Localization of RNA binding proteins in the control of cell survival.

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The research program in my laboratory is focused on understanding how distinct RNA binding proteins (RBP) regulate cellular stress response. Over the past 15 years we focused our attention on one such protein, hnRNP A1, which plays critical roles in RNA metabolism, including telomere repair, mRNA splicing, mRNA export, stress granule formation and selective mRNA translation. We have shown that in response to diverse cellular stresses, such as ultraviolet irradiation, osmotic shock or viral infection, hnRNP A1 accumulates in the cytoplasm of affected cells and regulates either mRNA stability or selective translation of mRNAs encoding key proteins that control cell death (e.g. cIAP1, XIAP, Bcl-xL and Apaf-1). In addition, cytoplasmic accumulation of hnRNP A1 is a critical step in the life cycle progression of Enteroviruses , such as poliovirus, enterovirus, and rhinovirus, all important human pathogens. Yet surprisingly, the identity and regulation of signaling pathways that control cytoplasmic accumulation of hnRNP A1 remain poorly understood. Advances in siRNA technologies and automated high throughput imaging technologies allowed for systematic interrogations of complex biological networks. We have applied these technologies to study subcellular localization of hnRNP A1 in the osmotic stress model and identified several novel kinases that modulate hnRNP A1 localization in cells. Our additional preliminary data further showed that one of these kinases, ARK5, interacts with and phosphorylates hnRNP A1 which causes cytoplasmic accumulation of hnRNP A1 during osmotic stress. This Discovery Ggrant will characterize novel hnRNP A1 kinases, first by exploring how ARK5 is involved in the phosphorylation and consequent subcellular localization of hnRNP A1, and what are the broad biological implication of this regulation on cell survival. Specifically, we will: 1.Characterize ARK5 interaction with hnRNP A1. 2.Identify and characterize the ARK5-mediated phosphorylation sites on hnRNP A1 and assess their functional relevance during osmotic stress. 3.Define how ARK5-mediated phosphorylation controls hnRNP A1 localization and assess the biological consequences of modulating this signaling pathway on cell survival and viral infection. By determining the impact of ARK5 on hnRNP A1 and consequent modulation of cellular stress response our work will help discover novel regulatory processes important for cellular stress response and survival. Although our initial focus is on ARK5, the strategies developed during this research will be applied to characterize other hnRNP A1 kinases identified in our screen. Furthermore, these kinases may have other RNA binding proteins as targets that could be important for cellular stress response. Our long term plan is to use our resources and expertise to study how cellular signaling pathways intersect with discrete RNA cis regulatory elements to selectively regulate gene expression under distinct physiological conditions.