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pre-mRNA聚腺苷化的机制和调控。研究旨在了解聚腺苷酸化机制的组成部分是如何被调节的；核内分裂是如何催化的；以及3'端加工是如何与转录相联系的。提出以下具体目标：1. 聚腺苷化的调节。对组织特异性聚A聚合酶（PAP）聚合化的机制和功能意义进行了研究。在细胞生长过程中，PAP的基本功能是否需要sumo化，以及它对PAP稳定性、亚细胞定位和酶活性的影响将被确定。体外试验将用于研究PAP聚合化的机制，并确定组织特异性的因素。Symplekin也通过sumo化修饰，这将使用类似的方法进行研究。研究结果表明，表达磷酸化缺陷PAP的细胞特异性过表达具有延长聚(a)尾部的c-jun mRNA。对过度表达PAP的肿瘤来源细胞系进行分析，以确定c-jun（和/或c-fos） poly(A) tail是否延长。2. 3′解理机理及控制。研究表明CPSF-73是负责3'切割的内切酶。CPSF-73在体外显示核酸酶活性的最新发现将得到进一步证实和扩展。p-内酰胺酶结构域的关键残基将发生突变，并确定对催化的影响。核酸酶的活性将以几种方式进行表征，包括分析新描述的CASP结构域的作用。CPSF-100具有与CPSF-73非常相似的结构域，但缺乏催化活性，其作用将被研究。sumoylation调节CPSF-73/100的可能性将被研究。与CPSF-73和CPSF-100相似的RC-68和RC-74在聚腺苷化中的潜在作用将被探讨。3. 转录和3'加工。最近的研究发现，RNA聚合酶II CTD选择性地结合RNA，这可以抑制转录偶联的3'端形成。将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的靶标细胞基因。我们将在体外研究3'加工抑制的机制，并在DT40细胞中通过遗传分析研究特异性CTD七肽重复序列的作用。Sub1/PC4功能有助于连接转录和3'加工，数据表明这反映了与CTD修饰酶的相互作用。这些功能在人类中是否保留还有待确定。PAF复合体的功能，在酵母中已知在连接转录与CTD和染色质修饰，以及可能的3'端形成中发挥作用，将在人类细胞中分析。将使用RNAi在体内消耗PAF成分，并确定对3'端形成的影响。最后，构建了一个重组的偶联转录-3‘加工系统，并利用染色质化模板分析了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.