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提供对前信使核糖核酸多腺苷化的机制和调节的见解。建议进行研究，以了解多聚腺苷化机制的组成部分是如何调节的；内切核裂是如何被催化的；以及3‘端加工是如何与转录联系在一起的。提出了以下具体目标。1.多聚腺苷酸化的调控。将对聚(A)聚合酶(PAP)的组织特异性和甲基化的机制和功能意义进行研究。PAP在细胞生长过程中的基本功能是否需要苏莫化，以及它对PAP的稳定性、亚细胞定位和酶活性的影响将被确定。体外试验将被用来研究PAP苏莫化的机制，并确定负责组织特异性的因素(S)。Symplekin也可以通过相加作用进行修饰，这将使用类似的方法进行研究。研究发现，表达磷酸化缺陷的PAP的细胞在延长的Poly(A)尾巴中特异性地过表达c-Jun mRNA。过度表达PAP的肿瘤细胞株将被分析，以确定c-jun(和/或c-fos)聚(A)尾巴是否延长。2.3‘解理的机理及控制。将继续进行研究，表明CPSF-73是负责3‘切割的内切酶。最近发现CPSF-73在体外显示核酸酶活性，这一发现将得到证实和推广。β-内酰胺酶结构域中的关键残基将发生突变，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。CPSF-100的结构域结构与CPSF-73非常相似，但缺乏催化活性，因此将对其作用进行研究。对CPSF-73/100受控的可能性进行了研究。RC-68和RC-74分别与CPSF-73和CPSF-100有相似之处，因此我们将探讨RC-68和RC-74在多聚腺苷化中的潜在作用。3.转录和3‘端加工。最近的发现是，RNA聚合酶II CTD选择性地与RNA结合，这可以抑制转录耦合的3‘末端的形成。作为可能靶点的细胞基因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。将在体外研究3‘加工抑制的机制，并利用遗传分析研究特定的CTD七肽重复序列在DT40细胞中的作用。Sub1/PC4的功能是帮助连接转录和3‘处理，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身上是保守的将会被确定。在人类细胞中，PAF复合体的功能将被分析。在酵母中，PAF复合体在连接转录、CTD和染色质修饰以及可能的3‘末端形成方面发挥作用。RNAi将被用于体内消耗PAF组分，并将确定其对3‘末端形成的影响。最后，我们将开发一个重组的转录-3‘处理系统，并利用色谱化模板分析PAF在连接转录、染色质修饰和3’处理中的功能。

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.