Phosphoinositide Signaling To and Within the Nucleus

描述(申请人提供)：肌醇磷脂信号调节所有真核细胞。在磷脂酰肌醇循环中，磷脂酰肌醇(PI)被磷脂酰肌醇(PI)激酶依次磷酸化到肌醇环的第四和第五个羟基上，然后是磷脂酰肌醇-磷酸酶(PIPKs)，形成磷脂酰肌醇-4，5-二磷酸(PIP2)。PIP2是许多细胞反应的直接脂质信使，也是许多其他磷脂酰肌醇衍生的第二信使的重要前体。值得注意的是，磷脂酰肌醇信号发生在细胞核内，PIP2是在称为核斑点的结构中空间产生的。斑点没有可识别的膜，但含有与mRNA加工有关的蛋白质和酶。PIPK通过与它们产生的PIPN的效应器相互作用来发出信号。PIPKI1是一种异构体，它使PIP2以斑点形式存在于细胞核中。PIP2也在斑点产生，可能调节mRNA处理酶的活性。在此背景下，我们发现PIPKI1与一种新的聚A聚合酶(PAP)相互作用，现在被称为Star-PAP。STAR-PAP被PIP2戏剧性地和特异性地刺激。StAR-PAP和PIPKI1受氧化应激反应调控，调节应激反应mRNAs的表达，包括血红素加氧酶-1(HO-1)、载脂蛋白E(APOE)和NAD(P)H：苯醌氧化还原酶(NQO1)。我们假设PIPKI1和Star-PAP作为一个多聚腺苷化复合体发挥作用，它与3‘-加工前mRNA转录物所需的转录机制有关。这种新型的多聚腺苷化复合体受磷脂酰肌醇信号的独特调节，在体内需要对选定的mRNA转录本进行3‘-加工，从而产生了一种新的调控基因表达的机制。我们将从以下几个方面对这一假说进行验证：1.研究Star-PAP的酶活性及磷脂酰肌醇的调节作用。Star-PAP中调节RNA底物特异性的功能结构域将被揭示。2.鉴定氧化应激信号下游的Star-PAP复合体组装。将定义Star-PAP中功能域的角色。3.研究Star-PAP对体内mRNAs的调控机制，重点是与基因和mRNAs的相互作用。将确定Star-PAP产生的Poly(A)尾部是否不同于经典PAP产生的Poly(A)尾部。应激反应蛋白HO-1、APOE和NQO1的表达调控对人类健康的许多方面具有重要的生物学意义，包括心血管疾病、移植、神经退行性变和中风、癌症治疗和肺部医学。磷脂酰肌醇信号通过PIPKI1和Star-PAP调节Pre-mRNA的多腺苷基化是一项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。与公共卫生相关：来自基因的细胞蛋白的表达通过每个基因制造的信使RNA(MRNAs)来实现，这就要求信使RNA有一个多聚腺苷的尾巴。在信使核糖核酸能够制造细胞蛋白质之前，这条尾巴是必需的。我们已经发现了一种新的酶，它可以独特地制造这些尾巴，这种酶可以产生mRNA和编码的蛋白质。最有趣和范式转变的是，这个过程是由一种名为磷脂酰肌醇-4，5-二磷酸的脂质信使调节的。这个新途径调控基因的表达是血红素加氧酶-1(HO-1)、载脂蛋白E(APOE)和NAD(P)H：苯醌氧化还原酶(NQO1)。这些基因的控制对人类健康的许多方面都有重大影响，包括心血管疾病、移植、神经退行性疾病、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.