Retroviruses have come to serve as an important model system for regulation of genes at the post-transcriptional level. Complex retroviruses such as HIV utilize viral proteins (e.g. Rev), which act on specific cis-acting elements to achieve expression of intron-containing RNAs. In contrast, simpler retroviruses such as MPMV utilize cis-acting elements (CTEs) that interact directly with cellular proteins. Results from 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 in mitosis. Recently, Sam68 was also identified as a substrate for Sik/BRK, a non-myristoylated member of the Src family. This kinase is mainly localized to the nucleus and was reported to co-localize with Sam68 in nuclear bodies. Although the exact functions of Sam68 are unknown, this protein appears to provide an important link between signal transduction and gene regulation. CTE mediated expression provides a tool by which this link can be further analyzed. Preliminary results show that over expression of """"""""constitutively"""""""" active Sik inhibits Sam68-mediated activation of CTE function, suggesting that phosphorylation of Sam68 regulates this activity.
The aim of this proposal is to further analyze the mechanism by which Sam68 promotes CTE function.
The specific aims are: 1) To determine the level at which Sam68 enhances CTE function, 2) To study how phosphorylation of Sam68 affects the ability of this protein to enhance CTE function, 3) To analyze if Sam68 has the capacity to shuttle actively between the nucleus and cytoplasm in mammalian cells and 4) To determine if Sam68 binds directly to RNA containing the CTE.
