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: 9199104
• 负责人: Richard A. Anderson
• 金额: $ 36.67万
• 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/9199104
• 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; Genes; Genetic Transcription; Histones; Human; Immunoprecipitation; Inositol; Lipids; MDM2 gene; Malignant Neoplasms; Maps; Membrane; Messenger RNA; Molecular Profiling; NQO1 gene; Names; Neurons; Nuclear; Oncogenes; PI3 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; mRNA Stability; 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（由适用提供）：mRNA的3'-End加工和多腺苷酸化对于基因表达至关重要。我们发现了一个非典型的聚（A）聚合酶Star-PAP（用于靶向靶向的PIPKI调节的聚合酶（A）聚合酶），该蛋白酶被脂质透磷脂磷脂酰氨基糖醇-4,5-双磷酸酯（PIP2）激活。 Star-PAP受细胞信号传导的调节，并通过使用基因/前MRNA上的特定裂解和聚腺苷酸化位点（PA）来控制基因表达。 3'UTR包含对于控制mRNA定位和翻译至关重要的序列。此外，超过70％的人类基因经历了替代聚腺苷酸化（APA），而APA的变化与干细胞的发育和癌症进展相关。我们发现，每个核PAP对于不同的APA和聚腺苷酸化（PA）位点具有独特的特异性，基因组宽，表明先前已经对3'-End加工调节的水平更高。这个事实表明，通过细胞信号传导对基因表达的3'末端控制水平高。我们假设Star-PAP和PAP/具有PA周围序列元素的PAS基因组的特异性。 Star-PAP由将共激活因激酶和RNA结合蛋白纳入其3'PROCOSESS络合物中的信号调节。 Star-PAP共激活因子（例如RBM10）是RNA结合蛋白，通过RNA识别确定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被激酶和磷酸酶循环以调节Star-PAP活性。在3'-Tail中添加我们的星-PAP调节mRNA表达。以下目的将检验以下假设：目标1。对PAS的PAP特异性将定义。由PAP控制的PA位点将由3'Reads定义，并通过交联，然后进行RNA免疫沉淀和深层测序。顺式元素将通过生物信息学识别，并使用记者分析进行验证。将研究A和U到3'-Tails的Star-PAP添加的作用，并定义后果。将揭示控制APA和3个OTSAIL更改的信号。 AIM 2。通过信号和共激活因子蛋白定义Star-PAP 3'UTR处理调节。通过定义的共激活因子（例如PI和蛋白激酶）以及确定特异性的RNA结合蛋白RBM10，将揭示对3'processing的星-PAP控制的机制。将评估RBM10在PA选择中的作用。我们将探索磷酸化如何调节星-PAP复合物组成和目标特异性。 AIM 3。恒星3'-End加工的空间和磷酸肌醇调节。 STAR-PAP具有PIP2载体蛋白的性能，我们将研究与Star-PAP的PIP2相互作用，并确定BONDEN PIPN是否由PIPK，PLC或PI3KS调节。我们将研究HO-1，NQO1和PTEN的Star-PAP空间3'处理，以划定裂解和聚腺苷酸化发生并探索空间mRNA处理的含义。