Targeted mRNA degradation in Drosophila spermatogenesis

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.

Pacman is a highly conserved cytoplasmic exoribonuclease which has been shown to processively degrade mRNAs in a 5'-3' direction after they have been decapped. Pacman/Xrn1 is not only involved in the cytoplasmic turnover of RNA but is also required during RNA interference, degradation via microRNAs and nonsense-mediated decay. Our previous results have shown that mutations in pacman result in reduced male fertility and morphologically abnormal testes. More recently, we have shown that pacman mutants have significantly fewer testis stem cells than wild-type controls. Since Pacman is a exoribonuclease, mutations in pacman would be expected to result in increased expression of candidate target mRNAs. Our preliminary results using micro-array analysis supported by quantitative RT-PCR experiments, show that a specific set of mRNAs in pacman mutant testes are expressed at levels more than 10-fold higher than in controls. We found that the set of mRNAs that are expressed at the highest levels in mutant testes are not the same as those expressed at highest levels in mutant imaginal discs. These results suggest that particular mRNAs, which are essential for stem cell renewal and maintenance, are susceptible to degradation by Pacman and that the targeting mechanism is tissue specific. The aim of the project is to understand how the conserved exoribonuclease Pacman, which shows no specificity in-vitro, can specifically target mRNAs in-vivo. Base on previous results, we have developed a hypothesis to explain this targeting. This proposal aims to test this hypothesis and so gain understanding of the molecular mechanisms involved. The complementary techniques to be used will make use of the wide range of tools and reagents available in the Drosophila model system. These innovative studies will help us to understand the mechanisms whereby particular RNAs can be targeted for degradation in a tissue-specific context not only in Drosophila, but also in mammalian cells.

Who will benefit from this research? This project is primarily 'basic, blue-sky research' which nevertheless is likely to lead to new insights important for new therapeutics in the future. Beneficiaries in the public sector already include clinicians, who are benefiting from the knowledge and techniques developed from our current BBSRC-funded research. Since the focus of the proposed project is on stem cells, it is relevant for many fields of clinical research. Other potential beneficiaries include Industrial collaborators and the general public, particularly school children. How will they benefit from this research? Clinicians will benefit from the proposed research because the project addresses the regulation of gene expression in stem cells. Since stem cells are important in many new therapeutics and in cancer, this project will provide fundamental insights which will be relevant to their research. The skills and expertise gained by the post-doc during the project will also benefit clinicians. The cross-fertilization between the BBSRC-funded research of my group and local clinicians/scientists has already resulted in collaborative projects. For example, local clinicians have already benefited from our expertise in microarray analysis in a joint project on the use of microRNAs as biomarkers in certain leukaemias. This research, which is funded by a small grant, has already shown excellent promise and we hope to develop a suitable biomarker within the next two years. In addition, in an existing collaboration with Dr. Liz James, from the University of Brighton, we are using our research skills to help her and her industrial partners to develop new therapeutics for wound healing, which involve seeding of cells on novel biomaterials. Therefore, the continuation of these collaborations, aided by the proposed BBSRC grant, is likely to enhance the quality of life in the UK. The research skills gained by both the post-doc and the research technician will be valuable to their future careers and applicable to Biomedical Industries. In addition, the post-doc will be in a position to teach high-level techniques to PhD students including clinical PhD and MD students. The proposed project will therefore benefit the biomedical knowledge of future NHS clinicians. What will be done to ensure that they benefit from this research? The existing collaborations will be fostered and research staff encouraged to share ideas and techniques with each other. For the biomarkers work, my collaborator and I intend to seek 'seedcorn' funding to develop our novel biomarker test; this will be through University of Sussex Enterprize schemes or suitable NHS funding. IP issues will be managed through the University of Sussex offices. For the wound healing work, my collaborator and I have already applied for a 'seedcorn' grant to fund a post-doc to develop the project; the work will be enhanced by her contacts with local Industrial Companies and my links with NHS physicians interested in wound healing, particularly in diabetic ulcers. Communication and engagement plans include presentation of our work at local events such as the Sussex Cancer stem cell Network meetings and the South Coast RNA network meetings which are attended by representative from Pharmaceutical Companies (both organised by the University of Sussex Business and Enterprize office). We will also continue to host school children in our labs to give them a 'taste of science' and encourage them to take up Biomedical Science as a career. We will also continue to participate in Open Days to showcase our research.