RNA localisation and transposition of a non-LTR retrotransposon

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.

Non-LTR retrotransposons, as their name implies, have no terminal repeats and transpose by reverse transcription of an RNA intermediate. This is not reverse transcribed in the cytoplasm using a tRNA as primer, as is the case for retroviruses and LTR retrotransposons, but is reverse transcribed in the nucleus using the 3'OH at a nick in the chromosomal DNA as primer, a process known as target primed reverse transcription. The transposition intermediate is also the messenger for the two elements encoded proteins, ORF1p and ORF2p, that are required for transposition. This RNA must therefore leave the nucleus to be translated, associate with its protein products and then return to the nucleus for reverse transcription and integration. The overall objective of the experiments described in this proposal is to gain a better understanding of the cellular events required for transposition of non-LTR retrotransposons. The specific aim of this proposal is to exploit a model system for studying transposition of non-LTR retrotransposons, transposition of the I factor in Drosophila oocytes, to investigate the interaction between the RNA transposition intermediate, element encoded proteins, and host proteins as the transposition complex finds and enters the nucleus. We have already shown that this involves proteins (dynein, Squid/hnRNP-A,) that are used to transport other RNAs within the oocyte, and in particular to move gurken RNA to the nucleus, and we have identified cis acting sequences that are required for these RNA to interact with the transport machinery. By comparing the behaviour of these two RNAs our experiments will also shed light on the mechanisms that move RNAs within cells and allow different RNAs to move to the same sub-cellular location but to achieve different biological ends. We shall investigate the behaviour of RNA and protein within oocytes using high resolution fluorescence microscopy (wide field deconvolution microscopy) and cryo-immuno electron