HOST CELL FACTORS CONTROLLING RETROVIRAL RNA PROCESSING

前体RNA的限制性剪接是细胞增殖的重要组成部分。 逆转录病毒生命周期 病毒初级转录本的剪接必须是 由于这种RNA是表达结构蛋白所必需的， 以及作为后代病毒体的基因组。 大量未拼接的 逆转录病毒RNA在受感染的细胞中积累。 两个不同的元素 在维持高水平的未剪接的 Rous肉瘤病毒中的RNA：i）gag基因中的元件，称为 剪接负调节子（NRS）控制从共同的5'端的剪接， ii）将第二剪接位点连接到env和src 3'剪接位点，和 Src 3'剪接位点附近的一个元件似乎控制Src剪接 具体来说 由于不需要病毒蛋白，因此宿主因子必须 调解这些事件。 这项工作的目标是确定和表征 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进行调控。 的蛋白质 将使用UV鉴定与NRS特异性相互作用的 交联、迁移率变动分析和色谱方法。 角色 NRS结合蛋白（NBP）的剪接抑制作用将在 体外和体内使用抑制缺陷的突变元件。 NRS- 结合蛋白将被纯化，测序，并编码它们的cDNA 确定，以允许随后的生物化学实验，以解决他们的 功能和亚细胞分布。 体内和体外相似 将用SRC元件进行实验以阐明其模式， 行动上 由于其功能可能取决于结构， SRC抑制元件的结构将通过化学方法确定 酶促地。 对src剪接抑制重要的蛋白质将被 寻求并与NRS活动所需的那些进行比较，以揭示潜在的 这两种元素之间机械上的相似之处。 Thr NRS也被 显示影响3 'LTR中的聚腺苷酸化效率; NRS突变 促进宿主DNA的通读，这可能导致癌基因 activation. 体外反应将用于进一步研究NRS聚（A） 推广. NRS募集多聚腺苷酸化因子的可能性 将使用设计用于检测 NBP和多聚腺苷酸化机制的组分。 理解 通过宿主细胞因子与病毒相互作用控制逆转录病毒剪接 顺式作用元件可能有助于了解细胞mRNA的调节 加工也是。

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.