Connecting the dots: Explaining ageing-driven alternative splicing dysregulation using dietary restriction and mTOR

Biological regulation is a highly complex process and involves many layers of complexity even at the RNA level.

Alternative splicing is crucial in the regulation of which components of a protein-coding gene are spliced into a translatable mRNA, and some level of alternative splicing occurs in most human genes. With age however, comes increasing levels of splicing dysregulation, leading to aberrant protein production.

Mammalian target of rapamycin (mTOR) suppression and dietary restriction (DR) are two treatments which have been shown to improve organismal longevity across many species and in fact both of these treatments have been shown to modulate alternative splicing. Surprisingly, this effect of these treatments is highly understudied in the field of ageing.

This project therefore aims to investigate the connection between these ageing-related processes, specifically identifying whether the lifespan elongation observed upon mTOR suppression and DR is a result of the splicing modulation these treatments produce.

This project will therefore improve understanding not only of alternative splicing regulation itself, but also of the global mechanisms through which ageing drives a reduction in health.

The proposed body of work is comprised of the following research questions: Research question 1: Which classes of splicing events correlate with longevity?

In order to answer this question, I will identify splicing events (such as modulated exon skipping or intron retention) which are shared between DR and mTOR suppression, utilising currently unpublished Drosophila transcriptome data from flies under these treatments.

I will use the extensive lifespan phenotyping of a large panel of inbred Drosophila lines already undertaken in the host lab to identify the 15 longest and 15 shortest living lines, before generating high-quality transcriptomes from these lines, thus identifying splicing events which correlate with increased longevity.

Research question 2: Which components of the spliceosome are essential for which splicing events? Here, I will manipulate expression of individual spliceosome components using a conditional GeneSwitch system.

By sequencing these transgenic flies I will be able to assess splicing event occurrence in each condition, thus enabling mapping of splicing event class to spliceosome component.

Research question 3: Are the splicing event modulations observed in flies under DR and mTOR suppression responsible for the observed increase to longevity?

To answer this final question, I will intentionally modulate shared splicing classes identified in Aim 1 using the spliceosome manipulations from Aim 2 in order to assess longevity under normal conditions, mTOR suppression and DR.

Through the utilisation of each of these conditions, I will be able to elucidate whether genome-wide alterations to each class of alternative splicing are mechanistically responsible for the reduced mortality observed through mTOR suppression and DR, as well as determining whether each splicing event class directly impacts ageing.

The proposed work will contribute to the fundamental understanding of RNA biology and splicing, whilst simultaneously improving our understanding of the underlying mechanisms of age-related disease.

Through the identification of promising therapeutic targets to improve health in the elderly, this work thus also has the potential to improve the quality of life for older people.