Structural investigation of the STAR protein Sam68

Sam68 is a member of the STAR family of proteins, linking post-transcriptional gene regulation with signal transduction pathways. Sam68 has been shown to specifically regulate the alternative splicing of many genes through interactions with the pre-mRNA and spliceosomal machinery. Selection of particular isoforms of several of these genes has been shown to contribute to neoplastic transformation and aberrant Sam68 expression and function has been implicated in the development of various genetic diseases and cancers. It is therefore important to understand Sam68 RNA recognition at the molecular level in order to design next generation drug therapies. This thesis describes the structural and biophysical techniques used to define the bipartite RNA consensus sequence specifically recognised by Sam68 and the mechanisms of interaction. This data provides a model of Sam68 contribution to alternative splicing regulation. Splice site selection is also influenced by posttranslational modifications of Sam68 including serine and threonine phosphorylation. In several cases, the phosphorylation state of Sam68 directly influences the outcome of splicing and leads to cancer development. Identification of phosphorylation sites in the STAR domain of Sam68 by NMR and radiolabelled kinase assays reveals how this post-translational modification may affect RNA binding at the molecular level.