The role of the transfer RNA repertoire in generating secretory phenotypes during epithelial homeostasis

Organs such as the intestine need to constantly renew their cells in order to maintain integrity. To do this, the intestine has a small population of stem cells which keep growing and dividing to provide a supply of new cells to support gut function. These stem cells are situated in a special niche in the crypts of the gut, and the supply of cells that they produce move out of the crypt and into other parts of the intestinal membrane. Upon leaving the crypt, gut cells stop dividing and start to perform other important functions, such as the production of secretions that assist digestion. All cells make proteins, and the catalogue of proteins that they make varies according to their function. Thus the population of gut stem cells that are rapidly proliferating must make proteins to support cell growth and division. However, when gut cells leave the crypt and stop growing, they cease to make proteins for cell growth and turn their hand to large-scale production of secretions. The cell's protein synthesis machinery must adapt to this major change in the menu of proteins it produces. This project will investigate the alterations to the protein synthesis machinery that occur as cells leave the stem cell niche in the intestinal crypt, stop dividing and start to make secreted proteins in large quantities. We will then make genetically-modified mice with altered protein synthesis machinery to determine how this influences the secretory function and integrity of the intestine. These genetically modified mice and the information that we gain from them will enable us to determine how a complex tissue like the gut is able to structure itself, and how it perform functions such as nutrient adsorption and the maintenance of the gut microbiota.

Much work has focussed on how regulation of mRNA translation can drive protein synthesis to support cell growth. However, less attention has been devoted to the role played by translational control during differentiation - when the protein synthesis machinery tends to reduce production of cellular components, and switches its attention to generation of large quantities of secreted proteins. For example, certain differentiated lineages of the intestinal epithelium produce copious quantities of anti-microbial proteins and the extracellular matrix (ECM) that constitutes the intestinal basement membrane.
There are differences in the transfer RNA (tRNA) repertoires of proliferating and differentiated cells - and it has been proposed that this might contribute to changing landscapes of protein synthesis during differentiation. We are interested in how tRNA repertoires might influence translational control, and have recently discovered that a transgenic animal with a defined alteration to its tRNA repertoire displays marked alterations to its translational landscape which are largely restricted to secreted proteins. Indeed, fibroblasts from mice with extra copies of the initiator methionine tRNA gene produce a markedly different profile of ECM components which leads to altered tumour angiogenesis.
This project is focussed on experimentally determining the tRNA repertoires that are associated with acquisition of secretory phenotypes in vivo, and to what extent these are responsible for dictating the complex secretomes of differentiated cells. We will characterise alterations to the tRNA repertoire and secretory translational landscape as intestinal stem cells differentiate in vivo and ex vivo. From this, we will investigate how genetic alterations to the tRNA repertoire influences intestinal homeostasis - in particular the generation of secretory phenotypes associated with differentiated intestinal lineages.

ACADEMIC IMPACT - UK AND WORLDWIDE
Secretomes and epithelial homeostasis - The mechanisms controlling protein expression are of fundamental importance to our understanding of cellular processes. Although there has been substantial international effort to understand how the selectivity of mRNA translation contributes to cellular protein production, little is known concerning how translational selectivity commands the production of complex secretomes. Knowledge of how cellular secretomes are generated and how their output and complexity is maintained is key to understanding a range of important physiological processes. In particular the differentiation of epithelial stem cells into their various lineages which is key to maintenance of epithelial function - and this project will focus on how this occurs in the intestinal epithelium. Indeed, the secretome of Paneth cells is important for maintenance of the stem cell niche at the base of intestinal crypts, but little is known about how RNA transition contributes to this. Furthermore, it is not yet known how differentiated lineages support the production of large quantities of the ECM proteins which constitute the epithelial basement membrane, and knowledge of how the translation machinery contributes to this will be invaluable to understanding how the architecture of epithelia is established and maintained.
The tRNA repertoire and translational selectivity - It is now being realised that the processes which match the tRNA repertoire with codon-usage underpin gene expression in complex organisms. However, experimental evidence supporting this is, so far, largely correlative. We have already made defined alterations to the tRNA repertoire in vivo and reported, using un-biased approaches, the consequences of this on expression of secretory proteomes. Our approach to continue to map alterations to tRNA repertoires during differentiation, and to experimentally manipulate these to determine the effect of this on secretory proteomes and epithelial homeostasis will provide important insight into a process which is now being acknowledged to be fundamental to life.

CLINICAL IMPACT
There are a number of disorders that are linked to secretory dysfunction in epithelia and activated fibroblasts. Wound repair is often compromised in the elderly and in diabetes and this is, in part, owing to reduced ability of fibroblasts to secrete sufficient ECM. Furthermore, fibrosis can often result from aberrant secretion of ECM proteins by fibroblasts and stellate cells which are present in tissues such as the liver and the lungs. The secretomes of activated fibroblasts present in tumours are closely linked to cancer progression, and an important part of this project is focussed on the role of the tRNA repertoire in generating the pro-invasive secretome of carcinoma-associated fibroblasts.
Cancers and dysplastic disease are the ones in which we have the most interest, and in which we have substantial expertise and resources. We have extensive experience in collaborating with clinicians to investigate aspects of our findings that have relevance to cancer and other diseases. Indeed, we have recently reported, in collaboration with a medical oncology group, that elevated levels of iMET and collagen II secretion are linked to the progression of serous ovarian carcinoma. We will continue to pursue any aspects of our findings that have relevance to disease and which exhibit the potential to lead to new therapeutic strategies.