RNA localisation and transposition of a non-LTR retrotransposon

转座因子是染色体DNA的片段，它在染色体内从一个地方移动到另一个地方，或者从一条染色体移动到另一条染色体。转座子主要有两类：转座子和反转座子。逆转录转座子首先将自身复制成RNA分子，即RNA转座子的中间物，然后将RNA转化回DNA，这一过程被称为逆转录。这就产生了一个新的反转录转座子拷贝，它可以插入到一个新的染色体位置。反转录转座子在这样做的过程中自我复制，从而增加拷贝数。因此，大多数生物体的染色体包含每个反转录转座子的许多拷贝。例如，人类基因组包含大约80万个特定类型的反转录转座子，这是一种称为L1元件的非ltr逆转录转座子。其中只有80-100个仍然能够转座，但这些有可能在短期内损害基因和染色体。从长远来看，它们对人类基因组的进化产生了深远的影响。本研究的目的是详细研究在果蝇染色体中发现的一种名为I因子的非ltr反转录转座子的RNA转座中间体的行为。由于果蝇作为实验生物的技术优势，而且I因子的转位可以很容易地增加，这比其他物种的类似元素更容易被研究。然而，这项工作的结果将与一般的非ltr反转录转座子相关。大多数非ltr逆转录转座子编码两种称为ORF1p和ORF2p的蛋白质。ORF1p可能以某种方式包装RNA转位中间体，而ORF2p则进行转位所需的化学反应。I因子的转位发生在雌性种系中，需要RNA转位中间体和ORF2p进入细胞核。我们之前已经展示过I因子RNA通过运输机器被运送到卵母细胞细胞核，同时也运送细胞RNA gurken，作为卵母细胞发育的一部分，RNA必须到达细胞核。rna转运到特定的亚细胞区室是许多细胞用来执行其功能的一种机制。尽管我们对这一过程了解很多，但仍有很多东西有待学习。本提案中描述的工作旨在解决两个一般的生物学问题，RNA如何在细胞内定位以及非ltr反转录转座子的RNA转座中间体及其相关蛋白如何到达转座的细胞核以及它们如何进入细胞核？第一个将通过比较I因子和断裂rna的行为来研究，因为它们移动到相同的亚细胞位置，以达到不同的生物目的。第二个将通过跟踪I因子RNA和I因子编码蛋白在卵母细胞内的运动来实现。这将使用高分辨率荧光显微镜和免疫电子显微镜的组合来完成。

Transposable elements are segments of chromosomal DNA that move from place to place within chromosomes, or from one chromosome to another. There are two main classes of transposable element, transposons and retrotransposons. Retrotransposons move by first copying of themselves into an RNA molecule, the RNA transposition intermediate, and then turning this RNA back into DNA, a process called reverse transcription. This makes a new copy of the retrotransposon that can insert at a new chromosomal position. Retrotransposons duplicate themselves as they do this and so increase in copy number. As a result the chromosomes of most organisms contain many copies of each retrotransposon. The human genome, for example, contains about 800,000 copies of a particular type of retrotransposon, a non-LTR retro transposon called an L1 element. Only 80-100 of these are still able to transpose but these have the potential to damage genes and chromosomes in the short term. In the long term they have had a profound effect on the evolution of the human genome. The aim of the work proposed in this application is to investigate in detail the behaviour of the RNA transposition intermediate of a non-LTR retrotransposon called the I factor that is found in the chromosomes of the fruit fly Drosophila. This is more amenable to investigation than similar elements in other species because of the of the technical advantages of Drosophila as an experimental organism, and because transposition of I factors can be increased easily. The results of this work will be of relevance to non-LTR retrotransposons in general, however. Most non-LTR retrotransposons code for two proteins known as ORF1p and ORF2p. ORF1p may package the RNA transposition intermediate in some way while ORF2p carries out the chemical reactions necessary for transposition. Transposition of the I factor takes place in the germ-line of females and requires that both the RNA transposition intermediate and ORF2p enter the nucleus. We have previously shown that I factor RNA is transported to the oocyte nucleus by a transport machine that also moves a cellular RNA, gurken, that has to reach the nucleus as part of oocyte development. The transport of RNAs to particular sub-cellular compartments is a mechanism used by many cells to allow them to carry out their functions. Although a good deal is known about this process much remains to be learnt. The work described in this proposal is directed towards addressing two general biological questions, how is RNA localised within cells and how does the RNA transposition intermediate of a non-LTR retrotransposon, and its associated proteins, reach the nucleus of a cell in which it is transposing and how do they enter the nucleus? The first will be investigated by comparing the behaviour the I factor and gurken RNAs as they move to the same sub-cellular location in order to achieve different biological ends. The second will be achieved by following the movement of I factor RNA and I factor encoded proteins within the oocyte. This will be done using a combination of high resolution fluorescence microscopy and immuno-electron microscopy.