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

流感病毒感染期间核斑点功能和剪接的激酶调节

• 批准号: 10491841
• 负责人: MELANIE H. COBB
• 金额: $ 56.88万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-25 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10491841
• 关键词: Alternative Splicing; Biochemical; Biochemistry; Biological Assay; Cell Nucleus; Cells; Chemicals; Complement; Data; Economic Burden; Event; Gene Expression; Genes; Genetic Transcription; Goals; Grant; Human; Immune response; Impairment; Infection; Influenza Virus Infected Cells; Integration Host Factors; Label; Link; Maintenance; Mass Spectrum Analysis; Mediating; Messenger RNA; Methods; Microscopy; Morphology; Nuclear; Nuclear Proteins; Nuclear RNA; Phosphorylation; Phosphotransferases; Positioning Attribute; Process; Protein Kinase; Proteins; RNA; RNA Processing; RNA Splicing; RNA-Binding Proteins; Regulation; Role; Shapes; Transcript; Viral; Viral Genes; Viral Proteins; Virus; Virus Diseases; Virus Replication; Western Blotting; Work; experience; genetic manipulation; health economics; high resolution imaging; influenza infection; influenzavirus; insight; knock-down; mRNA Precursor; therapeutic target; trafficking; transcriptome sequencing; viral RNA

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.

项目摘要 该提案的目标是揭示如何控制mRNA剪接和核斑点 激酶TAO2，并确定为什么成功的流感病毒（IAV）复制需要TAO2。 最近的工作已经确定了研究研究的蛋白激酶TAO2是剪接和 IAV M RNA的斑点定位。 TAO2池本地化为核斑点及其损失， 化学抑制或蛋白质消耗，改变核斑点组成，剪接和IAV M的出口 RNA，因此会损害IAV复制。 TAO2的抑制也破坏了宿主子集的剪接 mRNA，没有更改散装宿主mRNA。这些数据发现了TAO2的新细胞活性，并确定 抑制TAO2作为控制IAV感染的潜在方法。初步数据表明TAO2 与几个剪接因子和其他RNA结合蛋白相互作用。该提案中概述的工作 寻求对TAO2在人类细胞和功能的核活动的全面理解 这些活性的宿主和病毒RNA表达的结果。具体而言，一种三管齐下的方法 将被带到：（1）表征功能性TAO2相互作用伙伴和磷酸化的底物 在核蛋白质中，确定IAV感染是否改变了这些相互作用或TAO2是否相互作用 直接使用任何IAV编码的蛋白质； （2）定义TAO2在维持核的完整性中的作用 Speckles和（3）表征TAO2对人剪接机械和 此功能用于替代剪接的结果。这些目标将通过组合实现 细胞，生物化学，质谱和显微镜的基因操纵。重要的是， 从这些研究获得的结论将对RNA剪接的一般机制产生影响 以及核斑点的形成和功能，并将进一步告知对寄主需求的理解 和流感感染的脆弱性。