Post-meiotic transcription and RNA localisation in Drosophila spermatid bundles

Within any organism different cells have specialised functions. To develop these specialisations they turn on some genes, and turn off other genes, by regulating transcription of DNA into messenger RNA (mRNA). Sperm are extremely specialist, so show dramatic changes in gene expression during their development. Most of this happens relatively early in sperm production. We are working on fruit fly sperm formation, and have found a set of genes (that we call comets and cups) that are transcribed very late in sperm formation (long after we thought that transcription was impossible). Within cells different regions have different functions, mediated by differential localisation of proteins within the cell. Many cells have an inherent polarity / sperm are long and thin, with their nucleus at one end and a motile tail at the other. The mRNAs from the comet and cup genes are specifically localised to the very end of the tail of the developing sperm tails / 1.5mm away from the nuclei where they are made. In this project we will examine the control of the very late gene transcription. We will determine exactly when in the process of sperm formation the transcription occurs, and test the importance of some known transcription factor proteins in the process. We will map the region of genomic DNA important for promoting the late transcription. On the mRNA transport side we will develop a system in which we will inject fluorescent mRNA into forming sperm, look at localisation, and see what sequences we have to change on the mRNA to stop it localising properly. To examine how the mRNA is transported we will determine what cytoskeletal elements are important (analogous to train-tracks or roads), and what does the transporting (trains? lorries? cars?). In the third aspect of the project will use genetics to determine what role the genes play in normal sperm biology. This will give us clues as to why their transcription and mRNA localisation patterns are so unusual.

Production of the protein product from a specific gene can be regulated at many different stages, including transcription, mRNA maturation and transport and translation. During development and cellular differentiation the transcriptional profile of cells changes dramatically as they take on more specialised roles. Asymmetric localisation of transcripts within a cell allows for localised production of the protein product. We have recently discovered a set of Drosophila genes (collectively referred to as comets and cups) whose transcription occurs very late in spermiogenesis / long after all other transcription has been switched off, and after chromatin compaction. Moreover, the mRNAs derived from these genes are translocated to the end of the elongated spermatids / a distance of more than 1.5 mm. We propose a targeted series of experiments to examine the mechanisms regulating the late transcription and mRNA transport of the comets and cups. We will determine the timing of transcription relative to cellular events of spermiogenesis, particularly nuclear shaping and protamine loading and test the importance of several candidate transcription factors in transcription of comets and cups. We will use reporter constructs in transgenic flies to map the important promoter regions. To analyse mRNA transport we will develop an in vitro injection system and use this to map the RNA localisation motifs. We will also use this system to determine whether the transport is microtubule or microfilament dependent. Examination of transcript localisation in mutants of candidate transport genes will begin to identify the transport machinery involved. To assess to biological role of these comet and cup genes we will identify loss of function mutants. Careful phenotypic analysis will allow us to identify any spermiogenesis defects, and also allow us to determine any interdependence relationships between the genes. By studying the transcription, localisation and function of these genes we can gain insight into transcriptional activation in compact chromatin regions and into the mechanisms of specific mRNA translocation in specialised polarised cells.