Star-PAP controls oncogene expression through primary miRNA 3'-end formation to regulate cellular proliferation and tumour formation.

Star-PAP is a non-canonical poly(A) polymerase that is down regulated in breast cancer. While Star-PAP down regulation impairs target mRNA polyadenylation, paradoxically, we see up regulation of a large number of oncogenes on Star-PAP knockdown. Using two breast cancer cells (MCF7 with high Star-PAP, and MDA-MB-231 with negligible Star-PAP level), we discover that Star-PAP negatively regulates oncogene expression and subsequently cellular proliferation. This regulation is compromised with Star-PAP mutant of 3'-end processing function (serine 6 to alanine, S6A phospho-mutation). Concomitantly, xenograft mice model using MDA-MB-231 cells reveals a reduction in the tumour formation on ectopic Star-PAP expression that is ameliorated by S6A mutation. We find that Star-PAP control of target oncogene expression is independent of Star-PAP-mediated alternative polyadenylation or target mRNA 3'-end formation. We demonstrate that Star-PAP regulates target oncogenes through cellular miRNAs (miR-421, miR-335, miR-424, miR-543, miR-205, miR-34a, and miR-26a) that are down regulated in breast cancer. Analysis of various steps in miRNA biogenesis pathway reveals that Star-PAP regulates 3'-end formation and synthesis of primary miRNA (host) transcripts that is dependent on S6 phosphorylation thus controlling mature miRNA generation. Using mimics and inhibitors of two target miRNAs (miR-421 and miR-424) after Star-PAP depletion in MCF7 or ectopic expression in MDA-MB-231 cells, we demonstrate that Star-PAP controls oncogene expression and cellular proliferation through targeting miRNAs that regulates tumour formation. Our study establishes a novel mechanism of oncogene expression independent of alternative polyadenylation through Star-PAP-mediated miRNA host transcript polyadenylation that regulates breast cancer progression.

Star-PAP controlled alternative polyadenylation coupled poly(A) tail length regulates protein expression in hypertrophic heart.

Alternative polyadenylation (APA)-mediated 3'-untranslated region (UTR) shortening is known to increase protein expression due to the loss of miRNA regulatory sites. Yet, mRNAs with longer 3'-UTR also show enhanced protein expression. Here, we identify a mechanism by which longer transcripts generated by the distal-most APA site leads to increased protein expression compared to the shorter transcripts and the longer transcripts are positioned to regulate heart failure (HF). A Star-PAP target gene, NQO1 has three poly(A) sites (PA-sites) at the terminal exon on the pre-mRNA. Star-PAP selects the distal-most site that results in the expression of the longest isoform. We show that the NQO1 distal-specific mRNA isoform accounts for the majority of cellular NQO1 protein. Star-PAP control of the distal-specific isoform is stimulated by oxidative stress and the toxin dioxin. The longest NQO1 transcript has increased poly(A) tail (PA-tail) length that accounts for the difference in translation potentials of the three NQO1 isoforms. This mechanism is involved in the regulation of cardiac hypertrophy (CH), an antecedent condition to HF where NQO1 downregulation stems from the loss of the distal-specific transcript. The loss of NQO1 during hypertrophy was rescued by ectopic expression of the distal- but not the proximal- or middle-specific NQO1 mRNA isoforms in the presence of Star-PAP expression, and reverses molecular events of hypertrophy in cardiomyocytes.

Phosphorylation regulates the Star-PAP-PIPKIα interaction and directs specificity toward mRNA targets.

Star-PAP is a nuclear non-canonical poly(A) polymerase (PAP) that shows specificity toward mRNA targets. Star-PAP activity is stimulated by lipid messenger phosphatidyl inositol 4,5 bisphoshate (PI4,5P2) and is regulated by the associated Type I phosphatidylinositol-4-phosphate 5-kinase that synthesizes PI4,5P2 as well as protein kinases. These associated kinases act as coactivators of Star-PAP that regulates its activity and specificity toward mRNAs, yet the mechanism of control of these interactions are not defined. We identified a phosphorylated residue (serine 6, S6) on Star-PAP in the zinc finger region, the domain required for PIPKIα interaction. We show that S6 is phosphorylated by CKIα within the nucleus which is required for Star-PAP nuclear retention and interaction with PIPKIα. Unlike the CKIα mediated phosphorylation at the catalytic domain, Star-PAP S6 phosphorylation is insensitive to oxidative stress suggesting a signal mediated regulation of CKIα activity. S6 phosphorylation together with coactivator PIPKIα controlled select subset of Star-PAP target messages by regulating Star-PAP-mRNA association. Our results establish a novel role for phosphorylation in determining Star-PAP target mRNA specificity and regulation of 3'-end processing.