RNA localisation and transposition of a non-LTR retrotransposon

转座因子是染色体DNA的片段，其在染色体内从一个位置移动到另一个位置，或从一个染色体移动到另一个染色体。转座因子主要有两类：转座子和反转录转座子。反转录转座子首先将自身复制到RNA分子中，即RNA转座中间体，然后将该RNA转回到DNA中，这一过程称为逆转录。这使得反转录转座子的新拷贝可以插入到新的染色体位置。反转录转座子在复制的同时也复制自己，因此拷贝数增加。因此，大多数生物的染色体含有每个逆转录转座子的许多拷贝。例如，人类基因组包含大约80万个拷贝的一种特定类型的反转录转座子，一种称为L1元件的非LTR反转录转座子。其中只有80-100个仍然能够转座，但它们有可能在短期内破坏基因和染色体。从长远来看，它们对人类基因组的进化产生了深远的影响。本申请中提出的工作的目的是详细研究在果蝇的染色体中发现的称为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，以及I因子的转座可以很容易地增加，这比其他物种中的类似元件更容易进行研究。然而，这项工作的结果通常与非LTR反转录转座子相关。大多数非LTR反转录转座子编码两种蛋白质，称为ORF 1 p和ORF 2 p。ORF 1 p可以以某种方式包装RNA转座中间体，而ORF 2 p进行转座所需的化学反应。I因子的转座发生在雌性的生殖系中，并且需要RNA转座中间体和ORF 2 p都进入细胞核。我们以前已经表明，I因子RNA被运输机运输到卵母细胞核，该运输机也移动细胞RNA，gurken，作为卵母细胞发育的一部分，必须到达细胞核。RNA向特定亚细胞区室的运输是许多细胞用于允许它们执行其功能的机制。尽管人们对这一过程了解很多，但仍有许多东西有待了解。本提案中描述的工作是针对解决两个一般的生物学问题，RNA如何定位在细胞内，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gurken RNA的行为来研究，因为它们移动到相同的亚细胞位置以实现不同的生物学目的。第二种是通过跟踪I因子RNA和I因子编码蛋白在卵母细胞内的运动来实现的。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.