important for protein binding. Whether such motifs or structures are commonly present in lncRNAs is not clear. If present, what are their defining features, and do specific signatures promote the binding of specific proteins? Using techniques such as crosslinking immunoprecipitation, point mutation studies and biochemical analyses, we seek to map the binding sites of ischemia-relevant proteins on ischemia-responsive lncRNAs and characterize these interactions to identify the underlying factors that enable protein binding. This work will improve our understanding of the fundamental features guiding lncRNA-protein functional interactions and provide valuable information for designing drugs, interference technologies and other therapeutic applications.