Gen. & Mol. Analysis of Human LINE-1 Retrotransposition

描述（由申请人提供）：长散布元件-1（LINE-1或L1）是一种丰富的非长末端重复序列（non-LTR）I反转录转座子，约占人类DNA的17%。平均人类基因组含有约80 - 100个有逆转录转座能力的L1（RC-L1），并且这些元件编码酶活性（例如，逆转录酶和核酸内切酶）的迁移（即，反转录转座）。L1反转录转座可以是致突变的，因为在生殖系和体细胞组织中有害的插入都引起疾病。RC-L1编码的蛋白偶尔也可以反式发挥作用，以动员非自主逆转录转座子（例如，Alu和SVA元件）或细胞mRNA，导致加工的假基因形成。有害的Alu和SVA插入也与人类疾病有关。因此，直接或通过细胞RNA的混杂动员，RC-L1是有效的诱变剂。尽管RC-Lls具有致突变潜力，但对其反转录转座机制知之甚少。我的实验室已经开发了一种高通量检测以及分子生物学和生物化学方法来监测培养的哺乳动物细胞中的L1反转录转座。在这里，我们将使用这些方法来阐明L1反转录转座的分子机制。本提案的具体目的是1）鉴定和表征L1 5' UTR、L1 RNA和L1编码的蛋白质中的序列，这些序列对于逆转录转座至关重要; 2）确定L1介导的反式互补的分子机制;和3）表征L1核糖核蛋白颗粒。远景目标 该项目的主要目的是从根本上了解L1逆向转运如何促进人类疾病和遗传多样性。

DESCRIPTION (provided by applicant): Long Interspersed Element-1 (LINE-1 or L1) is an abundant non-Long Terminal Repeat (non-LTR) I retrotransposon that comprises about 17% of human DNA. The average human genome contains -80-100 retrotransposition-competent Lls (RC-Lls) and these elements encode enzymatic activities (e.g., reversel transcriptase and endonuclease) required for their mobility (i.e., retrotransposition). L1 retrotransposition can be mutagenic, as deleterious insertions in both germ line and somatic tissues have caused disease. The RC-L1 encoded proteins also occasionally can function in trans to mobilize either non-autonomous retrotransposons (e.g., Alu and SVA elements) or cellular mRNAs, resulting in processed pseudogene formation. Deleterious Alu and SVA insertions also are implicated in human disease. Thus, either directly or by the promiscuous mobilization of cellular RNAs, RC-Lls are potent mutagens. Despite the mutagenic potential of RC-Lls, relatively little is known about their retrotransposition mechanism. My lab has developed a high-throughput assay as well as molecular biological and biochemical approaches to monitor L1 retrotransposition in cultured mammalian cells. Here, we will use these approaches to elucidate the molecular mechanism of L1 retrotransposition. The Specific Aims of this proposal are 1) To identify and characterize sequences in the L1 5' UTR, L1 RNA, and the Ll-encoded proteins, which are critical for retrotransposition; 2) To determine the molecular mechanism of Ll-mediated trans-complementation; and 3) To characterize L1 ribonucleoprotein particles. The long-term goal of this project is to gain a fundamental understanding of how L1 retrotransportation contributes to human disease and genetic diversity.