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

我实验室的研究项目专注于了解不同的RNA结合蛋白(RBP)如何调节细胞应激反应。在过去的15年里，我们将注意力集中在这样一种蛋白质上，即hnRNP A1，它在RNA代谢中发挥关键作用，包括端粒修复、mRNA剪接、mRNA输出、应激颗粒形成和选择性mRNA翻译。我们已经证明，为了应对不同的细胞应激，如紫外线照射、渗透性休克或病毒感染，hnRNP A1积聚在受影响细胞的细胞质中，并调节mRNA的稳定性或编码控制细胞死亡的关键蛋白(如cIAP1、XIAP、BclxL和APAF-1)的mRNAs的选择性翻译。此外，hnRNP A1的细胞质积累是肠道病毒生命周期发展的关键步骤，如脊髓灰质炎病毒、肠道病毒和鼻病毒，这些都是人类的重要病原体。然而，令人惊讶的是，控制hnRNP A1细胞质积累的信号通路的识别和调控仍然知之甚少。SiRNA技术和自动化高通量成像技术的进步使系统地询问复杂的生物网络成为可能。我们应用这些技术研究了hnRNP A1在渗透压模型中的亚细胞定位，并鉴定了几种新的调节hnRNP A1在细胞中定位的激酶。我们另外的初步数据进一步表明，其中一种激酶ARK5与hnRNP A1相互作用并磷酸化，从而在渗透胁迫期间导致hnRNP A1的细胞质积累。这篇发现报告将描述新的hnRNP A1激酶的特征，首先通过探索ARK5如何参与hnRNP A1的磷酸化和随后的亚细胞定位，以及这一调控对细胞生存的广泛生物学意义。具体地说，我们将：1.表征ARK5与hnRNP A1的相互作用。2.鉴定和鉴定ARK5介导的hnRNP A1上的磷酸化位点，并评估它们在渗透胁迫中的功能相关性。3.确定ARK5介导的磷酸化如何控制hnRNP A1的定位，并评估调节这一信号通路对细胞存活和病毒感染的生物学后果。通过确定ARK5对hnRNP A1的影响和随后对细胞应激反应的调节，我们的工作将有助于发现对细胞应激反应和生存重要的新的调控过程。虽然我们最初的重点是ARK5，但在这项研究中开发的策略将应用于我们屏幕中确定的其他hnRNP A1激酶的特征。此外，这些激酶可能还有其他RNA结合蛋白作为靶标，这些蛋白可能对细胞应激反应起重要作用。我们的长期计划是利用我们的资源和专业知识来研究细胞信号通路如何与离散的RNA顺式调控元件相交，以选择性地调控不同生理条件下的基因表达。

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.