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

 描述（由申请人提供）：mRNA的3 '末端加工和多聚腺苷酸化对于基因表达至关重要。我们发现了一种非经典的多聚腺苷酸聚合酶Star-PAP（斑点靶向PIPKI调节的多聚腺苷酸聚合酶），它被脂质信使磷脂酰肌醇-4，5-二磷酸（PIP 2）激活。Star-PAP受细胞信号传导调节，并通过独特地使用基因/前mRNA上的特异性切割和多聚腺苷酸化位点（PA）来控制基因表达。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（阿帕），阿帕的变化与干细胞发育和癌症进展相关。我们发现，每个核PAP对不同的阿帕和多聚腺苷酸化（PA）位点具有独特的特异性，这表明3 '端加工调节的水平高于以前的水平。这一事实表明通过细胞信号传导的基因表达的3 '端控制的高水平。 我们假设Star-PAP和PAP α/β通过围绕PA的序列元件对全基因组范围的PA具有特异性。Star-PAP受信号调节，该信号将辅激活物激酶和RNA结合蛋白并入其3 '加工复合物中。Star-PAP共激活剂如RBM 10（一种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PIP 2控制，Star-PAP是PIP 2载体蛋白，其中结合的PIP → PIP 2 → PIP 3通过激酶和磷酸酶循环以调节Star-PAP活性。Star-PAP向3 '-尾添加Us调节mRNA表达。以下目标将检验这一假设：目标1。将定义PAP对PA的特异性。PAP控制的pA位点将通过3 'READS和交联，然后通过RNA免疫沉淀和深度测序来确定。将通过生物信息学鉴定顺式元件，并使用报告基因测定法进行验证。将研究Star-PAP向3 '-尾部添加A和U的作用，并定义结果。控制阿帕和3 '尾变化的信号将被揭示。目标二。通过信号和辅激活蛋白来定义Star-PAP 3 'UTR加工调节。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RBM 10来揭示。将评估RBM 10在PA选择中的作用。我们将探讨磷酸化如何调节Star-PAP复合物的组成和靶特异性。目标3。Star-PAP 3 '-末端加工的空间和磷酸肌醇调节。Star-PAP具有PIP 2载体蛋白的性质，我们将研究PIP 2与Star-PAP的相互作用，并确定结合的PIPn是否受PIPKs、PLC或PI 3 Ks调节。我们将研究Star-PAP对HO-1、NQO 1和PTEN的空间3 '加工，以描绘切割和多聚腺苷酸化发生的位置，并探索空间mRNA加工的意义。

 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.