Long non-coding RNA function during cellular ageing

Ageing is the largest risk factor for most human diseases in developed countries, including progressive diseases such as Alzheimer's and Parkinson's, diseases like cancer that show variable rates of onset, and catastrophic system failures such as heart-attack and stroke. As we are getting older, these age-associated diseases are becoming an ever increasing burden for our society. While the study of specific disease processes has long been a major focus of biomedical research, there is a growing realisation of the importance to study the normal ageing process itself as an essential part of the problem, and to explore ways to slow or reverse its effects. Ageing is a complex process that can be seen as an inevitable feature of the ravages of time. Recent discoveries, however, demonstrate that ageing can be modified in dramatic ways by simple interventions. For example, genetic manipulations or drugs can delay ageing and improve health late in the life of laboratory animals. The processes involved in ageing are similar in different organisms, including humans. A central challenge of ageing research remains to understand biological factors regulating healthy lifespan.

Biological processes such as ageing are influenced by the genetic information encoded in the genome. Genes coding for proteins have long been the main focus of research, while recent findings reveal that genomes also contain many genes that do not code for proteins but for so-called non-coding RNAs. The non-coding RNAs with known functions play critical roles in regulating other genes in different ways. But the roles of most non-coding RNAs are not yet known. Notably, several examples are emerging where non-coding RNAs are involved in ageing and associated diseases. However, how genome regulation and non-coding RNAs affect the healthy lifespan is largely unknown. It is therefore important to find out what roles ageing-associated non-coding RNAs play in the information flow from the genome to longevity.

Using fission yeast as a simple and cost-effective model organism, we have recently discovered a fascinating non-coding RNA that becomes up-regulated during ageing and prolongs the lifespan of yeast cells. We now want to study the detailed function of this non-coding RNA in gene regulation and ageing. Remarkably, gene regulation and ageing are similar from yeast to human, but are much easier to study in the simple yeast. Yeast cells enter a dormant, non-dividing state under limiting nutrients. The transition to this dormant state provides a compelling cellular system to analyse our ageing-associated non-coding RNA and gene-regulatory processes affecting the lifespan in this state. We have created genetically manipulated mutant cells in which the non-coding RNA is either over-abundant, leading to long-lived cells, or completely absent, leading to short-lived cells. In order to study the role of our non-coding RNA during ageing, we will apply complementary genome-wide methods that can systematically determine different regulatory aspects for all genes. We will also combine cells that lack our non-coding RNA with cells that lack different proteins to study the effects of such double mutants on lifespan. This potent approach can point to biological processes that are important for non-coding RNA function. Moreover, we will use current methods to identify genes, other RNAs or proteins that interact with our non-coding RNA and thus may determine its function. Combining these complementary approaches will provide vital clues on biological functions, which we will then test with further detailed analyses to obtain rich information on how our non-coding RNA regulates genes and lifespan.

We anticipate that this project will provide a valuable platform to better understand universal principles of non-coding RNA function for gene regulation during ageing, which could help to eventually develop interventions that extend healthy lifespan in humans.

We have recently discovered a long non-coding RNA, aal1, which extends the chronological lifespan of ageing fission yeast cells. Here we propose an integrated, multi-dimensional project to study how aal1 affects gene expression and longevity. We will analyse different aspects of genome regulation in aal1 overexpression and deletion mutants, along with control cells, before and after entry into a non-dividing, ageing state. To test for aal1-dependent changes in RNA levels or processing, we will use RNA-seq. To quantify rates of RNA synthesis, splicing and degradation, we will analyse transcriptomes of 4-thiouracil labelled cells using mathematical modelling. Depending on results, further genome-wide methods will reveal any roles of aal1 in chromatin or translation. To uncover functional relationships of aal1 with all non-essential proteins, we will systematically screen genetic interactions for both growth and lifespan traits by pioneering a parallel-profiling assay that combines Synthetic Genetic Arrays with Barcode sequencing. To identify RNA-binding proteins, other RNAs or genomic sites to which aal1 binds, we will adopt the ChIRP approach to isolate crosslinked aal1 with biotinylated oligos, followed by mass spectrometry or sequencing of the pull-downs. These complementary, cutting-edge approaches will provide rich insights into regulatory processes affected by aal1. We will follow up our findings with further analyses to elucidate how aal1 regulates genes during ageing, or whether it controls gene-expression fidelity or noise. These analyses will also reveal biological processes by which aa1-mediated genome regulation promotes longevity. This research will thus exploit genetic, genomic, proteomic, cellular and biochemical methods to help uncover fundamental aspects of aal1 function in a simple yet potent model organism, which in turn may reveal general principles of lncRNA function that affect longevity and age-associated genome regulation in other organisms.

Who will benefit from this research?
The proposed research is basic by its nature, and the immediate impacts from this work relate to scientific and knowledge advancement and the development of skills, capacity and capability. In the longer term, this research has the potential to impact areas of wealth and health. Beneficiaries beyond academia therefore are the commercial private sector and the wider public.

How will they benefit from this research?
The proposed project takes state-of-the-art experimental approaches to address fundamental questions relating to non-coding RNA function in genome regulation and cellular ageing. The research will deliver increased capacity and capability in strategically relevant areas of genetics, genomics, proteomics, and sequencing-based approaches with associated computational data mining, through the provision of broad training and the further development of novel genetic screening methods and resources. Establishment of these methods is significant as they have a wide range of applications that reach beyond basic science into fields relating to human healthy ageing, the commercial (pharmaceutical) sector and beyond.

The commercial sector might benefit by recruiting a highly skilled and experienced scientist trained through this project. Ultimately, the pharmaceutical sector will benefit from all the publicly available experimental data that we will make available. Moreover, non-coding RNAs are emerging as useful molecules to diagnose diseases as they are easily detected in body fluids and are easier to manipulate by drugs than proteins. Thus, they might also benefit from fresh drug targets by exploiting principles of non-coding RNA function during ageing established in this project (ageing-associated RNAs that slow ageing when up-regulated). Success in this endeavour could reduce the effects of ageing as the major risk factor for multiple diseases.

The ageing population is a huge and increasing problem in our society, with enormous cost implications due to the economic and social burden of the rise in associated diseases and diminished quality of life for both patients and carers. It is evident that any measures that promote healthy ageing will be of massive, broad-ranging benefit to our society with respect to economy, quality of life, health and creative output. In the longer term, the general public may thus benefit from our fundamental contribution to the understanding of regulatory mechanisms and conserved principles involved in ageing-related processes that will guide and empower research in humans, and could help to develop safe broad-spectrum, preventative measures against age-associated diseases.

Immediate and concrete deliverables with respect to impact beyond academia will be in public engagement, which we recognize as an important responsibility of scientists. We already have experience and established links that will facilitate outreach and effective communication of the research outputs. Details of our specific plans and timelines with respect to public engagement are outlined in the Pathways to Impact.