This proposal focuses on the mechanism underlying the regulation of specific mRNAs controlled by a novel nuclear poly(A) polymerase (PAP) and a nuclear phosphatidylinositol phosphate kinase (PIPK). At this time there is no direct evidence in the literature that nuclear phosphoinositide signals directly modulate the 3'-polyadenylation of messenger RNA (mRNA). Recently, a link between nuclear PIPKIalpha and the regulation of 3'-processing of mRNAs by a novel PAP was established. Our lab has identified a unique nuclear PAP that is targeted to nuclear speckles called Star-PAP, for nuclear speckle targeted PIPKIalpha regulated poly (A) polymerase, which interacts with nuclear PIPKIalpha in vivo.
The aim of this proposal is to provide new mechanistic information about the function of Star-PAP and PIPKIalpha as a signaling complex, which regulates the polyadenylation of specific mRNAs. The central hypothesis of this study proposes a model for Star-PAP and PIPKIalpha in 3' processing of specific mRNAs. The hypothesis to be tested is that Star-PAP and PIPKIalpha function together in a polyadenylation complex that regulates 3'-modification of specific mRNAs. This polyadenylation complex is uniquely regulated by PIP2 and polyadenylates selected mRNA transcripts, resulting in a novel mechanism to regulate the metabolism of a subset of mRNA. The following two focused aims will guide this investigation: 1.) To elucidate the underlying mechanism by which Star-PAP controls specific mRNA levels and 2.) To determine how PIPKIalpha modulates Star-PAP, thereby regulating the function of Star-PAP. These studies will employ a combination of methodologies to dissect and explore the mechanism regulating the polyadenylation of specific mRNAs by Star-PAP. These methods include multiple small interfering RNA (siRNA) and mutant constructs for Star-PAP and PIPKIalpha, polyadenylation assays, in vitro transcription assays, and chromatin immunoprecipitation assays (ChIP). Previous studies showed that both Star-PAP and PIPKIalpha are required for the expression of the stress response mRNA levels of heme oxygenase 1 (HO-1) and NAD(P)H: quinone oxidoreductase (NQO1). Regulation of stress response mRNAs, such as HO-1 and NQ01, has dramatic implications for numerous aspects of human health. The proposed experiments have the potential to reveal a very novel mechanism for gene regulation. Thus, understanding the additional layers of regulation will be critical for developing therapeutic approaches for controlling these gene and their products. ? ? ?
|Laishram, Rakesh S; Barlow, Christy A; Anderson, Richard A (2011) CKI isoforms ? and ? regulate Star-PAP target messages by controlling Star-PAP poly(A) polymerase activity and phosphoinositide stimulation. Nucleic Acids Res 39:7961-73|
|Barlow, Christy A; Laishram, Rakesh S; Anderson, Richard A (2010) Nuclear phosphoinositides: a signaling enigma wrapped in a compartmental conundrum. Trends Cell Biol 20:25-35|