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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 酿酒酵母含有五个与动物逆转录病毒密切相关的LTR逆转录ty1-​​5家族。 像逆转录病毒一样，酵母TY元素编码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中间体进行转p，以制造TY cDNA。将逆转录病毒和LTR逆转座子的RNA分组为LTR恢复元素，而不是被动中间体。他们是LTR恢复复制的多个步骤的动态参与者，包括促进自己的二聚体，包装和逆转录，在某些情况下，他们自己的转录和核出口。 最重要的是，全长LTR retroement RNA扮演两个核心角色：用作元素mRNA，这些元素mRNA被翻译成蛋白质以及元素基因组RNA，它们被包装到病毒体或VLP中。 RNA如何通过隔离或可互换的RNA种群发挥这两个作用，仅在少数LTR返回元素的大纲中才知道。由于LTR的恢复元素取决于其宿主来执行其传播所需的大多数功能，因此很可能在大多数恢复的RNA动力学中涉及宿主因子，包括区分基因组和Messenger RNA群体的机制。 在TY1和TY3逆转录中起作用的宿主因子的遗传筛选已鉴定出数百个基因。但是，对于这些宿主因素中的大多数在TY元素复制中的作用知之甚少。为了补充这些遗传方法，我们采用了一种蛋白质组学方法，鉴定了与TY1 VLP相关的宿主蛋白。 我们假设某些与VLP物理相关的宿主蛋白在逆转录中起着直接（？近端？）作用，尤其是那些在先前的遗传筛选中鉴定出的TY宿主因子的蛋白质。我们将注意力集中在可能与TY1 RNA相互作用的宿主蛋白上，并有助于TY1 RNA动力学的键方面。 我们的研究涉及TY1 VLP的亲和力纯化，然后进行质谱分析（MUDPIT）。到目前为止，我们已经确定了约200种与TY1 VLP相关联的宿主蛋白。 其中包括PUF6P和KHD1P，两个RNA结合蛋白抑制其靶mRNA的翻译。 我们已经表明，PUF6和KHD1突变体在TY1转座中有缺陷，并且至少KHD1P结合TY1 RNA。 我们目前正在探索这些蛋白质在TY1换位中的作用。