Function and structure of specialised ribosomes in the testis

All proteins in animals, plants and bacteria are made by a machine called the ribosome. This is one of the largest complexes in any cell, consisting of ~90 different components. Until recently it was thought that all ribosomes were the same, responsible for synthesising protein without exerting any regulatory activity. But fruit fly genetics and human diseases caused by mutations in the ribosome components suggest that not every component is essential all of the time. Mutations to specific components result in specific disease and developmental problems. For example, some components are important during development (e.g. mouse nervous system) and others during viral infection. So the current thinking is that some ribosomal components can be substituted by alternative components, allowing the ribosome to control which protein it makes. This is termed 'ribosome specialisation'. However, we do not yet understand how this works and what the functional consequences are.

Our recent experiments and data from other researchers indicate that this specialisation of the ribosome is particularly common and extensive in the testis. In particular, we have seen differences in the components within the ribosomes in the fruit fly testis. Since protein production by the ribosome is particularly important during sperm production, we hypothesise that changes in ribosome composition are important during sperm production. Mutations in ribosomal components in the fruit fly result in male sterility. Therefore, understanding this ribosome specialisation has the potential to provide insight into the causes of male infertility in humans.

This project aims to understand the composition of these specialised ribosomes in the testis and how the alterations in composition affect the production of protein. We will do this by understanding 1) how changes in composition affect the overall shape and structure of the ribosome; 2) how specialised ribosomes control the production of specific proteins; and 3) how the ribosome changes during sperm development in the testis. By shedding light into these two downstream effects we will be able understand the importance of specialised ribosomes.

This project is a new collaboration between two experts in two different fields. This allows us to use the most appropriate and cutting-edge approaches to tackle the structure and function of specialised ribosome, including:
- Fruit fly genetics: The fruit fly testis is a great model for studying specialised ribosomes because we can genetically modify the flies and dissect testes to generate enough material for sequencing and structural analysis.
- Cryo-electron microscopy, which allows us to image ribosomes at a close-to-atomic resolution.
- Next Generation Sequencing: To characterise how protein synthesis is affected by the different specialised ribosomes, we will use a novel sequencing approach to identify all the RNAs that the specialised ribosomes are decoding into protein. This is a high-throughput technique to look at all the protein production taking place in cells.

This is a very novel area of research and this project has the potential to change the way we think about how protein production is controlled. It also has the potential to provide a basis for therapeutic targeting of a range of human diseases and disorders. A group of diseases are caused by mutations to the different ribosomal components, termed ribosomopathies, including Diamond-Blackfan disease. By understanding changes in ribosome composition and the functional importance of the different components we will shed light on the causes of these ribosomopathies.

Our understanding of 'the ribosome' and mRNA translation has been transformed by the discovery that specialised ribosomes exist and regulate the translation of specific mRNAs. It is now evident that not all ribosomal proteins are equally important to the translation of all mRNAs. For example, RpL38 is required for the specific translation of Hox mRNAs, which is essential for neural specification in mice. Here, for the first time, we will determine the underlying structural implications of changes to ribosomal protein composition and how this facilitates specialised ribosome function. Using an exciting combination of genetics, biochemistry, translatomics and structural biology we will uncover the structure-function relationship of specialised ribosomes. We aim to determine 1) specialised ribosome composition, 2) translational output, 3) structural modifications resulting from altered protein constitution and 4) biological importance during spermatogenesis. This will enable us to dissect how changes in ribosome composition result in ability to translate specific pools of mRNA, and how differences in the ribosome structure mechanistically regulate mRNA translation events, contributing to differentiation in testes. We will use the Drosophila testis as a model because i) of the importance of mRNA translation to spermatogenesis, ii) mutations to ribosomal ribosomes result in male infertility, iii) numerous ribosomal proteins are enriched in testes. Most importantly we have identified altered ribosomal protein composition in the D. melanogaster testis, which provides evidence for the existence of several different ribosome populations in the testis. Our cutting-edge strategy to address one of the most important and exciting questions in gene expression at the moment, has the potential to shed light on molecular mechanisms underlying human diseases caused by mutations to ribosomal proteins e.g. Diamond-Blackfan, and the molecular mechanisms contributing to male infertility.

This exciting and cutting-edge proposal will dissect the structure-function relationship of specialised ribosomes in the Drosophila testis. This work will provide insight into i) how translation is regulated in the testis, ii) the complexity of ribosome composition, iii) differences in ribosome structure, iv) mechanisms of translation regulation by ribosomes and v) biological importance of specialised ribosomes. In the future the discoveries that we make will contribute to improved understanding of human diseases caused by mutations in ribosome proteins and molecular causes of male infertility. Here we outline who will benefit and how;

General public: This research project will enable us to develop a new program of public engagement. This will improve our communication of our science with the public in a number of ways; i) Astbury Conversation conference public engagement event stand on ribosomes, ii) Discovery Zone stand (for school children), and iii) articles in Frontiers for Young Minds. We aim to improve understanding of the ribosome, the importance of translation and its potential to go wrong and result in human disease (within project time-frame and beyond).

Other academics: Academics from several fields will benefit from this project. Researchers in the ribosome field with benefit from additional high-resolution structures of ribosomes. The mRNA translation field will benefit from improved understanding of mRNA translational regulation by specialised ribosomes. The Ribo-Seq community will profit from open access tools and protocols that we will develop. The Drosophila and testis communities will also gain from additional tools and improved knowledge of how gene expression is regulated during spermatogenesis. Protocols of using Drosophila tissues for complex biochemistry and genomics will also be of benefit (within project time-frame and beyond).

Highly-skilled workforce: The PDRA and RA on this project are critical beneficiaries. We pride ourselves in providing a positive environment for the furthering of scientific knowledge and the acquisition of new skills. These researchers will be trained in exciting new approaches and given scope to develop as well-rounded scientists with opportunities for dissemination of the project nationally and internationally, engagement with wider audiences and further training to prepare them for future employment. They will access the world-class continued professional development training available at the University of Leeds. We will also aim to recruit PhD students (e.g. BBSRC DTP) (within project time-frame).

Healthcare professionals: Mutations in ribosome proteins, termed ribosomopathies, are the underlying cause of several poorly understood human diseases, e.g. Diamond Blackfan. Our research will provide insight into why specific mutations result in such specific diseases. Further insight into variations in ribosome structure and composition will be extremely valuable in providing potential therapeutic targets for the future. There are also many genetic contributions to human male fertility that are under appreciated. Our work on translation in the testis may provide insight into key proteins and mRNA translation events, which could be essential for sperm production in flies and humans. (beyond project time-frame).

Communication: To ensure our research reaches its impact potential we will strive to communicate our questions and results effectively to these groups. This will involve a) publishing in open-access journals, b) presenting our work at conferences, c) training researchers in LeedsOmics and Astbury Centre, d) public engagement area of website, e) establishing ribosomopathy network, e) taking part in public engagement events, f) writing articles for Frontiers for Young Minds.

In summary, several sectors of society will benefit by our proposed research plans and we have developed ways in which to maximise this and measure the impact over time.