Biology of LINE retrotransposition

长散布核元件（LINEs）是一类连续地 突变基因组，包括哺乳动物基因组。目前在人类中活跃的LINE家族被称为 生产线-1（L1）。为了复制，L1 mRNA和两个L1编码的蛋白质（称为ORF1p和ORF2p）形成了一个复制区。 胞质核糖核蛋白颗粒（RNP）中间体。L1 RNP进入细胞核， 在新的染色体位点转录L1 mRNA。这一过程被称为反转录转座， 在人类中非常成功，已经产生了超过三分之一的人类基因组。此外，本发明还提供了一种方法， 人和/或模式生物中L1 RNP的表达与突变、DNA损伤、衰老 以及各种疾病，如癌症、不育症和神经系统疾病。在大多数情况下， 知道L1是否只是与这些疾病相关，或者L1是否在这些疾病中发挥着致病作用。 我们无法令人信服地解决这个问题是由于我们对LINE的基本理解存在差距 这限制了我们以特定方式操纵内源性L1活性的能力。的简单性 典型的LINE元件掩盖了LINE反转录转座的复杂性，表明LINE RNP利用宿主 蛋白质（下文称为"宿主因子"），以帮助它们的复制。尽管一些宿主因素 与L1 RNP相互作用，几乎所有这些因子都是RNA结合抑制剂， 逆转录转座，或者是非特异性RNA结合的人工产物。我们对核心主机仍然知之甚少 这些因素对于LINE逆转录转座的成功至关重要。这种偏向于提取L1 抑制剂可能是由于方法学，需要替代策略来鉴定核心LINE宿主 因素我们实验室的一个长期目标是解决这个差距-我们想了解LINE是如何复制的， 特别是通过鉴定和表征LINE RNP/宿主蛋白相互作用， 反转录转位为此，我们开发了一种LINE逆转录转座的酵母模型，基于 白色念珠菌L1样元件称为Zorro3。我们已经使用这种酵母模型发现， 转运所需的内体分选复合物（ESCRT）与LINE RNP物理相互作用， 在酵母和人类中进行反转录转座。在本提案中，我们将进一步描述这种相互作用， 确定L1/ESCRT相互作用发生的位置、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的一种表型不同的状态，称为不透明 状态这一发现是一种新的蛋白质组学策略（目标2）的基础，以确定生物学相关的主机 与LINE RNP相互作用的因素。目标2中的战略解决了以前研究的一个主要缺点 - 从大量的宿主蛋白中鉴定与L1 RNP结合的生物学上重要的宿主因子， 生化下拉

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.