FACTORS WHICH RECOGNIZE MAMMALIAN POLYADENYLATION SIGNAL

识别哺乳动物多聚腺苷酸信号的因素

基本信息

批准号：
2181646
负责人：
CLAIRE L MOORE
金额：
$ 19.99万
依托单位：
TUFTS UNIVERSITY BOSTON
依托单位国家：
美国
项目类别：
财政年份：
1989
资助国家：
美国
起止时间：
1989-09-01 至 1995-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2181646
关键词：
RNA binding protein affinity chromatography autoradiography gel electrophoresis gene expression genetic library genetic translation immunochemistry laboratory mouse nucleic acid probes polyadenylate protein signal sequence transcription factor

项目摘要

The process of polyadenylation is essential in the biogenesis of eukaryotic mRNA. The poly(A) tall is thought to participate in mRNA translation and stability. Defects in the formation of the poly(A) tail decrease the amount of mRNA available for translation into protein, and thus interfere with normal cell function. In addition, polyadenylation can play a role in the regulation of gene expression, especially in cases with alternative selection of poly(A) sites. In this way, it becomes part of a cell's response to stimuli governing growth, differentiation, and tissue-specific gene expression. The goals of this proposal are to determine the factors responsible for this processing and how they interact with each other to give an active processing complex. Understanding the basic mechanism of polyadenylation will make it feasible to ask how the process is regulated as the physiological. state of the cell changes, and how this regulation affects mRNA levels globally or specifically. This research will characterize the factors which recognize the signal sequences on polyadenylation precursor. It will focus on two proteins, pl55 and p68, which require the AAUAAA sequence to bind to precursor RNA, are found in polyadenylation-specific complexes, and dissociate from the RNA once it is cleaved and polyadenylated. These observations are consistent with a role for these proteins in the polyadenylation process, possibly in signal recognition and assembly of the processing complex. Their relationship to the enzymes responsible for cleavage and poly(A) addition will be determined by whether they chromatographically cofractionate. Other experiments using an in vitro system will explore how polyadenylation specific factors interact with each other and with precursor RNA and products during the polyadenylation reaction. Three approaches will be taken to clone polyadenylation-specific factors: a) screening of lambda gt11 human cDNA expression libraries with RNA probes containing polyadenylation signal sequences; b) use of ultraviolet-crosslinked ribonucleoprotein complexes as immunogens to produce antibodies to polyadenylation-specific proteins; and c) sufficient purification of these proteins so as to use them either as antigens for specific antibody production or to obtain protein sequence to generate DNA probes. Either reagent would then be used to screen expression libraries. Using an in vitro system, the 3' end processing of mRNAs from bovine leukemia virus, an oncogenic retravirus, and the role of viral specific factors in this processing will be examined. Finally, the role of polyadenylation in transcription termination will be investigated. Transcriptional templates which encode a self-cleaving RNA sequence and a termination site will be used to determine the role of cleavage of the nascent transcript on termination of RNA polymerase 11 transcription. This will be studied in vivo using transient expression assays, and if possible, with an in vitro system capable of transcription initiation and elongation, polyadenylation, and termination.

聚腺苷酸化过程在真核的生物发生中至关重要 mRNA。 poly（a）高被认为参与mRNA翻译和稳定。 poly（a）尾巴形成的缺陷减少了数量可用于转化为蛋白质的mRNA，从而干扰正常的细胞功能。另外，聚腺苷酸化可以在基因表达的调节，尤其是在替代的情况下选择poly（a）位点。这样，它变成了单元的一部分对刺激的响应，刺激的生长，分化和组织特异性基因表达。该提案的目标是确定因素负责此处理以及他们如何相互互动给出主动处理复合物。了解的基本机制聚腺苷酸化将使询问如何调节该过程是可行的作为生理。细胞的状态发生变化，以及该调节如何全球或具体影响mRNA水平。这项研究会表征识别信号序列的因素聚腺苷酸前体。它将专注于两种蛋白质PL55和P68，需要AAUAAA序列与前体RNA结合，在聚腺苷酸化特异性复合物，并与RNA分离。切割和聚腺苷酸化。这些观察与角色一致对于这些蛋白质在聚腺苷酸过程中，可能在信号中识别和组装处理复合物。他们与负责裂解的酶和poly（a）的添加将是由它们是否在色谱上进行辅助分解确定。其他使用体外系统的实验将探讨如何聚腺苷酸化特定因素彼此相互作用，并与前体RNA和聚腺苷酸化反应期间的产物。三种方法将是采用克隆多腺苷酸化特异性因素：a）筛选lambda GT11人cDNA表达文库，带有RNA探针聚腺苷酸信号序列； b）使用紫外线链接核糖核蛋白复合物作为免疫原子，可产生抗体聚腺苷酸化特异性蛋白；和c）足够纯化这些蛋白质将它们用作特定抗体的抗原生产或获得蛋白质序列以生成DNA探针。任何一个然后，试剂将用于筛选表达式库。使用一个in 体外系统，来自牛白血病病毒mRNA的3'末端加工，一种致癌性金属病毒和病毒特异性因素在此中的作用将检查处理。最后，聚腺苷酸化在将研究转录终止。转录模板哪个编码自我切换的RNA序列和终止位点将是用于确定新生转录本在 RNA聚合酶11转录的终止。这将在体内使用瞬态表达测定，并在可能的情况下进行体外能够转录启动和伸长，聚腺苷酸化的系统和终止。