Kinase Regulation of Nuclear Speckle Function and Splicing during Influenza Virus Infection

项目总结 该提案的目标是揭示前信使核糖核酸剪接和核斑点是如何由 并确定成功复制流感病毒(IAV)需要TAO2的原因。 最近的工作发现，未被研究的蛋白激酶TAO2是一种对剪接和 IAV M RNA的斑点定位。TAO2池定位于核斑点及其损失，由 化学抑制或蛋白质耗尽，改变核斑点组成、剪接和IAV M的输出 RNA，因此会损害IAV的复制。抑制TAO2也会扰乱宿主子集的剪接 MRNAs，不改变主体宿主的mRNAs。这些数据揭示了TAO2的一种新的细胞活动，并确定了 抑制TAO2可作为控制IAV感染的潜在方法。初步数据显示，TAO2 与多种剪接因子和其他RNA结合蛋白相互作用。这项提案中概述的工作 寻求全面了解TAO2在人类细胞中的核活动和功能 这些活动对宿主和病毒RNA表达的影响。具体地说，三管齐下 将采取：(1)表征功能TAO2相互作用伙伴和底物的磷酸化 并确定IAV感染是否改变了这些相互作用或TAO2是否相互作用 直接与任何IAV编码的蛋白质结合；(2)确定TAO2在维持核完整性中的作用 SPECKLES和(3)表征了TAO2对人类剪接机械和 此功能对备用剪接的影响。这些目标将通过联合起来实现 细胞的遗传操作、生物化学、质谱学和显微镜。重要的是， 这些研究得出的结论将对RNA剪接的一般机制产生影响 和核斑点的形成和作用，将进一步促进对宿主需求的理解 以及易受流感感染的风险。

PROJECT SUMMARY The goals of this proposal are to uncover how pre-mRNA splicing and nuclear speckles are controlled by the kinase TAO2, and to determine why TAO2 is required for successful Influenza virus (IAV) replication. Recent work has identified the understudied protein kinase TAO2 as a host factor essential for splicing and speckle localization of the IAV M RNA. A pool of TAO2 localizes to nuclear speckles and its loss, by chemical inhibition or protein depletion, alters nuclear speckle composition, splicing and export of IAV M RNA, and therefore impairs IAV replication. Inhibition of TAO2 also disrupts splicing of a subset of host mRNAs, without altering bulk host mRNA. These data uncover a new cellular activity for TAO2 and identify inhibition of TAO2 as a potential approach for controlling IAV infection. Preliminary data suggest that TAO2 interacts with several splicing factors and other RNA binding proteins. The work outlined in this proposal seeks a comprehensive understanding of the nuclear activities of TAO2 in human cells and the functional consequence of these activities for host and viral RNA expression. Specifically, a three-pronged approach will be taken to: (1) characterize functional TAO2 interaction partners and substrates of phosphorylation amongst nuclear proteins and determine if IAV infection alters these interactions or if TAO2 interacts directly with any IAV-encoded proteins; (2) define the role of TAO2 in maintaining the integrity of nuclear speckles and (3) characterize the global impact of TAO2 on the human splicing machinery and the consequence of this function for alternative splicing. These goals will be achieved through a combination of genetic manipulation of cells, biochemistry, mass spectrometry and microscopy. Importantly, the conclusions obtained from these studies will have implications for general mechanisms of RNA splicing and nuclear speckle formation and function and will further inform the understanding of host requirements and vulnerabilities for influenza infection.