Phosphoinositide Signaling To and Within the Nucleus

描述（由申请人提供）：磷酸肌醇信号转导调节所有真核细胞。在磷酸肌醇循环中，磷脂酰肌醇（PI）在肌醇环的第四和第五个羟基上依次被PI激酶磷酸化，然后被磷脂酰肌醇磷酸激酶（PIPK）磷酸化，形成磷脂酰肌醇-4，5-二磷酸（PIP 2）。PIP 2是许多细胞反应的直接脂质信使，并且是许多其他磷脂酰肌醇衍生的第二信使的必需前体。值得注意的是，磷酸肌醇信号发生在细胞核内，其中PIP 2在称为核斑点的结构处空间生成。斑点没有识别的膜，但含有在mRNA加工中起作用的蛋白质和酶。PIPK通过与它们产生的PIPn的效应物相互作用来发信号。PIPKI 1是一种产生PIP 2的同种型，PIP 2以斑点形式存在于细胞核中。PIP 2也在斑点处产生，并且可以调节mRNA加工酶的活性。在这种情况下，我们发现PIPKI 1与一种新的多聚腺苷酸聚合酶（PAP）相互作用，现在称为Star-PAP。PIP 2显著且特异性地刺激Star-PAP。Star-PAP和PIPKI 1受氧化应激反应调节，这调节应激反应mRNA的表达，包括血红素加氧酶-1（HO-1）、载脂蛋白E（APOE）和NAD（P）H：醌氧化还原酶（NQO 1）。我们假设PIPKI 1和Star-PAP作为多聚腺苷酸化复合物发挥作用，与前体mRNA转录物的3 '-加工所需的转录机制相关。这种新的多聚腺苷酸化复合物独特地受磷酸肌醇信号调节，并且在体内需要用于选择mRNA转录物的3 '-加工，从而产生调节基因表达的新机制。我们将测试这个假设与以下集中的具体目标：1。研究星星- PAP的酶活性及肌醇磷脂对其的调节作用。Star-PAP中调节RNA底物特异性的功能结构域将被揭示。2.表征氧化应激信号下游的Star-PAP复合物组装。将定义Star-PAP中功能结构域的作用。3.将研究Star-PAP在体内调节mRNA的机制，重点是与基因和mRNA的相互作用。将确定Star-PAP产生的poly（A）尾是否与标准PAP不同。应激反应蛋白HO-1、APOE和NQO 1的表达调控发挥着关键的生物学作用，对人类健康的许多方面具有重大意义，包括心血管疾病、移植、神经变性和中风、癌症治疗和肺部医学。PIPKI 1和Star-PAP通过磷酸肌醇信号调节前mRNA多聚腺苷酸化是一个令人难以置信的新发现，对核信号转导和基因表达具有许多意义。公共卫生相关性：来自基因的细胞蛋白质的表达通过由每个基因产生的信使RNA（mRNA）发生，这需要mRNA具有聚腺苷尾。这条尾巴是mRNA制造细胞蛋白质所必需的。我们已经发现了一种新的酶，它独特地制造了这些尾巴，这种酶可以产生mRNA和编码的蛋白质。最有趣和范式转变的事实是，这一过程是由一种称为磷脂酰肌醇-4，5-二磷酸的脂质信使调节的。这种新途径控制表达的基因是血红素加氧酶-1（HO-1）、载脂蛋白E（APOE）和NAD（P）H：醌氧化还原酶（NQO 1）。这些基因的控制对人类健康的许多方面具有重大意义，包括心血管疾病、移植、神经变性、癌症治疗和肺部医学。

DESCRIPTION (provided by applicant): 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.