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

 描述(申请人提供)：mRNAs的3‘端加工和聚腺苷酸化是基因表达的关键。我们发现了一种非规范的聚(A)聚合酶Star-PAP(针对斑点靶向PIPKI调节的聚(A)聚合酶)，它被脂质信使磷脂酰肌醇-4，5-二磷酸(PIP2)激活。StAR-PAP受细胞信号调节，通过独特地利用基因/前-mRNAs上的特定切割和多聚腺苷酸化位点(PA)来控制基因的表达。3‘端非编码区包含对控制信使核糖核酸定位和翻译至关重要的序列。此外，超过70%的人类基因经历了选择性多聚腺苷酸化(APA)，APA的变化与干细胞发育和癌症进展相关。我们发现，每个核PAP对基因组范围内不同的APA和多聚腺苷酸化(PA)位点都有独特的特异性，这表明3‘端加工调节比以前所认识的水平更高。这一事实表明，通过细胞信号转导，基因表达的3‘端控制水平很高。我们推测，Star-PAP和PAP/通过PA周围的序列元件对Pas全基因组具有特异性。StAR-PAP受信号调控，将共激活蛋白和RNA结合蛋白整合到其3‘处理复合体中。StAR-PAP共激活子，如RBM10，一种RNA结合蛋白，通过RNA识别决定PA位点选择的特异性。StAR-PAP活性受PIP2控制，是一种PIP2载体蛋白，其中结合的PIP⇄、PIP2、⇄、PIP3通过激酶和磷酸酶来调节其活性。在3‘-Tail上加入我们的Star-PAP可以调节mRNA的表达。以下目的将验证这一假说：目的1.将定义PAP对PAS的特异性。由PAPs控制的PA位点将通过3‘阅读和交联、RNA免疫沉淀和深度测序来确定。顺式元件将通过生物信息学进行鉴定，并使用报告分析进行验证。将研究A和U在3‘-尾巴上的Star-PAP加法的作用并定义其后果。控制APA和3‘尾部变化的信号将被揭示。目的2.明确Star-PAP 3‘端非编码区的信号和共激活蛋白的加工调控。Star-PAP控制3‘加工的机制将通过定义确定特异性的共激活因子，如PI和蛋白激酶以及RNA结合蛋白RBM10来揭示。将评估RBM10在PA选择中的作用。我们将探索磷酸化如何调节Star-PAP复合体的组成和靶标特异性。目的3.Star-PAP 3‘-末端加工的空间和磷酸肌醇调节。Star-PAP具有PIP2载体蛋白的性质，我们将研究PIP2与Star-PAP的相互作用，并确定结合的PIPn是否受PIPKs、PLC或PI3Ks的调控。我们将研究Star-PAP对HO-1、NQO1和PTEN的空间3‘处理，以描绘发生切割和多腺苷基化的位置，并探索空间mRNA处理的意义。

 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.