Human transposable element

描述（由申请人提供）：LINE-1或L1元素是哺乳动物基因组中最多产和最突出的可移动元素。在人类和小鼠基因组中，活性L1s及其遗迹占基因组质量的17-20%。通过为Alu和其他元件的迁移提供必要的活性，它们间接占这些基因组的另外10- 12%。L1逆转录转座子通过复制的“复制和粘贴”机制移动。在这个更新提案中，我们攻击了一些悬而未决的问题，包括逆转录转位的时间和细胞类型特异性，L1插入基因组的机制，以及双向L1启动子产生的siRNA在控制逆转录转位中的作用。在特异性目标1中，我们试图确定发生在男性生殖细胞、女性生殖细胞和胚胎发育早期阶段的总逆转录转位事件的比例。我们还希望确定逆转录转位是否随着年龄的增长而减少，在几代之后被抑制，并且由于甲基化或DMA双链断裂修复的缺陷而增加。在Specific Aim 2中，我们进一步证明了来自L1双向启动子的siRNA的作用。我们还表征了源自L1 RNA的小RNA序列，并确定L1转录物水平的特异性敲低是否会改变转化细胞的表型。在具体目标3中，我们将测试L1整合的新机制。该机制的假设特征是1)5‘截断是由于L1 RNA的5’降解，2)逆转录酶的模板跳跃是该机制的关键特征，3)DMA第二链的合成是由L1逆转录酶的DNA聚合酶活性进行的。在Specific Aim 3A中，我们确定了哪种RNA聚合酶转录L1以及RNA如何加工（盖帽和聚腺苷化）。在Specific Aim 3B中，我们确定了L1 RNA降解所涉及的过程，特别是，是否涉及死基化，脱帽和5‘降解，以及L1 RNA是否有很大一部分被快速5’降解。这些研究将大大增加我们对L1逆转位生物学的认识。此外，它们将为在哺乳动物中使用L1作为随机插入突变原提供重要的新信息。

DESCRIPTION (provided by applicant): LINE-1, or L1, elements, are the most prolific and prominent mobile elements in mammalian genomes. In the human and mouse genomes, active L1s and their relics account for 17-20% of the genome mass. By providing activities necessary for mobility of Alu and other elements, they indirectly account for another 10- 12% of these genomes. L1 retrotransposons move through a duplicative "copy and paste" mechanism. In this renewal proposal, we attack a number of unanswered questions concerning the timing and cell type specificity of retrotransposition, the mechanism of L1 insertion into the genome, and the role of siRNA produced by the bidirectional L1 promoter in the control of retrotransposition. In Specific Aim 1, we seek to determine the fraction of total retrotransposition events that occur in male germ cells, female germ cells, and in the early stages of embryonic development. We also wish to determine whether retrotransposition decreases with age, is suppressed after a number of generations, and is increased by defects in methylation or DMA double strand break repair. In Specific Aim 2 we develop further evidence of the role of siRNA derived from the L1 bidirectional promoter. We also characterize the small RNA sequences derived from L1 RNA, and determine whether specific knockdown of L1 transcript levels alters the phenotype of transformed cells. In Specific Aim 3, we will test a novel mechanism of L1 integration. Postulated features of this mechanism are 1) 5' truncation is due to 5' degradation of L1 RNA, 2) template jumping of reverse transcriptase is a key feature of the mechanism, and 3) synthesis of DMA second strand is carried out by a DNA polymerase activity of L1 reverse transcriptase. In Specific Aim 3A we determine which RNA polymerase transcribes L1 and how the RNA is processed (capped and polyadenylated). In Specific Aim 3B we determine the processes involved in L1 RNA degradation, in particular, whether deadenylation, decapping and 5' degradation are involved and whether a significant fraction of L1 RNA is rapidly 5' degraded. These studies will increase greatly our knowledge of the biology of L1 retrotransposition. In addition, they will provide important new information towards the effort to use L1 as a random insertional mutagen in mammals.