RNA helicases are a class of enzymes that modulate RNA structure in living cells, functioning in every aspect of RNA biology from transcription to decay. However, the precise biological function of the vast majority of the ~40 eukaryotic RNA helicases is largely unknown. In the previous funding cycle, we utilized a powerful multidisciplinary approach to provide biological and biochemical characterization of the RNA helicase Dbp2 in S. cerevisiae. We specifically focused on this enzyme because the human ortholog of Dbp2, DDX5, is functionally enigmatic oncogene. These studies revealed a novel role for an RNA helicase in cellular metabolism and elucidated timely insights into lncRNA and R-loop biology. However, key questions remain regarding the precise function of Dbp2/hDDX5 and, by extension, the functions of all of the ~40 DEAD-box helicases. These questions are: 1. What are the RNA structures that Dbp2 targets? 2. How does Dbp2 sense the nutritional status of the cell? 3. How does a single RNA helicase regulate a specific gene expression network? Our central hypothesis is that Dbp2 modulates the secondary structure of specific RNA Pol II transcripts to promote termination and repression of R-loop formation in response to nutritional availability. The objective of this application is to determine how Dbp2 couples nutritional status to metabolic gene regulation as a direct extension of our prior insights. We propose to test this hypothesis with three, focused Specific Aims, which integrate newly established and innovative strategies with proven experimental techniques.
In Aim 1, we will define the precise RNA substrates for enzymatic rearrangement by Dbp2.
In Aim 2, we will determine how subcellular localization of Dbp2 is regulated in response to nutritional status.
In Aim 3, we will determine the mechanism for metabolic gene regulation by Dbp2 and lncRNAs. This research is relevant to multiple aspects of RNA biology, gene regulation, and cancer.
RNA helicases are a class of enzymes that modulate RNA structure in living cells, functioning in every aspect of RNA biology from RNA splicing to translation. Dbp2/hDDX5 is an RNA helicase that functions in gene regulation and whose misregulation results in cancer, resistance to chemotherapies, and metabolic disorders. Knowledge regarding the biochemical and biological function of this enzyme is central to human disease intervention.
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