Sam 68 and the Function of Mason Pfizer Monkey Virus CTE

描述（由申请人提供）：逆转录病毒已成为转录后水平基因调控的重要模型系统。复杂逆转录病毒利用反式作用病毒蛋白作用于病毒基因组中特定的顺式作用元件来实现含内含子rna的表达。相反，更简单的逆转录病毒，如梅森-辉瑞猴病毒（MPMV）利用直接与细胞蛋白相互作用的顺式作用元件（cte）。我们实验室最近的实验表明，细胞蛋白Sam68具有显著增强MPMV CTE功能的能力。Sam68是一种rna结合蛋白，是有丝分裂中Src和其他激酶磷酸化酪氨酸的主要靶点。Sam68也是Sik/Brk的底物，Sik/Brk是Src家族的非肉豆蔻酰基化成员，主要表现为核定位。我们的实验表明，组成活性Sik/Brk的表达以剂量依赖的方式抑制SAM68对CTE功能的增强。我们还证明了sam68相关蛋白（SLM-2/T-STAR）能够增强CTE功能。尽管Sam68的确切功能尚不清楚，但该蛋白似乎在哺乳动物细胞的信号转导和转录后基因调控之间提供了重要的联系。Sam68调节CTE功能和病毒基因表达的事实为我们提供了一个功能分析系统，通过该系统可以进一步分析这一重要环节。本课题的主要目标是进一步分析Sam68促进CTE功能的机制，并利用该系统进一步了解Sam68在细胞代谢中的作用，具体目标为：目的1：确定Sam68增强含CTE RNA的细胞质利用的机制。目的2：分析磷酸化和选择突变如何影响Sam68和SLM-2/T-STAR增强CTE功能和绘制Sam68中特定磷酸化位点的能力。目的3：分析Sam68是直接与含有RNA的CTE结合起作用，还是通过与其他宿主细胞蛋白的相互作用间接起作用。目的4：鉴定和表征Sam68的体内细胞mRNA靶点。

DESCRIPTION (provided by applicant): Retroviruses have come to serve as important model systems for regulation of genes at the post-transcriptional level. Complex retroviruses utilize trans acting viral proteins which act on specific cis-acting elements in the viral genome to achieve expression of intron-containing RNAs. In contrast, simpler retroviruses such as Mason-Pfizer Monkey Virus (MPMV) utilize cis-acting elements (CTEs) that interact directly with cellular proteins. Recent experiments in our laboratory indicate that the cellular protein Sam68 has the capacity to dramatically enhance the function of the MPMV CTE. Sam68 is an RNA-binding protein that is a major target for tyrosine phosphorylation by Src and other kinases in mitosis. Sam68 is also a substrate for Sik/Brk, a non-myristoylated member of the Src family that shows a mainly nuclear localization. Our experiments have shown that expression of constitutively active Sik/Brk inhibits SAM68 enhancement of CTE function in a dose dependent manner. We have also demonstrated that a Sam68-related protein (SLM-2/T-STAR) is able to enhance CTE function. Although the exact functions of Sam68 remain unknown, this protein appears to provide an important link between signal transduction and post-transcriptional gene regulation in mammalian cells. The fact that Sam68 regulates CTE function and viral gene expression provides us with a functional assay system through which this important link can be further analyzed. The major goals of this proposal are to further analyze the mechanism by which Sam68 promotes CTE function and to utilize this system to gain further insight into the role that Sam68 plays in cellular metabolism, The specific aims are: Aim1: To determine the mechanism by which SAM68 functions to enhance cytoplasmic utilization of CTE containing RNA. Aim 2: To analyze how phosphorylation and selected mutations affect the ability of Sam68 and SLM-2/T-STAR to enhance CTE function and to map specific phosphorylation sites in Sam68. Aim 3: To analyze whether Sam68 works directly by binding to CTE containing RNA or indirectly through interactions with other host-cells proteins. Aim 4: To identify and characterize in vivo cellular mRNA targets of Sam68.