Regulation of Retrotransposition in S. cerevisiae

描述(申请人提供)：逆转录病毒和长末端重复(LTR)反转录转座子由一系列可移动的元件组成，它们编码逆转录酶并将其RNA基因组复制到整合到宿主基因组中的cDNA中。这些多产的基因组寄生虫几乎构成了所有真核生物基因组的重要组成部分。它们作为寄生虫的成功依赖于一系列宿主功能，因为逆转录病毒和反转录转座子的基因组很小，复制模式复杂。逆转录酶元件复制所需的宿主因子是小分子治疗艾滋病的潜在靶点，但到目前为止还没有几个被表征。酿酒酵母中活跃的Ty1反转录转座子家族为探索真核宿主-反转录转座子的关系提供了一个独特的模型系统。使用合成基因阵列筛选和二级分子筛选，我的实验室已经确定了积累Ty1基因所需的48个逆转座子宿主因子(RHFs)。其中许多RHFs是翻译和mRNA定位和周转的全球调节因子，包括核糖体蛋白辅基、核糖体生物发生因子、翻译起始因子eIF4G的辅基、无义介导的衰变蛋白UPF1-Upf3、解帽激活剂Dhh1、解帽整酶Dcp1/Dcp2和5‘至3’外切核酸酶Xrn1。与逆转录病毒一样，Ty1的RNA基因组可以作为病毒样颗粒(VLP)蛋白的翻译模板和VLP内的逆转录模板。我们最近发现，eIF4G1和两个核糖体蛋白平行对齐是积累主要衣壳蛋白Tya所必需的，而Xrn1影响Ty1 RNA包装到VLP中。此外，我们还发现Ty1 RNA在蔗糖致密的核糖核蛋白颗粒中被翻译抑制，Tya与细胞质mRNA加工体(P体)相关，翻译调控因子和mRNA衰变因子集中在细胞质mRNA加工体。我们建议研究Ty1 RNA的翻译调控机制，并确定该调控是否在VLP中Ty1 RNA在翻译和包装之间的分配中发挥作用。此外，我们还将研究mRNA衰变蛋白在VLP形成中的作用，VLP对逆转录具有功能。其具体目的是：(1)确定在Ty1基因表达水平降低的rhf突变体中被阻断的逆转录转座步骤；(2)研究核糖体蛋白辅基、核糖体生物发生因子和eIF4G1在Ty1 RNA和蛋白质的翻译调控和定位中的作用；以及(3)表征Ty1 RNPs并确定信使核糖核酸衰减因素是否影响Ty1 RNA或蛋白质定位、VLP中的RNA包装和/或逆转录。该项目将阐明真核宿主因子促进逆转录转座子和逆转录病毒复制的具体机制。

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.