DESCRIPTION (provided by applicant): The 3'-end processing and polyadenylation of mRNAs is critical for gene expression. We discovered a non-canonical poly(A) polymerase Star-PAP (for speckle targeted PIPKI regulated-poly(A) polymerase) that is activated by the lipid messenger phosphatidylinositol-4,5-bisphosphate (PIP2). Star-PAP is regulated by cell signaling and controls gene expression by uniquely using specific cleavage and polyadenylation sites (pAs) on genes/pre-mRNAs. The 3'UTR contains sequences that are critical for controlling mRNA localization and translation. Further, over 70% of human genes undergo alternative polyadenylation (APA) and changes in APA correlate with stem cell development and cancer progression. We discovered that each of the nuclear PAPs has unique specificity for distinct APA and polyadenylation (pA) sites genome wide indicating a greater level of 3'-end processing regulation then had been previously appreciated. This fact indicates a high level of 3'-end control of gene expression by cell signaling. We hypothesize that Star-PAP and PAP/ have specificity toward pAs genome wide though sequence elements around the pA. Star-PAP is regulated by signals that incorporate co-activator kinases and RNA binding proteins into its 3'processing complex. Star-PAP co-activators such as RBM10, an RNA binding protein, determine specificity of pA site selection by RNA recognition. Star-PAP activity is controlled by PIP2 and Star-PAP is a PIP2 carrier protein where bound PIP⇄PIP2⇄PIP3 is cycled by kinases and phosphatases to regulate Star-PAP activity. Star-PAP addition of Us to the 3'-tail modulates mRNA expression. The following aims will test this hypothesis: Aim 1. PAP specificity toward pAs will be defined. pA sites controlled by PAPs will be defined by 3'READS and by crosslinking followed by RNA immunoprecipitation and deep sequencing. Cis elements will be identified by bioinformatics and validated using reporter assays. The role of Star-PAP addition of both A and U to 3'-tails will be studied and the consequences defined. Signals that control APA and 3'tail changes will be revealed. Aim 2. Define Star-PAP 3'UTR processing regulation by signals and co-activator proteins. Mechanisms for Star-PAP control of 3'processing will be revealed by defining co-activators, such as PI and protein kinases and the RNA binding protein RBM10, that determine specificity. The role of RBM10 in pA selection will be assessed. We will explore how phosphorylation regulates Star-PAP complex composition and target specificity. Aim 3. Spatial and phosphoinositide regulation of Star-PAP 3'-end processing. Star-PAP has properties of a PIP2 carrier protein and we will study PIP2 interactions with Star-PAP and determine if bound PIPn is modulated by PIPKs, PLC or PI3Ks. We will study Star-PAP spatial 3'processing of HO-1, NQO1 and PTEN to delineate where cleavage and polyadenylation occur and explore implications of spatial mRNA processing.

PUBLIC HEALTH RELEVANCE: The 3'-end processing of pre-mRNA is essential for gene expression. About 70% of human genes contain alternative 3'-end cleavage and polyadenylation sites. The usage of these alternative sites is poorly defined but is implicated in development and human diseases including cancer. We have discovered a poly (A) polymerase named Star-PAP that controls the alternative polyadenylation and expression of the PTEN tumor suppressor, AKT1/3 and MDM2 oncogenes, and the NQO1 antioxidant gene and others. The products of these genes all play pivotal roles in cancer, cardiovascular and neuronal diseases. Understanding how these genes are regulated will be key for defining novel therapeutics to target diseases that are induced by aberrant expression of these genes. We will further develop a genome wide map of PAP regulation of APA sites, which would provide a rapid genome wide expression profile with clear diagnostic and therapeutic applications.