Biology of LINE retrotransposition

LINE 逆转录转座的生物学

• 批准号: 9980955
• 负责人: Jeffrey S Han
• 金额: $ 30.1万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-02 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9980955
• 关键词: Address; Affinity; Aging; Animal Model; Binding Proteins; Biochemical; Biological; Biology; Candida albicans; Cell Nucleus; Cells; Complex; DNA Damage; Data; Development; Disease; Elements; Eukaryota; Family; Gene Rearrangement; Genome; Goals; Human; Human Genome; Infertility; Integration Host Factors; Knowledge; Malignant Neoplasms; Membrane; Messenger RNA; Methodology; Morphologic artifacts; Mutate; Mutation; Nuclear; Phenotype; Play; Process; Proteins; Proteomics; RNA Binding; Regulation; Retrotransposition; Ribonucleoproteins; Role; Sea; Site; Sorting - Cell Movement; Yeast Model System; Yeasts; base; genetic element; in vivo; inhibitor/antagonist; innovation; insight; mammalian genome; nervous system disorder; novel; particle

Long Interspersed Nuclear Elements (LINEs) are a class of retrotransposable elements that continually mutate genomes, including mammalian genomes. The currently active family of LINEs in humans is called LINE-1 (L1). To replicate, L1 mRNA and two L1-encoded proteins (called ORF1p and ORF2p) form a cytoplasmic ribonucleoprotein particle (RNP) intermediate. The L1 RNP enters the nucleus and reverse transcribes L1 mRNA at new chromosomal sites. This process, called retrotransposition, has been spectacularly successful in humans and has generated over one-third of the human genome. In addition, expression of L1 RNPs in humans and/or model organisms is associated with mutations, DNA damage, aging, and various disorders, such as cancer, infertility, and neurologic disease. In most of these cases we do not know whether L1 is simply correlated with these disorders, or if L1 plays a causative role in these disorders. Our inability to convincingly address this question is due to a gap in our rudimentary understanding of LINE biology, which limits our ability to manipulate endogenous L1 activity in a specific manner. The simplicity of a typical LINE element belies the complexity of LINE retrotransposition, suggesting that LINE RNPs utilize host proteins (henceforth referred to as “host factors”) to assist in their replication. Although some host factors that interact with L1 RNPs have been identified, almost all of these factors are RNA-binding inhibitors that block retrotransposition, or are artifacts of non-specific RNA binding. We still have little knowledge of the core host factors that are crucial for a successful round of LINE retrotransposition. This bias towards retrieving L1 inhibitors is likely due to methodology, and alternative strategies are needed for identifying core LINE host factors. A long term goal in our lab is to address this gap – we want to understand how LINEs replicate, specifically by identifying and characterizing LINE RNPs/host proteins interactions important for active retrotransposition. To this end, we have developed a yeast model of LINE retrotransposition, based on the Candida albicans L1-like element called Zorro3. We have already used this yeast model to discover that the endosomal sorting complex required for transport (ESCRT) physically interacts with LINE RNPs and is critical for retrotransposition in both yeast and humans. In this proposal we will further characterize this interaction by determining where, how, and why the L1/ESCRT interaction takes place (Aim 1). We have also discovered that LINE retrotransposition occurs in a phenotypically distinct state of Candida albicans called the opaque state. This finding is the basis for a novel proteomics strategy (Aim 2) to identify biologically relevant host factors that interact with LINE RNPs. The strategy in Aim 2 addresses a major shortcoming of previous studies – identifying the biologically significant host factors from the sea of host proteins that bind to L1 RNPs in biochemical pulldowns.

长散布核元件 (LINE) 是一类逆转录转座元件，持续存在 突变基因组，包括哺乳动物基因组。目前人类中活跃的 LINE 家族称为 LINE-1（L1）。为了复制，L1 mRNA 和两个 L1 编码的蛋白质（称为 ORF1p 和 ORF2p）形成一个 细胞质核糖核蛋白颗粒（RNP）中间体。 L1 RNP进入细胞核并逆转 在新的染色体位点转录 L1 mRNA。这个过程称为逆转录转座， 在人类身上取得了惊人的成功，并产生了超过三分之一的人类基因组。此外， L1 RNP 在人类和/或模型生物体中的表达与突变、DNA 损伤、衰老、 以及各种疾病，例如癌症、不孕症和神经系统疾病。在大多数情况下，我们不 了解 L1 是否仅与这些疾病相关，或者 L1 是否在这些疾病中起因果作用。 我们无法令人信服地解决这个问题是因为我们对 LINE 的基本理解存在差距 生物学，这限制了我们以特定方式操纵内源性 L1 活性的能力。简单的一个 典型的 LINE 元件掩盖了 LINE 逆转录转座的复杂性，表明 LINE RNP 利用宿主 蛋白质（以下称为“宿主因子”）来协助其复制。尽管一些主机因素 已鉴定出与 L1 RNP 相互作用的因子，几乎所有这些因子都是 RNA 结合抑制剂，可阻断 逆转录转座或非特异性 RNA 结合的产物。我们对核心主机还知之甚少 对于成功进行 LINE 逆转录转座至关重要的因素。这种偏向于检索 L1 抑制剂可能是由于方法论造成的，需要替代策略来识别核心 LINE 宿主 因素。我们实验室的一个长期目标是解决这一差距——我们想了解 LINE 是如何复制的， 特别是通过识别和表征对活性至关重要的 LINE RNP/宿主蛋白相互作用 逆转录转座。为此，我们基于 LINE 逆转录转座的酵母模型开发了 白色念珠菌 L1 样元件称为 Zorro3。我们已经使用这个酵母模型发现 运输所需的内体分选复合物 (ESCRT) 与 LINE RNP 发生物理相互作用，并且至关重要 用于酵母和人类的逆转录转座。在本提案中，我们将进一步描述这种相互作用： 确定 L1/ESCRT 交互发生的地点、方式和原因（目标 1）。我们还发现 LINE 逆转录转座发生在白色念珠菌表型独特的状态（称为不透明状态）中 状态。这一发现是识别生物学相关宿主的新型蛋白质组学策略（目标 2）的基础 与 LINE RNP 相互作用的因素。目标 2 中的策略解决了先前研究的一个主要缺点 – 从与 L1 RNP 结合的宿主蛋白海洋中识别具有生物学意义的宿主因子 生化下拉。