PROTEOMIC ANALYSIS OF TY RETROTRANSPOSON HOST FACTORS IN YEAST

酵母TY逆转录转座子宿主因子的蛋白质组学分析

• 批准号: 8171346
• 负责人: THOMAS M MENEES
• 金额: $ 0.24万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8171346
• 关键词: Affinity Chromatography; Animals; Attention; Binding; Complement; Computer Retrieval of Information on Scientific Projects Database; Dimerization; Elements; Family; Funding; Genes; Genetic; Genetic Screening; Genetic Transcription; Genomics; Grant; Homologous Gene; Institution; Integration Host Factors; Length; Messenger RNA; Nuclear Export; Play; Population; Proteins; Proteomics; RNA; RNA-Binding Proteins; RNA-Directed DNA Polymerase; Research; Research Personnel; Resources; Retroelements; Retrotransposition; Retrotransposon; Retroviridae; Reverse Transcription; Role; Saccharomyces cerevisiae; Source; Translating; Translations; United States National Institutes of Health; Virion; Virus-like particle; Yeasts; gag-pol Fusion Proteins; mutant

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Saccharomyces cerevisiae contains five families of LTR retrotranspososns, Ty1-5, that are closely related to animal retroviruses. Like retroviruses, yeast Ty elements encode homologs of Gag and Pol proteins, form virus-like particles (VLPs), and transpose through RNA intermediates using reverse transcriptases to make Ty cDNAs. The RNAs of both retroviruses and LTR retrotransposons, grouped together as LTR retroelements, are more than passive intermediates; they are dynamic players in multiple steps of LTR retroelement replication, including promoting their own dimerization, packaging, and reverse transcription, and in some cases their own transcription and nuclear export. On top of all this, full-length LTR retroelement RNAs play two central roles: serving as element mRNAs, which are translated into proteins, as well as element genomic RNAs, which are packaged into virions or VLPs. How the RNA serves these two roles, either via segregated or interchangeable RNA populations, is only known in broad outline for a few LTR retroelements. Since LTR retroelements depend on their hosts to perform most of the functions necessary for their propagation, it is very likely that host factors are involved in most retroelement RNA dynamics, including the mechanism by which genomic and messenger RNA populations are distinguished. Genetic screens for host factors that play roles in Ty1 and Ty3 retrotransposition have identified hundreds of genes. However, little is known about how most of these host factors act in Ty element replication. To complement these genetic approaches we took a proteomic approach, identifying host proteins associated with Ty1 VLPs. We hypothesized that some of the host proteins physically associated with VLPs play direct (?proximal?) roles in retrotransposition, especially those proteins identified in previous, genetic screens for Ty host factors. We focused our attention on host proteins that might interact with Ty1 RNA and assist in keys aspects of Ty1 RNA dynamics. Our study involved affinity purification of Ty1 VLPs followed by mass spectrometric analysis (MudPIT). So far we have identified ~200 host proteins that specifically associate with Ty1 VLPs. Among these are Puf6p and Khd1p, two RNA binding proteins that inhibit translation of their target mRNAs. We have shown that puf6 and khd1 mutants are defective for Ty1 transposition and that at least Khd1p binds Ty1 RNA. We are currently exploring the roles of these proteins in Ty1 transposition.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 酿酒酵母含有 5 个 LTR 反转录转座子家族 Ty1-5，与动物反转录病毒密切相关。 与逆转录病毒一样，酵母 Ty 元件编码 Gag 和 Pol 蛋白的同源物，形成病毒样颗粒 (VLP)，并使用逆转录酶通过 RNA 中间体转座以生成 Ty cDNA。逆转录病毒和 LTR 逆转录转座子的 RNA 统称为 LTR 逆转录元件，它们不仅仅是被动中间体；它们在LTR逆转录元件复制的多个步骤中是动态的参与者，包括促进自身的二聚化、包装和逆转录，以及在某些情况下促进自身的转录和核输出。 最重要的是，全长LTR逆转录元件RNA发挥着两个核心作用：作为元件mRNA，被翻译成蛋白质，以及元件基因组RNA，被包装成病毒颗粒或VLP。 RNA 如何通过分离或可互换的 RNA 群体发挥这两个作用，目前仅对少数 LTR 逆转录因子有大致了解。由于 LTR 逆转录元件依赖于其宿主来执行其繁殖所需的大部分功能，因此宿主因素很可能参与大多数逆转录元件 RNA 动力学，包括区分基因组和信使 RNA 群体的机制。 对在 Ty1 和 Ty3 逆转录转座中发挥作用的宿主因子的遗传筛选已鉴定出数百个基因。然而，人们对大多数宿主因子如何在 Ty 元件复制中发挥作用知之甚少。为了补充这些遗传方法，我们采用了蛋白质组学方法，鉴定了与 Ty1 VLP 相关的宿主蛋白。 我们假设一些与 VLP 物理相关的宿主蛋白在逆转录转座中发挥直接（？近端？）作用，特别是在之前的 Ty 宿主因子遗传筛选中鉴定的那些蛋白。我们将注意力集中在可能与 Ty1 RNA 相互作用并协助 Ty1 RNA 动力学关键方面的宿主蛋白上。 我们的研究涉及 Ty1 VLP 的亲和纯化，然后进行质谱分析 (MudPIT)。到目前为止，我们已经鉴定出约 200 种与 Ty1 VLP 特异性相关的宿主蛋白。 其中包括 Puf6p 和 Khd1p，这两种 RNA 结合蛋白可抑制其靶 mRNA 的翻译。 我们已经证明 puf6 和 khd1 突变体在 Ty1 转座方面存在缺陷，并且至少 Khd1p 结合 Ty1 RNA。 我们目前正在探索这些蛋白质在 Ty1 转座中的作用。