PROCESSING AND FUNCTION OF POLYOMA RNA

多聚瘤 RNA 的加工和功能

• 批准号: 3188494
• 负责人: Gordon G Carmichael
• 金额: $ 16.28万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 1992-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3188494
• 关键词: Polyomavirus muris 1; chromosome deletion; gel electrophoresis; gene deletion mutation; gene expression; genetic manipulation; genetic mapping; genetic promoter element; genetic regulation; genetic transcription; genetic translation; genome; immunofluorescence technique; messenger RNA; molecular genetics; neoplastic cell culture for noncancer research; nucleic acid hybridization; nucleic acid sequence; oligonucleotides; point mutation; tissue /cell culture; transfection; transposon /insertion element; virus RNA; virus infection mechanism

Our goal is to understand better the relationship between polyoma virus genome structure and gene expression. Our particular interests are in the late leader unit and in the late promoter. Polyoma late mRNA molecules have multiple, tandem copies of a 57-base, untranslated sequence the "late leader" at their 5'-ends. The function of the leader in late gene expression is somewhat unclear. We shall study its role in RNA synthesis, processing and translation. Our recent work has shown that the length of the leader, but not its sequence, is important for both late RNA splicing and stability. We shall extend these studies in order to elucidate the molecular events involved in late leader splicing. We shall use deletion and point mutants to test the role of leader length and of the 3' splice site at its 5' boundary in splicing and RNA stability. We shall insert the ALM leader region into other transcripts, upstream or downstream of other exons, to determine whether it can confer instability on other pre-mRNAs. We shall use chimeric double-genome plasmids and DNA transfections to study the order of exon splicing in polyoma late transcripts and we shall examine the possibility of trans splicing of polyoma late RNAs. We shall establish in vitro splicing of late viral pre- mRNAs in HeLa cell extracts and use this system to test leader region mutants for splicing, RNA stability, and splicing complex formation. In order to investigate the significance of leader multiplicity we shall study a mutant genome with the 3'-end processing/polyadenylation region of SV40 inserted into the 3'-end region of polyoma. This virus should only generate single leader units on all late messages. We shall test the leader region for a role in translation. We shall use reticulocyte lysates for in vitro translation of VP1 or VP2 from capped, T7 transcripts. We shall translate RNAs individually or use them in competition experiments with rabbit beta-globin. Experiments will be done with RNAs containing deleted, substituted or reiterated leader units. In in vivo experiments, we shall use S1 analysis to determine whether larger polysomes form infected cells contain viral mRNAs with more leader units. Another of our goals is to define cis-acting regulatory sequences that are involved in the initiation and control of transcription from the late promoter. Toward this end we shall use CAT assays, nuclear run-off experiments and S1 analysis to measure and compare early and late promoter activity in a large number of mutants, many made by site-directed oliqonucleotide deletion mutagenesis. We shall perform these experiments in the presence or absence of large T antigen or DNA replication inhibitors. Nuclear run-off experiments will be done as a function of time after wild type or mutant transfection/infection in order to determine the time course of late promoter activation and its relationship to large T, DNA replication and specific cis-acting DNA sequences.

我们的目标是更好地了解多瘤之间的关系 病毒基因组结构和基因表达。 我们的特殊 兴趣是已故领导者部门和已故的发起人。 多瘤晚期mRNA分子具有多个A的串联副本 57基数，未翻译的序列是他们5'末端的“已故领导者”。 领导者在晚期基因表达中的功能有些 不清楚。 我们将研究其在RNA合成，加工和 翻译。 我们最近的工作表明 领导者，但不是其序列，对两个晚RNA都很重要 剪接和稳定性。 我们将扩展这些研究，以便 阐明了晚期领导者剪接所涉及的分子事件。 我们将使用删除和点突变体来测试领导者的角色 长度和3'剪接位点在其5'边界处的剪接和 RNA稳定性。 我们将插入ALM领导区域 成绩单，其他外显子的上游或下游，以确定 它是否可以赋予其他前MRNA的不稳定。 我们将 使用嵌合双基因组质粒和DNA转染 研究多瘤晚期的外显子剪接的顺序，并 我们将检查多瘤迟发剪接的可能性 RNA。 我们将在病毒前的晚期建立体外剪接 Hela细胞提取物中的mRNA，并使用该系统测试领导者 用于剪接，RNA稳定性和剪接复合物的区域突变体 形成。 为了研究领导者多样性的重要性，我们 应研究3'-末端的突变基因组 插入3'-End的SV40的加工/多腺苷酸化区域 多瘤区域。 该病毒只能产生单身领导者 所有较晚消息的单位。 我们将测试领导区域在翻译中的作用。 我们将 使用网状细胞裂解物进行VP1或VP2的体外翻译 从上限，T7成绩单。 我们将单独翻译RNA 或在与兔β-珠蛋白的竞争实验中使用它们。 实验将使用包含已删除的RNA进行， 替换或重申的领导单位。 在体内实验中，我们 应使用S1分析来确定是否形成了较大的多聚体 感染的细胞包含具有更多领导单元的病毒mRNA。 我们的另一个目标是定义顺式作用调节序列 参与转录的启动和控制 来自已故的发起人。 为此，我们将使用猫 测定，核径流实验和S1分析以测量 并比较大量的早期和晚期启动子活动 突变体，许多由位于定向的oliqonucleotide删除的突变体 诱变。 我们将在存在的情况下进行这些实验 或没有大的T抗原或DNA复制抑制剂。 核径流实验将随着时间的变化而进行 野生型或突变转染/感染之后 确定晚期启动子激活及其的时间过程 与大T，DNA复制和特定顺式作用的关系 DNA序列。