tRNA biology in healthy ageing: Functional differentiation and expression of tRNAiMet loci in Drosophila.

The proportion of older people in our societies is rapidly increasing. For many, older age comes with increased frailty, impaired function and increased susceptibility to disease. The ageing of our populations is incurring massive personal and socioeconomic costs that need to be urgently addressed.

Ageing itself can be modulated. Understanding this plasticity presents us with an opportunity to devise interventions to improve human health into old age. Importantly, directly targeting ageing has the potential for broad health improvements not limited to a single disease.

This research project will answer a fundamental question in the biology of transfer RNAs (tRNAs) with strong relevance to ageing. tRNAs are the adaptor molecules universally used to translate the genetic code into proteins. Our interest in tRNA biology comes from our work on understanding the molecular mechanisms whereby the activity of a key regulator of cellular protein synthesis, the Target of Rapamycin Kinase Complex 1 (TORC1), promotes animal ageing. We found that TORC1 acts though RNA polymerase III, which itself is responsible for generating tRNAs. Hence, understanding tRNA biology will advance our knowledge of the ageing process.

100s of tRNA genes are present in animal genomes, often as copies of identical sequence, complicating the analysis of their biology. Historically, such identical copies were thought to be simply redundant, providing multiple templates to facilitate high expression levels required for protein synthesis. However, there is growing appreciation that placing tRNA copies in different genomic contexts has allowed animals to finetune tRNA expression patterns thus diversifying organismal functions of identical or similar tRNAs. This hypothesis remains experimentally unaddressed due to lack of suitable animal models and genetic reagents, despite its fundamental importance.

The fruit fly is a small animal but a powerful experimental model that has proven utility in understanding the basic biology of animals, including humans, and how they age. To start probing into the organismal roles of tRNAs, we focused on a tRNA specialised for initiation of protein synthesis, the initiator tRNAMet (tRNAiMet). We generated a set of fly mutants deleting copies of tRNAiMet from four different genomic locations. Our preliminary phenotyping revealed at least one tRNAiMet locus that contributes to ageing and indicated an interplay of unique and redundant organismal functions for the four loci. We will use this set of mutants, together with a set of reporter lines we have generated, to answer a fundamental question in tRNA biology, which will help us understand ageing: Why are multiple, identical copies of tRNA genes present throughout an animal's genome?

Firstly, we will perform extensive phenotyping of single mutants and their combinations to identify organismal functions that are unique to certain loci as well as those that are redundant, with focus on ageing. Secondly, we will use reporter lines to assess the expression from each locus during development and in different adult tissues and organs. We will correlate the two sets of findings as well as integrate them by formally testing if unique expression patterns drive unique organismal functions. Thirdly, we will examine the role of tRNAiMet loci in the plasticity of ageing. Specifically, we will determine if longevity resulting from a reduction in nutrient intake or TORC1 inhibition is in part caused by changes in tRNAiMet levels.

The project will answer a fundamental question in tRNA biology to provide us with a step change in understanding of tRNAs in the context of animal physiology. It will provide us with a pioneering insight into their role in animal ageing, as well as the knowledge required to decipher their organismal functions downstream of TORC1. In turn, this knowledge will inform interventions aimed at ensuring human health throughout the life course.

Ageing and the associated functional decline are of growing medical, social and economic importance. Ageing can be modulated, for example by the activity of Target of Rapamycin Complex 1 (TORC1). However, the basic mechanistic understanding is often lacking.

tRNAs are universal adaptor molecules that translate nucleic acid code into proteins. Our interest in tRNA biology stems from our work on TORC1: We found that TORC1 acts though RNA polymerase III, itself responsible for transcription of tRNAs. Here, we propose to unpick some of the complex biology of tRNAs to understand their role in ageing.

100s of tRNA genes are present in animals, often as identical copies. Such copies were thought to be redundant, however, there is growing appreciation that different expression patterns may underlie a differentiation of their organismal functions. We will experimentally and systematically test this idea here, focusing on the initiator tRNA (tRNAiMet) and ageing.

We generated a unique set of mutants deleting copies of tRNAiMet from four different loci in Drosophila melanogaster. Preliminary phenotyping revealed that at least one tRNAiMet locus contributes to ageing and the four have both unique and redundant organismal functions. Using mutant and reporter lines we have generated, we will answer three fundamental questions in tRNA biology and ageing: 1) Can identical tRNAs have different organismal functions? 2) What are the spatiotemporal expression patters that underlie these functions? 3) Can the reduced expression from one of many of these loci account for the beneficial effects of diet or TORC1 inhibition?

Answering these questions will give us a pioneering insight into why multiple, identical copies of tRNA genes are present throughout an animal's genome and decipher the roles of tRNAiMet genes in development and ageing. This will deepen our understanding of the fundamental mechanisms of ageing to ensure human health and wellbeing throughout the life course.