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.
