Phosphoinositide signaling regulates all eukaryotic cells. In the phosphoinositide cycle, phosphatidylinositol (PI) is sequentially phosphorylated on the fourth and fifth hydroxyl of the myo-inositol ring by PI kinases and then phosphatidylinositol-phosphate kinases (PIPKs), forming phosphatidylinositol-4,5-bisphosphate (PIP2). PIP2 is a direct lipid messenger for many cellular responses and is an essential precursor to many other phosphatidylinositol-derived second messengers. Remarkably phosphoinositide signaling occurs within the nucleus where PIP2 is spatially generated at structures called nuclear speckles. Speckles have no identified membrane, but contain proteins and enzymes with roles in mRNA processing. PIPKs signal by interacting with effectors of the PIPn that they generate. PIPKI1 is an isoform that makes PIP2 that is present in the nucleus at speckles. PIP2 is also generated at speckles and may regulate activities of mRNA processing enzymes. In this context, we have discovered that PIPKI1 interacts with a novel poly A polymerase (PAP) now called Star-PAP. Star-PAP is dramatically and specifically stimulated by PIP2. Star-PAP and PIPKI1 are regulated by oxidative stress response and this regulates the expression of stress response mRNAs, including heme oxygenase-1 (HO-1), apolipoprotein E (APOE) and NAD(P)H:quinone oxidoreductase (NQO1). We hypothesize that PIPKI1 and Star-PAP function as a polyadenylation complex that associates with the transcriptional machinery required for 3'-processing of pre-mRNA transcripts. This novel polyadenylation complex is uniquely regulated by phosphoinositide signals and is required in vivo for 3'- processing of select mRNA transcripts, resulting in a novel mechanism to regulate gene expression. We will test this hypothesis with the following focused specific aims: 1. Study enzymatic activity of Star- PAP and regulation by phosphoinositides. Functional domains in Star-PAP that modulate specificity toward RNA substrates will be revealed. 2. Characterize Star-PAP complex assembly down stream of oxidative stress signals. The role of functional domains in Star-PAP will be defined. 3. The mechanisms for Star-PAP regulation of mRNAs in vivo will be investigated with an emphasis on the interaction with genes and mRNAs. It will be determined if the poly(A) tail generated by Star-PAP different than that by canonical PAP. The regulation of expression of the stress response proteins HO-1, APOE, and NQO1 play key biological roles that have dramatic implications for many aspects of human health including cardiovascular disease, transplantation, neurodegeneration and stroke, cancer therapy, and pulmonary medicine. The regulation of pre-mRNA polyadenylation by phosphoinositide signals via PIPKI1 and Star-PAP is an incredibly novel finding that has many implications for nuclear signal transduction and gene expression. Public Health Relevance: The expression of cellular proteins from genes occurs through messenger RNAs (mRNAs) made by each gene and this requires that the mRNA have a polyadenosine tail. This tail is required before the mRNA can make cellular proteins. We have discovered a new enzyme that uniquely makes these tails and this enzyme works to generate mRNAs and the encoded proteins. Most interesting and paradigm shifting is the fact that this process is regulated by a lipid messenger called phosphatidylinositol-4,5-bisphosphate. The genes whose expression this novel pathway controls are heme oxygenase-1 (HO-1), apolipoprotein E (APOE) and NAD(P)H:quinone oxidoreductase (NQO1). The control of these genes has dramatic implications for many aspects of human health including cardiovascular disease, transplantation, neurodegeneration, cancer therapy, and pulmonary medicine.

Public Health Relevance

The expression of cellular proteins from genes occurs through messenger RNAs (mRNAs) made by each gene and this requires that the mRNA have a polyadenosine tail. This tail is required before the mRNA can make cellular proteins. We have discovered a new enzyme that uniquely makes these tails and this enzyme works to generate mRNAs and the encoded proteins. Most interesting and paradigm shifting is the fact that this process is regulated by a lipid messenger called phosphatidylinositol-4,5-bisphosphate. The genes whose expression this novel pathway controls are heme oxygenase-1 (HO-1), apolipoprotein E (APOE) and NAD(P)H:quinone oxidoreductase (NQO1). The control of these genes has dramatic implications for many aspects of human health including cardiovascular disease, transplantation, neurodegeneration, cancer therapy, and pulmonary medicine.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM051968-14
Application #
8017412
Study Section
Nuclear Dynamics and Transport (NDT)
Program Officer
Gerratana, Barbara
Project Start
1994-12-01
Project End
2012-11-30
Budget Start
2010-12-01
Budget End
2011-11-30
Support Year
14
Fiscal Year
2011
Total Cost
$356,766
Indirect Cost
Name
University of Wisconsin Madison
Department
Pharmacology
Type
Schools of Medicine
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Li, Weimin; Li, Wencheng; Laishram, Rakesh S et al. (2017) Distinct regulation of alternative polyadenylation and gene expression by nuclear poly(A) polymerases. Nucleic Acids Res 45:8930-8942
Choi, Suyong; Anderson, Richard A (2016) IQGAP1 is a phosphoinositide effector and kinase scaffold. Adv Biol Regul 60:29-35
Thapa, Narendra; Tan, Xiaojun; Choi, Suyong et al. (2016) The Hidden Conundrum of Phosphoinositide Signaling in Cancer. Trends Cancer 2:378-390
Choi, Suyong; Hedman, Andrew C; Sayedyahossein, Samar et al. (2016) Agonist-stimulated phosphatidylinositol-3,4,5-trisphosphate generation by scaffolded phosphoinositide kinases. Nat Cell Biol 18:1324-1335
Tan, Xiaojun; Lambert, Paul F; Rapraeger, Alan C et al. (2016) Stress-Induced EGFR Trafficking: Mechanisms, Functions, and Therapeutic Implications. Trends Cell Biol 26:352-366
Choi, Suyong; Thapa, Narendra; Tan, Xiaojun et al. (2015) PIP kinases define PI4,5P?signaling specificity by association with effectors. Biochim Biophys Acta 1851:711-23
Mohan, Nimmy; Sudheesh, A P; Francis, Nimmy et al. (2015) Phosphorylation regulates the Star-PAP-PIPKI? interaction and directs specificity toward mRNA targets. Nucleic Acids Res 43:7005-20
Li, W; Anderson, R A (2014) Star-PAP controls HPV E6 regulation of p53 and sensitizes cells to VP-16. Oncogene 33:928-32
Li, Weimin; Laishram, Rakesh S; Anderson, Richard A (2013) The novel poly(A) polymerase Star-PAP is a signal-regulated switch at the 3'-end of mRNAs. Adv Biol Regul 53:64-76
Sun, Yue; Thapa, Narendra; Hedman, Andrew C et al. (2013) Phosphatidylinositol 4,5-bisphosphate: targeted production and signaling. Bioessays 35:513-22

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