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

项目摘要 这项计划的目标是揭示前体mRNA剪接和核斑点是如何被控制的， 激酶TAO 2，并确定为什么TAO 2是成功的流感病毒（IAV）复制所必需的。 最近的工作已经确定了未充分研究的蛋白激酶TAO 2作为剪接和 IAV M RNA的斑点定位。TAO 2池定位于核斑点及其损失， 化学抑制或蛋白质缺失，改变IAV M的核斑点组成、剪接和输出 RNA，因此削弱IAV复制。TAO 2的抑制也破坏了宿主细胞的一个亚群的剪接。 mRNA，而不改变大量宿主mRNA。这些数据揭示了TAO 2的新细胞活性，并确定了 抑制TAO 2作为控制IAV感染的潜在途径。初步数据表明，TAO 2 与几种剪接因子和其他RNA结合蛋白相互作用。本提案中概述的工作 旨在全面了解TAO 2在人类细胞中的核活性和功能 这是宿主和病毒RNA表达的这些活性的结果。具体而言，三管齐下的办法 将采取：（1）表征功能TAO 2相互作用伴侣和磷酸化底物 并确定IAV感染是否改变了这些相互作用，或者TAO 2是否相互作用， 直接与任何IAV编码的蛋白质;（2）确定TAO 2在维持核完整性中的作用 散斑和（3）表征TAO 2对人类剪接机制的全球影响， 这是选择性剪接功能的结果。这些目标将通过以下方式实现： 细胞遗传操作、生物化学、质谱和显微镜。重要的是 从这些研究中得到的结论将对RNA剪接的一般机制有意义 和核斑点的形成和功能，并将进一步告知宿主要求的理解 和流感感染的脆弱性。

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.