Phosphoinositide Signaling To and Within the Nucleus

描述（由申请人提供）：磷酸肌醇信号传导调节所有真核细胞。在磷酸肌醇循环中，磷脂酰肌醇（PI）在肌醇环的第四个和第五个羟基上依次被PI激酶和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（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，磷酸肌醇信号传导发生在细胞核内，其中 PIP2 在称为核斑点的结构中空间生成。斑点没有确定的膜，但含有在 mRNA 加工中发挥作用的蛋白质和酶。 PIPK 通过与它们生成的 PIPn 效应器相互作用来发出信号。 PIPKI1 是产生 PIP2 的同种型，PIP2 存在于细胞核中的斑点处。 PIP2 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 PIPKI1 与一种新型多聚 A 聚合酶 (PAP) 相互作用，现在称为 Star-PAP。 Star-PAP 受到 PIP2 显着且特异性的刺激。 Star-PAP 和 PIPKI1 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 (NQO1)。我们假设 PIPKI1 和 Star-PAP 作为聚腺苷酸化复合物发挥作用，与前 mRNA 转录本的 3' 加工所需的转录机制相关。这种新型聚腺苷酸化复合物独特地受磷酸肌醇信号调节，并且是体内特定 mRNA 转录物 3'- 加工所需的，从而产生调节基因表达的新机制。我们将通过以下重点具体目标来检验这一假设： 1. 研究 Star-PAP 的酶活性和磷酸肌醇的调节。 Star-PAP 中调节 RNA 底物特异性的功能域将被揭示。 2. 表征氧化应激信号下游的 Star-PAP 复合体组装。 Star-PAP 中功能域的作用将被定义。 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 生成的 Poly(A) 尾是否与规范 PAP 生成的不同。应激反应蛋白 HO-1、APOE 和 NQO1 的表达调节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影响，包括心血管疾病、移植、神经退行性和中风、癌症治疗和肺医学。磷酸肌醇信号通过 PIPKI1 和 Star-PAP 调节前体 mRNA 多腺苷酸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。公共健康相关性：基因中细胞蛋白的表达是通过每个基因产生的信使 RNA (mRNA) 进行的，这要求 mRNA 具有多聚腺苷尾部。在 mRNA 制造细胞蛋白质之前，需要这条尾巴。我们发现了一种新的酶，可以独特地制造这些尾巴，并且这种酶可以生成 mRNA 和编码的蛋白质。最有趣和范式转变的事实是，该过程受到称为磷脂酰肌醇-4,5-二磷酸的脂质信使的调节。该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 (NQO1)。这些基因的控制对人类健康的许多方面具有重大影响，包括心血管疾病、移植、神经退行性变、癌症治疗和肺医学。

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.