Gene expression in eukaryotes is one of the most complex, highly orchestrated processes in living organisms. Numerous studies have shown that steps such as transcription, messenger RNA (mRNA) processing and mRNA decay, are highly coupled, forming an intricate molecular circuit for regulation of protein-coding genes. Central to this process, are a class of RNA helicases called DEAD-box proteins that perform essential roles in all aspects of RNA biology. Dbp2 is a largely uncharacterized member of the DEAD-box RNA helicase family in the budding yeast Saccharomyces cerevisiae. Whereas numerous studies have shown that the human (h) ortholog of Dbp2, hDDX5 or p68, functions as a transcriptional regulator, no transcriptional role has been described for Dbp2 and the precise molecular function of hDDX5 in this process is not understood. Our studies now provide the first demonstration that Dbp2 is required for nuclear gene expression steps in budding yeast, functioning at the interface between mRNA biogenesis and chromatin remodeling. Furthermore, our work demonstrates that Dbp2 functions at sites of non-protein coding RNA synthesis by RNA polymerase II. Herein, we propose to obtain a detailed understanding of Dbp2 using a combination of biochemical, molecular and genetic approaches.
In Aim I, we will utilize a series of in vitro assays to biochemically characterize Dbp2 and subsequently analyze the enzymatic requirements for normal cell growth and gene expression.
In Aim II, we will determine how Dbp2 functions in gene expression through a combination of molecular and genetic approaches.
In Aim III, we will define and characterize the in vivo molecular interactions that enable Dbp2 to 'sense'nascent transcripts. Our long term working model is that Dbp2 is an enzymatic 'toggle'in the gene expression circuit that regulates the transcriptome. Thus, these studies have the potential to reveal novel mechanisms for genome-wide epigenetic regulation, an NIH strategic initiative reflective of the current challenges facing biomedical research. Importantly, numerous DEAD-box protein genes have been linked to human disease states including cancer, neurological disorders and AIDS. Therefore, uncovering the physiological role of individual DEAD-box proteins is a major challenge to basic biological research and the medical community.
The ability to control the expression of specific genes is essential to cellular growth, adaptation and survival. Dbp2/hDDX5 is an RNA helicase enzyme whose over-expression promotes tumor formation and resistance to chemotherapeutics. Knowledge regarding the function of hDDX5 in gene expression is key to defining the underlying molecular basis for cancer and other human diseases.
|Cloutier, Sara C; Wang, Siwen; Ma, Wai Kit et al. (2016) Regulated Formation of lncRNA-DNA Hybrids Enables Faster Transcriptional Induction and Environmental Adaptation. Mol Cell 61:393-404|
|Ma, Wai Kit; Paudel, Bishnu P; Xing, Zheng et al. (2016) Recruitment, Duplex Unwinding and Protein-Mediated Inhibition of the Dead-Box RNA Helicase Dbp2 at Actively Transcribed Chromatin. J Mol Biol 428:1091-106|
|Zhang, Hao; Xing, Zheng; Mani, Saravana Kumar Kailasam et al. (2016) RNA helicase DEAD box protein 5 regulates Polycomb repressive complex 2/Hox transcript antisense intergenic RNA function in hepatitis B virus infection and hepatocarcinogenesis. Hepatology 64:1033-48|
|Ma, Wai Kit; Tran, Elizabeth J (2015) Measuring helicase inhibition of the DEAD-box protein Dbp2 by Yra1. Methods Mol Biol 1259:183-97|
|Tran, Elizabeth J; King, Megan C; Corbett, Anita H (2014) Macromolecular transport between the nucleus and the cytoplasm: Advances in mechanism and emerging links to disease. Biochim Biophys Acta 1843:2784-2795|
|Beck, Zachary T; Cloutier, Sara C; Schipma, Matthew J et al. (2014) Regulation of glucose-dependent gene expression by the RNA helicase Dbp2 in Saccharomyces cerevisiae. Genetics 198:1001-14|
|Cloutier, Sara C; Wang, Siwen; Ma, Wai Kit et al. (2013) Long noncoding RNAs promote transcriptional poising of inducible genes. PLoS Biol 11:e1001715|
|Ma, Wai Kit; Cloutier, Sara C; Tran, Elizabeth J (2013) The DEAD-box protein Dbp2 functions with the RNA-binding protein Yra1 to promote mRNP assembly. J Mol Biol 425:3824-38|
|Cloutier, Sara C; Ma, Wai Kit; Nguyen, Luyen T et al. (2012) The DEAD-box RNA helicase Dbp2 connects RNA quality control with repression of aberrant transcription. J Biol Chem 287:26155-66|