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

我实验室的研究项目集中在了解不同的RNA结合蛋白（RBP）如何调节细胞应激反应。在过去的15年里，我们把注意力集中在这样一个蛋白质，hnRNP A1，它在RNA代谢中起着关键作用，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。我们已经表明，在响应不同的细胞应激，如紫外线照射，渗透压休克或病毒感染，hnRNP A1积累在受影响的细胞的细胞质中，并调节mRNA的稳定性或选择性翻译的mRNA编码的关键蛋白，控制细胞死亡（如cIAP 1，XIAP，Bcl-xL和Apaf-1）。此外，hnRNP A1的细胞质积累是肠道病毒（如脊髓灰质炎病毒、肠道病毒和鼻病毒，所有重要的人类病原体）生命周期进展中的关键步骤。然而令人惊讶的是，控制hnRNPA 1细胞质积累的信号通路的身份和调节仍然知之甚少。 siRNA技术和自动化高通量成像技术的进步允许对复杂的生物网络进行系统的询问。我们已经应用这些技术来研究亚细胞定位的hnRNP A1的渗透压模型，并确定了几个新的激酶，调节hnRNP A1定位在细胞中。我们额外的初步数据进一步表明，这些激酶之一，ARK 5，相互作用和磷酸化hnRNP A1，导致细胞质积累的hnRNP A1在渗透胁迫。这个发现Ggrant将表征新的hnRNP A1激酶，首先通过探索ARK 5如何参与hnRNP A1的磷酸化和随后的亚细胞定位，以及这种调节对细胞存活的广泛生物学意义。具体而言，我们将： 1.表征ARK 5与hnRNP A1的相互作用。 2.鉴定和表征ARK 5介导的hnRNP A1磷酸化位点，并评估其在渗透胁迫期间的功能相关性。 3.定义ARK 5介导的磷酸化如何控制hnRNP A1定位，并评估调节此信号通路对细胞存活和病毒感染的生物学后果。 通过确定ARK 5对hnRNP A1的影响以及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存活重要的新的调节过程。虽然我们最初的重点是ARK 5，在这项研究中开发的策略将被应用到表征其他hnRNP A1激酶在我们的屏幕上。此外，这些激酶可能有其他RNA结合蛋白作为靶点，这些靶点对细胞应激反应可能很重要。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顺式调控元件交叉，以在不同的生理条件下选择性地调控基因表达。

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.