Targeted mRNA degradation in Drosophila spermatogenesis

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Stem cells have a vast potential in regenerative medicine for the replacement of defective tissue. They therefore offer a potential cure for injuries and also for degenerative diseases such as Alzheimers and Duchenne muscular dystrophy. In the testis, stem cells are required to maintain the supply of sperm, so that the male has potential to produce offspring for much of his life. In recent years, there have been rapid advances in the understanding of the genes which are required to be switched ON to produce self-renewing stem cells that are capable of developing into a wide range of tissues e.g. skin or nervous tissue. However, it is now known that it is also crucially important to make sure that certain genes are switched OFF to prevent stem cells from spontaneously differentiating into particular cell types or developing into cancer cells. We have recently discovered that an enzyme named Pacman, which is involved in the destruction of messenger RNA molecules, is necessary for stem cell function in testis cells of the fruit fly Drosophila. Messenger RNAs are the molecules which instruct the cell to make particular proteins. By comparing mutant flies with normal flies, we have found out that there are fewer testis stem cells in the pacman mutant leading to fewer sperm and offspring. Using various genetic and molecular techniques we have found out that Pacman is likely to destroy particular RNAs that would otherwise prevent stem cell division or cell death. This is interesting as it shows that these mRNAs must somehow be 'tagged' allowing Pacman and its partners to identify, 'hunt down' and destroy these particular RNAs. The specific aim of this project is to understand, in molecular terms, exactly how Pacman and its partners can identify and destroy its correct target RNAs and how this is controlled. The ability of Pacman to selectively destroy target RNAs is not intrinsic to itself as isolated Pacman protein in the test tube cannot distinguish between different RNAs. We know that different RNAs are selected in different tissues so how does Pacman and its partners do this? Our hypothesis is that a particular protein (or a tiny regulatory RNA) binds to a specific feature or the target RNA and 'tags' it for destruction. This tag is then recognised by Pacman and its partner proteins. We propose that Pacman, which is a large protein, can act as a scaffold to assemble other proteins upon it. When the correct partners are assembled, in the correct 3-dimensional shape, then a decapitation enzyme snips off the end of the RNA and the rest is rapidly chewed up. In this project, we aim to find out the details of this destruction pathway, including the ways that the target is tagged. This work will increase our general understanding of the ways that genes are specifically switched off in response to their cellular context. Since Pacman is known to be important in other important cellular events such as wound healing and migration of cell sheets, this work may also shed light on the molecular mechanisms of these processes. Although this project will be carried out using the fruit fly Drosophila, it also has relevance for stem cell function in humans as the cellular processes are surprisingly similar in both organisms. In addition, the Pacman enzyme is extremely similar between flies and humans therefore the insights we gain during this project may help us to improve treatment for fertility and also help us to understand the ways that stem cells remain virtually immortal for the lifetime of the organism. This project will therefore provide valuable insights into the ways in which specific RNAs are targeted in a particular cell type which can be used in the development of new therapeutics.