PROJECT SUMMARY/ABSTRACT Prostate cancer is the most frequently diagnosed male cancer and second leading cause of male cancer deaths. A key biological property of prostate cancer cells is that their growth is dependent on a transcription factor called the androgen receptor (AR). The AR is activated by androgen, the male sex hormone. Accordingly, an effective treatment for patients with advanced prostate cancer is androgen deprivation therapy, which blocks the effects of androgens, inhibits the AR, and halts the growth of prostate cancer cells. The limitation of androgen deprivation therapy is that prostate cancer cells eventually develop resistance through mechanisms that allow AR to become reactivated. This lethal stage is referred to as castration-resistant prostate cancer (CRPC). In this proposal, we have identified aberrant mRNA polyadenylation as a regulatory mechanism that promotes AR re-activation in CRPC. Splicing of pre-RNA is a biological process regulated by the core spliceosome and splicing factors. Alternative mRNA splicing is a mechanism underlying proteomic diversification, enabling several hundreds of thousands of different protein products to be synthesized from the approximately 20,000 genes encoded in the human genome. mRNA splicing is critical for normal development and tissue homeostasis, and is known to be altered in pathologies including cancer. One key decision point in mRNA splicing is recognition of the last exon, which must be cleaved at the 3’ end before addition of the poly(A) tail. This process, termed cleavage and polyadenylation, is directed by binding of the consensus AAUAAA poly(A) site in the last exon by a polypeptide complex called the cleavage and polyadenylation specificity factor (CPSF). Our preliminary data demonstrates that expression of core components of the CPSF complex display altered expression in prostate cancer, which is associated with aggressive disease features including metastasis. We have uncovered a new prostate cancer regulatory mechanism whereby the CPSF complex, mediates utilization of an alternative AAUAAA poly(A) site in intron 3 of the AR gene, which coordinates upstream splicing events that drive expression of multiple constitutively active AR variant proteins in CRPC cells. The hypothesis of this study is that aberrant AR mRNA polyadenylation via de-regulated CPSF action promotes expression of multiple AR variants that collectively promote CRPC and resistance to AR- targeted therapies. To test this hypothesis, we will 1) study the expression and activity of CPSF complex components in clinical prostate cancer; 2) elucidate the mechanisms by which the CPSF complex binds AR pre-RNA and regulates expression of constitutively active AR variants, and 3) test efficacy of nucleic acid- based therapeutics we have developed that block CPSF interaction with the AR gene locus and inhibit expression of AR variant proteins. Overall, this work is expected to advance and link the broad fields of mRNA...