Assessing androgen sensitive RNA splicing patterns in LNCaP cells.

Our previous studies of proteomic-coupled-network analysis of AR protein interaction complexes (Paliouras et al., Integrative Biology, 2011) identified a number of proteins involved in RNA metabolism, specifically alternative RNA splicing. We selected two interacting RNA splicing proteins, SAM68 and DDX5 to examine RNA splicing events in prostate cancer (PCa). This analysis suggests a much more robust effect on RNA splicing with AR dictating either an exon-inclusion or -exclusion pathway. To establish the true physiological roles of AR in alternative RNA splicing, we opted to further examine the changes in global splicing profiles of LNCaP PCa cells, stimulated with and without androgens in conjunction with overexpression studies of SAM68 and DDX5. LNCaP cells transfected with SAM68 or DDX5 expression contructs were stimulated with 10nM Mibolerone (a synthetic non-metabolizable androgen) and RNA collected after 2hrs, 6hrs or 18hrs stimulation. Controls include untransfected and uinstimulated cells. All experiments were performed in triplicate (n=3).