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（LTR）逆转录转座子包含一系​​列可移动元件，这些元件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较小的基因组和复杂的复制模式。逆转录元件复制所需的宿主因子是小分子治疗艾滋病的潜在靶标，但迄今为止很少有特征被表征。 酿酒酵母中的活性 Ty1 LTR 逆转录转座子家族为探索真核宿主-逆转录转座子关系提供了一个独特的模型系统。通过使用合成基因阵列筛选和二级分子筛选，我的实验室已鉴定出 Ty1 cDNA 积累所需的 48 种逆转录转座宿主因子 (RHF)。许多 RHF 是翻译和 mRNA 定位和周转的全局调节因子，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 eIF4G 的旁系同源物、无义介导的衰变蛋白 Upf1-Upf3、脱帽激活剂 Dhh1、脱帽全酶 Dcp1/Dcp2 和 5' 至 3' 核糖核酸外切酶 Xrn1。 Ty1 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，主要衣壳蛋白 TyA 的积累需要 eIF4G1 和两个核糖体蛋白旁系同源物，而 Xrn1 会影响 Ty1 RNA 包装到 VLP 中。此外，我们发现Ty1 RNA在蔗糖致密核糖核蛋白颗粒中受到翻译抑制，并且TyA与细胞质mRNA加工体（P体）相关，其中翻译调节因子和mRNA衰减因子集中。我们建议检查 Ty1 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分配 Ty1 RNA 中发挥作用。此外，我们将研究 mRNA 衰变蛋白在具有逆转录功能的 VLP 形成中的作用。具体目标是： (1) 确定在 Ty1 cDNA 水平降低的 rhf 突变体中被阻断的逆转录转座步骤； (2) 研究核糖体蛋白旁系同源物、核糖体生物发生因子和eIF4G1在Ty1 RNA和蛋白的翻译调控和定位中的作用； (3) 表征 Ty1 RNP 并确定 mRNA 衰减因子是否影响 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.