HOST CELL FACTORS CONTROLLING RETROVIRAL RNA PROCESSING

前体RNA的有限剪接是 逆转录病毒的生命周期。病毒初级转录本的剪接必须是 由于该RNA是结构蛋白表达所必需的，因此受到控制 作为子代病毒粒子的基因组。大量未拼接的 逆转录病毒RNA在受感染的细胞中积聚。两个截然不同的元素 从剪接位点起到维持高水平非剪接的作用 Rous肉瘤病毒中的RNA：I)Gag基因中的一个元件，称为 剪接负调控因子(NRS)控制常见的5‘端剪接 与env和src 3‘剪接位点的剪接位点，以及ii)第二个 位于src 3‘剪接位点附近的元件似乎控制src的剪接。 具体地说。由于病毒蛋白不是必需的，宿主因素必须 调解这些事件。这项工作的目标是识别和描述 在这种新的基因表达形式中重要的细胞蛋白质 采用体内和体外策略的调节。蛋白质是指 与NRS的具体交互将使用UV进行识别 交联法、迁移率变化分析和层析方法。角色 对于NRS结合蛋白(Nbps)在剪接抑制中的作用将在 在体外和体内使用缺陷突变元件进行抑制。国税局- 结合蛋白将被提纯、测序和编码它们的cDNA 以允许后续的生化实验解决他们的 功能和亚细胞分布。体内和体外相似 将对src元素进行实验，以阐明其模式。 行动。由于其功能可能取决于结构，次要的 Src抑制元件的结构将通过化学方法和 酶促作用。抑制src剪接的重要蛋白质将是 寻找并与NRS活动揭示潜力所必需的那些进行比较 这两个元素之间存在机械上的相似之处。THR NRS也一直在 显示影响3‘LTR中的多聚腺苷酸化效率；NRS突变 促进对宿主DNA的读取，这可能导致癌基因 激活。体外反应将被用于进一步研究NRS聚(A) 升职。NRS招募多聚腺苷化因子的可能性 将使用旨在检测相互作用的分析进行调查 Nbps和聚腺苷酸化装置的组件。理解 宿主细胞因子与病毒相互作用控制逆转录病毒剪接 顺式作用元件可能对细胞mRNA的调控有深入的了解 处理过程也是如此。

Limited splicing of precursor RNA is an essential component of the retrovirus life cycle. Splicing of viral primary transcripts must be controlled since this RNA is required for expression of structural proteins and as genome for progeny virions. A substantial amount of unspliced retrovirus RNA accumulates in an infected cells. Two elements distinct from the splice sites play a role in maintaining high levels of unspliced RNA in Rous sarcoma virus: i) An element in the gag gene termed the negative regulator of splicing (NRS) controls splicing from the common 5' splice site to both the env and src 3' splice sites and ii) A second element near the src 3' splice site appears to control src splicing specifically. Since viral proteins are not required, host factors must mediate these events. The goal of the work is to identify and characterize cellular proteins important in this novel form of gene expression regulation employing in vivo and in vitro strategies. Proteins that specifically interact with the NRS will be identified using UV crosslinking, mobility shift assays, and chromatographic approaches. Roles for NRS-binding proteins (NBPs) in splicing inhibition will be assessed in vitro and in vivo using mutant elements defective for inhibition. The NRS- binding proteins will be purified, sequenced, and cDNAs encoding them identified to allow subsequent biochemical experiments to address their function and subcellular distribution. Similar in vivo and in vitro experiments will be performed with the src element to elucidate its mode of action. Since its function may depend on structure, the secondary structure of the src inhibitory element will be determined chemically and enzymatically. Proteins important for src splicing inhibition will be sought and compared to those necessary for NRS activity to reveal potential mechanistic parallels between the two elements. Thr NRS has also been shown to influence polyadenylation efficiency in the 3'LTR; NRS mutations facilitate read through into host DNA, which can result in oncogene activation. In vitro reactions will be used to further study NRS poly(A) promotion. The possibility that the NRS recruits polyadenylation factors will be investigated using assays designed to detect interactions between NBPs and components of the polyadenylation machinery. Understanding retroviral splicing control by interactions of host-cell factors with viral cis-acting elements may yield insights into regulation of cellular mRNA processing as well.