Poster abstracts
Poster number 11 submitted by Zachary Beck
Using high-throughput microscopy coupled with machine learning to decipher regulatory pathways for the RNA helicase Dbp2
Zachary T. Beck (Department of Biochemistry, Purdue University), Ben Grys (Department of Molecular Genetics, University of Toronto), Brenda Andrews (Department of Molecular Genetics, University of Toronto), Elizabeth J. Tran (Department of Biochemistry, Purdue University)
Abstract:
The ability of an organism to sense the current status of available nutrients is key to survival and fecundity. Responses to changes in the environment require highly specific and coordinated alterations in gene expression. Dbp2 is a bona fide ATP-dependent RNA helicase in Saccharomyces cerevisiae that associates directly with actively transcribed chromatin to promote co-transcriptional packaging and processing of mRNAs and ribosomal RNAs.1,2 Under normal growth conditions when carbon sources are readily available, Dbp2 is concentrated in the nucleus. However, when carbon sources are limited, such as during glucose deprivation, Dbp2 is rapidly relocalized to cytoplasm.3 Utilizing high-throughput confocal microscopy (HTC) of a synthetic gene array (SGA), we were able to track the localization of Dbp2 in response to +/- glucose conditions in over 3000 mutant yeast strains. Images were analyzed with CellProfiler to quantitatively determine the localization of Dbp2 in relation to reference markers in the cytoplasm and nucleus. Known genetic and physical interaction data of genes identified as hits from the screen were obtained from the Saccharomyces Genome Database. This list was then compiled into interaction pathways using Cytoscape to construct a visual representation of the glucose responsive network for Dbp2. Interestingly, our analyses show three general cellular processes associated with glucose-dependent Dbp2 localization: kinase signaling, sugar metabolism, and ribosome assembly. We are in the process of independently validating the results and constructing a model of nutrition-based environmental sensing. The glucose-dependent redistribution of Dbp2 represents the first time a DEAD-box helicase has been tied directly to sensing extracellular carbon source cues. Importantly, the human counterpart of Dbp2, termed hDDX5, has recently been linked to normal glucose metabolism, a process that is drastically altered in cancer cells. Thus, our studies, have direct implications to our understanding of DDX5 and mechanisms which promote tumor cell metabolism.
References:
1. Cloutier SC, Ma WK, Nguyen LT, Tran EJ. The DEAD-box RNA helicase Dbp2 connects RNA quality control with repression of aberrant transcription. J Biol Chem 2012; 287:26155–66.
2. Ma WK, Cloutier SC, Tran EJ. The DEAD-box protein Dbp2 functions with the RNA-binding protein Yra1 to promote mRNP assembly. J Mol Biol 2013; 425:3824–38.
3. Beck ZT, Cloutier SC, Schipma MJ, Petell CJ, Ma WK, Tran EJ. Regulation of glucose-dependent gene expression by the RNA helicase Dbp2 in Saccharomyces cerevisiae. Genetics 2014; 198:1001–14.
Keywords: Dbp2, helicase, metabolism