mRNA synthesis in animal cells - 3' end formation

描述（由申请人提供）：本提案中描述的实验旨在深入了解前体mRNA聚腺苷酸化的机制和调控。研究提出，以了解多聚腺苷酸化机制的组件是如何调节;如何内切核酸裂解催化;以及3'端加工是如何连接到转录。提出了以下具体目标。1.多聚腺苷酸化的调节。本研究旨在探讨多聚腺苷酸聚合酶（PAP）组织特异性类小泛素化的机制及其功能意义。细胞生长过程中是否需要类小泛素化对PAP的基本功能，以及其对PAP稳定性、亚细胞定位和酶活性的影响将被确定。将使用体外试验研究PAP类小泛素化的机制，并鉴定负责组织特异性的因子。Symplekin也通过sumoylation修饰，这将使用类似的方法进行研究。将探索表达磷酸化缺陷PAP的细胞特异性过表达具有延长的poly（A）尾的c-jun mRNA的发现。将分析过表达PAP的肿瘤衍生细胞系以确定c-jun（和/或c-fos）poly（A）尾是否延长。2. 3'切割的机制和控制。将继续进行表明CPSF-73是负责3'切割的内切核酸酶的研究。CPSF-73在体外显示核酸酶活性的最新发现将得到证实和扩展。β-内酰胺酶结构域中的关键残基将被突变，并确定对催化的影响。核酸酶活性将以几种方式表征，包括分析新描述的CASP结构域的作用。将研究CPSF-100的作用，其包含与CPSF-73非常相似的结构域结构，但缺乏催化活性。将研究sumoylation调节CPSF-73/100的可能性。将探索分别与CPSF-73和CPSF-100具有相似性的RC-68和RC-74在聚腺苷酸化中的潜在作用。3.转录和3'加工。最近的研究发现，RNA聚合酶II CTD选择性结合RNA，这可以抑制转录偶联的3'端形成将继续进行。通过可疑序列的突变和确定CTD是否与这些序列相互作用来检查可能成为靶点的细胞基因。将在体外研究3'加工抑制的机制，并在DT 40细胞中使用遗传分析研究特异性CTD七肽重复的作用。Sub1/PC 4的功能是帮助连接转录和3'加工，数据表明这反映了与CTD修饰酶的相互作用。这些功能是否在人类中是保守的将被确定。已知在酵母中PAF复合物在将转录与CTD和染色质修饰以及可能的3'末端形成联系起来中起作用，其功能将在人类细胞中进行分析。RNAi将用于体内消耗PAF组分，并将确定对3'末端形成的影响。最后，我们将建立一个重组的偶联转录-3 '加工系统，并利用染色质化的模板分析PAF在连接转录、染色质修饰和3'加工中的功能。

DESCRIPTION (provided by applicant): The experiments described in this proposal are designed to provide insights into the mechanism and regulation of pre-mRNA polyadenylation. Studies are proposed to understand how components of the polyadenylation machinery are regulated; how endonucleolytic cleavage is catalyzed; and how 3' end processing is linked to transcription. The following Specific Aims are proposed. 1. Regulation of polyadenylation. The mechanism and functional significance of the tissue-specific sumoylation of poly(A) polymerase (PAP) will be investigated. Whether sumoylation is required for PAP's essential function during cell growth will be determined, as will its effects on PAP stability, subcellular localization and enzymatic activity. In vitro assays will be used to investigate the mechanism of PAP sumoylation and to identify factor(s) responsible for tissue specificity. Symplekin is also modified by sumoylation, and this will be investigated using similar methods. Findings that cells expressing a phosphorylation-defective PAP specifically overexpress c-jun mRNA with lengthened poly(A) tails will be explored. Tumor-derived cell lines that overexpress PAP will be analyzed to determine whether c-jun (and/or c-fos) poly(A) tails are lengthened. 2. Mechanism and control of 3' cleavage. Studies indicating that CPSF-73 is the endonuclease responsible for 3' cleavage will be pursued. Recent findings that CPSF-73 displays nuclease activity in vitro will be confirmed and extended. Key residues in the p-lactamase domain will be mutated, and effects on catalysis determined. Nuclease activity will be characterized in several ways, including analysis of the role of the newly described CASP domain. The role of CPSF-100, which contains a domain structure very similar to that of CPSF-73 but lacks catalytic activity, will be studied. The possibility that sumoylation regulates CPSF-73/100 will be investigated. The potential role in polyadenylation of RC-68 and RC-74, which share similarity with CPSF-73 and CPSF-100, respectively, will be explored. 3. Transcription and 3' processing. Recent findings that the RNA polymerase II CTD binds RNA selectively and that this can suppress transcription-coupled 3' end formation will be pursued. Cellular genes that are possible targets will be examined by mutation of suspected sequences and by determining whether the CTD interacts with these sequences. The mechanism of 3' processing inhibition will be investigated in vitro, and the role of specific CTD heptad repeats studied using genetic analysis in DT40 cells. Sub1/PC4 functions to help connect transcription and 3' processing, and data suggesting that this reflects interactions with CTD modifying enzymes will be pursued. Whether these functions are conserved in humans will be determined. The function of the PAF complex, known in yeast to play a role in linking transcription with CTD and chromatin modification, and perhaps 3' end formation, will be analyzed in human cells. RNAi will be used to deplete PAF components in vivo and the effects on 3' end formation will be determined. Finally, a reconstituted coupled transcription-3' processing system will be developed and, using chromatinized templates, the function of PAF in linking transcription, chromatin modification and 3' processing analyzed.