Nuclear Phosphoinositide Control of 3'-end mRNA Processing and Gene Expression

核磷酸肌醇控制 3 端 mRNA 加工和基因表达

• 批准号: 9027153
• 负责人: Richard A. Anderson
• 金额: $ 37.99万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-12-25 至 2019-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9027153
• 关键词: 1-Phosphatidylinositol 3-Kinase; 1-Phosphatidylinositol 4-Kinase; 3' Untranslated Regions; AKT1 gene; Address; Antioxidants; Binding; Binding Proteins; Bioinformatics; Biological Assay; Cardiovascular system; Carrier Proteins; Cell Nucleus; Cells; Cleaved cell; Complex; Development; Diagnostic; Diglycerides; Disease; Elements; Enzymes; Essential Genes; Eukaryota; Gene Expression; Gene Expression Process; Genes; Histones; Human; Immunoprecipitation; Inositol; Lipids; MDM2 gene; Malignant Neoplasms; Maps; Membrane; Messenger RNA; Molecular Profiling; NQO1 gene; Names; Neurons; Nuclear; Oncogenes; PI3 gene; PIP5K2A gene; PTEN gene; Pathway interactions; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phospholipase C; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiological; Play; Poly A; Polyadenylation; Polyadenylation Pathway; Polynucleotide Adenylyltransferase; Process; Property; Protein Kinase; Protein Kinase C; Proteins; RNA; RNA Binding; RNA Sequences; RNA-Binding Proteins; Recombinants; Regulation; Regulator Genes; Reporter; Role; Signal Pathway; Signal Transduction; Site; Specificity; Stem Cell Development; Tail; Testing; Therapeutic; Translations; Tumor Suppressor Proteins; crosslink; deep sequencing; gene product; genome-wide; human disease; interest; mRNA Expression; mRNA Precursor; novel therapeutics; nucleotidyltransferase; protein expression; public health relevance; reconstitution; tumor progression

 DESCRIPTION (provided by applicant): The 3'-end processing and polyadenylation of mRNAs is critical for gene expression. We discovered a non-canonical poly(A) polymerase Star-PAP (for speckle targeted PIPKI regulated-poly(A) polymerase) that is activated by the lipid messenger phosphatidylinositol-4,5-bisphosphate (PIP2). Star-PAP is regulated by cell signaling and controls gene expression by uniquely using specific cleavage and polyadenylation sites (pAs) on genes/pre-mRNAs. The 3'UTR contains sequences that are critical for controlling mRNA localization and translation. Further, over 70% of human genes undergo alternative polyadenylation (APA) and changes in APA correlate with stem cell development and cancer progression. We discovered that each of the nuclear PAPs has unique specificity for distinct APA and polyadenylation (pA) sites genome wide indicating a greater level of 3'-end processing regulation then had been previously appreciated. This fact indicates a high level of 3'-end control of gene expression by cell signaling. We hypothesize that Star-PAP and PAP/ have specificity toward pAs genome wide though sequence elements around the pA. Star-PAP is regulated by signals that incorporate co-activator kinases and RNA binding proteins into its 3'processing complex. Star-PAP co-activators such as RBM10, an RNA binding protein, determine specificity of pA site selection by RNA recognition. Star-PAP activity is controlled by PIP2 and Star-PAP is a PIP2 carrier protein where bound PIP⇄PIP2⇄PIP3 is cycled by kinases and phosphatases to regulate Star-PAP activity. Star-PAP addition of Us to the 3'-tail modulates mRNA expression. The following aims will test this hypothesis: Aim 1. PAP specificity toward pAs will be defined. pA sites controlled by PAPs will be defined by 3'READS and by crosslinking followed by RNA immunoprecipitation and deep sequencing. Cis elements will be identified by bioinformatics and validated using reporter assays. The role of Star-PAP addition of both A and U to 3'-tails will be studied and the consequences defined. Signals that control APA and 3'tail changes will be revealed. Aim 2. Define Star-PAP 3'UTR processing regulation by signals and co-activator proteins. Mechanisms for Star-PAP control of 3'processing will be revealed by defining co-activators, such as PI and protein kinases and the RNA binding protein RBM10, that determine specificity. The role of RBM10 in pA selection will be assessed. We will explore how phosphorylation regulates Star-PAP complex composition and target specificity. Aim 3. Spatial and phosphoinositide regulation of Star-PAP 3'-end processing. Star-PAP has properties of a PIP2 carrier protein and we will study PIP2 interactions with Star-PAP and determine if bound PIPn is modulated by PIPKs, PLC or PI3Ks. We will study Star-PAP spatial 3'processing of HO-1, NQO1 and PTEN to delineate where cleavage and polyadenylation occur and explore implications of spatial mRNA processing.

 描述（由申请人提供）：mRNA 的 3' 端加工和聚腺苷酸化对于基因表达至关重要。 ）由脂质信使磷脂酰肌醇-4,5-二磷酸 (PIP2) 激活，并受细胞信号传导和控制基因调节。通过独特地使用基因/前体 mRNA 上的特定切割和聚腺苷酸化位点 (PA) 来表达。 3'UTR 包含对控制 mRNA 定位和翻译至关重要的序列。此外，超过 70% 的人类基因经历选择性聚腺苷酸化 (APA) 和我们发现每个核 PAP 对全基因组范围内的不同 APA 和聚腺苷酸化 (pA) 位点具有独特的特异性，表明 APA 的变化水平较高。 3'端加工调控此前已被认识到，这一事实表明细胞信号传导对基因表达的高水平控制，我们发现Star-PAP和PAP/对pAs基因组范围内的序列具有特异性。 Star-PAP 周围的元件受将共激活激酶和 RNA 结合蛋白纳入其 3' 加工复合物（例如 RBM10，一种 RNA 结合蛋白）的信号调节。通过 RNA 识别进行 pA 位点选择的特异性。Star-PAP 活性由 PIP2 控制，Star-PAP 是一种 PIP2 载体蛋白，其中结合的 PIP⇄PIP2⇄PIP3 通过激酶和磷酸酶循环来调节 Star-PAP 活性。我们的 3' 尾调节 mRNA 表达的目的如下： 目标 1. 将定义 PAP 对 pA 位点的特异性。由 PAP 控制的序列将通过 3'READS 进行定义，然后通过 RNA 免疫沉淀和深度测序来确定顺式元件，并通过生物信息学进行鉴定，并使用报告分析来验证 Star-PAP 添加到 3'- 的作用。将研究尾部并定义控制 APA 和 3' 尾部变化的信号。 目标 2. 通过信号和 定义 Star-PAP 3'UTR 处理调节。 Star-PAP 控制 3' 加工的机制将通过定义共激活剂（例如 PI 和蛋白激酶以及 RNA 结合蛋白 RBM10）来揭示，RBM10 在 pA 选择中的作用将是。我们将探讨磷酸化如何调节 Star-PAP 复合物的组成和靶标特异性。 目标 3. Star-PAP 3' 端的空间和磷酸肌醇调节。 Star-PAP 具有 PIP2 载体蛋白的特性，我们将研究 PIP2 与 Star-PAP 的相互作用，并确定结合的 PIPn 是否受到 PIPK、PLC 或 PI3K 的调节。我们将研究 HO-1 的 Star-PAP 空间 3' 处理。 、NQO1 和 PTEN 来描绘切割和聚腺苷酸化发生的位置并探索空间 mRNA 加工的影响。