Regulation of Retrotransposition in S. cerevisiae

描述（由申请人提供）：逆转录病毒和长末端重复序列（LTR）逆转录转座子包含一个移动的元件家族，其编码逆转录酶并将其RNA基因组复制成整合到宿主基因组中的cDNA。这些多产的基因组寄生虫构成了几乎所有真核生物基因组的重要部分。它们作为寄生虫的成功依赖于一系列宿主功能，因为逆转录病毒和逆转录转座子具有小的基因组和复杂的复制模式。逆转录因子复制所需的宿主因子是治疗艾滋病的小分子的潜在靶点，但迄今为止很少有特征。 酵母中的活性Ty 1 LTR反转录转座子家族，S. cerevisiae提供了一个独特的模型系统来探索真核宿主与逆转录转座子的关系。使用合成的遗传阵列屏幕和二级分子屏幕，我的实验室已经确定了48反转录转座宿主因子（RHFs）的Ty 1 cDNA的积累所需的。这些RHF中的许多是翻译和mRNA定位和周转的全局调节剂，包括核糖体蛋白旁系同源物、核糖体生物发生因子、翻译起始因子eIF 4G的旁系同源物、无义介导的衰变蛋白Upf 1-Upf 3、去帽激活剂Dhh 1、去帽全酶Dcp 1/Dcp 2和5'至3'核糖核酸外切酶Xrn 1。 Ty 1的RNA基因组与逆转录病毒的RNA基因组一样，充当病毒样颗粒（VLP）蛋白翻译和VLP内逆转录的模板。我们最近发现，eIF 4G 1和两个核糖体蛋白旁系同源物的积累所需的TyA，主要的衣壳蛋白，而Xrn 1的影响Ty 1 RNA包装成VLP。此外，我们已经发现，Ty 1 RNA被抑制在蔗糖致密的核糖核蛋白颗粒和TyA是与细胞质mRNA加工机构（P机构），翻译调节和mRNA衰变因子集中。我们建议研究Ty 1 RNA的翻译调控机制，并确定这种调控是否在VLP中的翻译和包装之间的Ty 1 RNA分区中发挥作用。此外，我们将研究mRNA衰变蛋白在VLP形成中的作用，VLP是逆转录的功能。具体目标是：（1）鉴定Ty 1 cDNA水平降低的rhf突变体中逆转录转座被阻断的步骤：（2）检测核糖体蛋白旁系同源物、核糖体生物发生因子和eIF 4G 1在Ty 1 RNA和蛋白的翻译调控和定位中的作用;和（3）表征Ty 1 RNP并确定mRNA衰变因子是否影响Ty 1 RNA或蛋白定位、VLP中的RNA包装和/或逆转录。本计画将阐明真核生物宿主因子促进反转录转座子及反转录病毒复制的特定机制。

DESCRIPTION (provided by applicant): Retroviruses and long terminal repeat (LTR) retrotransposons comprise a family of mobile elements that encode reverse transcriptase and copy their RNA genome into a cDNA that is integrated into the host genome. These prolific genomic parasites constitute a significant fraction of virtually all eukaryotic genomes. Their success as parasites relies on an array of host functions, since retroviruses and retrotransposons have small genomes and complex modes of replication. Host factors that are required for retroelement replication are potential targets for small molecules to treat AIDS, yet few have been characterized thus far. The family of active Ty1 LTR-retrotransposons in the yeast, S. cerevisiae, provides a unique model system to explore the eukaryotic host-retrotransposon relationship. Using a synthetic genetic array screen and secondary molecular screens, my lab has identified 48 retrotransposition host factors (RHFs) that are required for accumulation of Ty1 cDNA. Many of these RHFs are global regulators of translation and mRNA localization and turnover, including ribosomal protein paralogs, ribosome biogenesis factors, a paralog of translation initiation factor eIF4G, nonsense-mediated decay proteins Upf1-Upf3, the activator of decapping, Dhh1, the decapping holoenzyme, Dcp1/Dcp2 and the 5' to 3' exoribonuclease, Xrn1. The RNA genome of Ty1, like that of retroviruses, functions as a template for both translation of virus- like particle (VLP) proteins and for reverse transcription within VLPs. We have recently found that eIF4G1 and two ribosomal protein paralogs are required for accumulation of TyA, the major capsid protein, while Xrn1 influences Ty1 RNA packaging into VLPs. Moreover, we have found that Ty1 RNA is translationally repressed in sucrose-dense ribonucleoprotein particles and that TyA is associated with cytoplasmic mRNA processing bodies (P bodies), where translational regulators and mRNA decay factors are concentrated. We propose to examine the mechanism of translational regulation of Ty1 RNA and determine whether this regulation plays a role in partitioning Ty1 RNA between translation and packaging in VLPs. In addition, we will examine the role of mRNA decay proteins in the formation of VLPs that are functional for reverse transcription. The specific aims are: (1) to identify the steps in retrotransposition that are blocked in rhf mutants with reduced levels of Ty1 cDNA; (2) to examine the role of ribosomal protein paralogs, ribosome biogenesis factors and eIF4G1 in the translational regulation and localization of Ty1 RNA and protein; and, (3) to characterize Ty1 RNPs and determine whether mRNA decay factors influence Ty1 RNA or protein localization, RNA packaging in VLPs and/or reverse transcription. This project will elucidate specific mechanisms by which eukaryotic host factors promote the replication of retrotransposons and retroviruses.