Sam 68 and the Function of Mason Pfizer Monkey Virus CTE

描述（由申请人提供）：逆转录病毒已成为在转录后水平调控基因的重要模型系统。复杂的逆转录病毒利用反式作用病毒蛋白，其作用于病毒基因组中的特定顺式作用元件以实现含内含子的RNA的表达。相比之下，更简单的逆转录病毒，如梅森-辉瑞猴病毒（MPMV），利用顺式作用元件（CTE）直接与细胞蛋白质相互作用。我们实验室最近的实验表明，细胞蛋白Sam 68具有显著增强MPMV CTE功能的能力。Sam 68是一种RNA结合蛋白，是有丝分裂中Src和其他激酶酪氨酸磷酸化的主要靶点。Sam 68也是Sik/Brk的底物，Sik/Brk是Src家族的非豆蔻酰化成员，其显示主要核定位。我们的实验已经表明，组成型活性Sik/Brk的表达以剂量依赖性方式抑制SAM 68对CTE功能的增强。我们还证明了Sam 68相关蛋白（SLM-2/T-STAR）能够增强CTE功能。虽然Sam 68的确切功能仍然未知，但该蛋白似乎在哺乳动物细胞中的信号转导和转录后基因调控之间提供了重要的联系。Sam 68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测定系统，通过该系统可以进一步分析这一重要联系。本研究的主要目的是进一步分析SAM 68促进CTE功能的机制，并利用该系统进一步了解SAM 68在细胞代谢中的作用。具体目标是：目的1：确定SAM 68增强细胞质对含有CTE的RNA利用的机制。目标二：分析磷酸化和选定的突变如何影响Sam 68和SLM-2/T-STAR增强CTE功能的能力，并定位Sam 68中的特异性磷酸化位点。目的3：分析Sam 68是通过与含有CTE的RNA结合直接起作用还是通过与其他宿主细胞蛋白相互作用间接起作用。目的4：鉴定和表征Sam 68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.