It is estimated that a fraction of the eukaryotic long non-coding RNAs are retained in the nucleus (nrRNAs). Several studies already have demonstrated crucial roles played by novel nrRNAs in vital cellular processes and their abnormal expression has been implicated in various diseases including cancer. The long-term goal of my laboratory is to understand how mammalian nrRNAs regulates vital gene regulatory pathways. As a model system, we are focusing on CTN-RNA, a paraspeckle-localized nrRNA that regulates the expression of Cat2, a receptor in the Nitric Oxide synthesis pathway. CTN-RNA is made as a transcript containing the Cat2 mRNA sequence plus an additional, long 3'UTR. The unique 3'UTR of CTN-RNA is required for its nuclear-retention and is modified by adenosine to inosine hyper-editing. Upon treating cells with Interferon-3 and lipopolysaccharide, CTN-RNA is post-transcriptionally cleaved at its 3'UTR to produce a mature Cat2 mRNA that is rapidly exported out of the nucleus and translated. However, the mechanisms those mediate the nuclear retention of nrRNAs like CTN-RNA and stress-induced post-transcriptional cleavage remain to be elucidated. Interestingly, ~300 human genes show similar structural features to CTN-RNA indicating that this may be a common mechanism shared by other genes to allow rapid gene induction. The objective of the present proposal is to determine the mechanism/s that regulate nuclear retention of CTN-RNA and its cleavage upon stress. The central hypothesis, based on preliminary data, is that post-transcriptional modifications and/or sequence elements in the 3'UTR of CTN-RNA and their interaction with specific factors regulate nuclear- retention and stress-induced processing of CTN-RNA. To test this hypothesis, we propose the following specific aims: 1) Identify the mechanism responsible for CTN-RNA nuclear retention;2) Determine the functional significance of the CTN-RNA association with paraspeckle sub-nuclear domains;and 3) Identify the factors responsible for stress-induced post-transcriptional processing and cleavage of CTN-RNA. Our approach is innovative because we will characterize a new gene regulatory mechanism utilized by CTN-RNA, which is likely shared by an entire class of genes. This mechanism may have evolved to accelerate the rapid induction of proteins encoded by long genes upon specific signals, which otherwise would take a long time to transcribe and process. This proposal is innovative in its combination of molecular genetics with state of the art live cell imaging of intranuclear RNA dynamics to address the dynamics of CTN-RNA and CAT2 gene regulation. My expertise in both RNA molecular biology / biochemistry and live cell imaging is quite unique and essential to this project. CTN-RNA regulates Cat2 synthesis, thereby modulates the cellular levels of NO, an important molecule involved in innate immune response. Thus, the proposed research is significant because understanding how CTN-RNA mediated gene regulatory mechanism operates in the cell would enable future pharmacological interventions aimed at modulating such stress and disease related response.
The long-term focus of my laboratory is to understand the roles played by mammalian nuclear-retained RNAs (nrRNAs) in gene regulation. In the present proposal we aim to study the mechanism that allows the nuclear-retention of nrRNA;CTN-RNA and characterize the components that facilitate its stress induced processing. Since CTN-RNA regulates the cellular levels inducible Nitric oxide (NO), part of the bodies innate immune response, understanding CTN-RNA mediated gene regulation would have direct implications for biomedical research.
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