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

描述（由申请人提供）：Long Interspersed Element-1 （LINE-1或L1）是一种丰富的非Long Terminal Repeat (non-LTR) I反转录转座子，约占人类DNA的17%。人类基因组平均含有-80-100个逆转录能力强的Lls (RC-Lls)，这些元件编码其迁移（即逆转录）所需的酶活性（例如逆转录酶和核酸内切酶）。L1逆转录可致突变，因为在生殖系和体细胞组织中有害的插入可引起疾病。RC-L1编码的蛋白偶尔也可以在反式中发挥作用，调动非自主的反转录转座子（如Alu和SVA元件）或细胞mrna，导致加工假基因的形成。有害的Alu和SVA插入也与人类疾病有关。因此，无论是直接的还是通过细胞rna的混杂动员，rc - ll都是有效的诱变剂。尽管RC-Lls具有致突变潜能，但对其逆转录转位机制的了解相对较少。我的实验室已经开发了一种高通量测定以及分子生物学和生化方法来监测培养的哺乳动物细胞中的L1逆转录转位。在这里，我们将使用这些方法来阐明L1反转录转位的分子机制。本提案的具体目的是：1)鉴定和表征L1 5' UTR， L1 RNA和ll编码蛋白中的序列，这些序列对逆转录至关重要；2)确定l介导的反式互补的分子机制；3)对L1核糖核蛋白颗粒进行表征。长期目标

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.