Title: How does ageing affect post-transcriptional gene regulation in articular cartilage?
Funder: Dunhill Medical Trust

Abstract: Post-transcriptional gene regulation is being increasingly viewed as a level of gene regulation that can be targeted therapeutically in articular cartilage to treat age-related diseases such as osteoarthritis. It is influenced by context dependent alterations in mRNA sequence content, such as the use of alternative polyadenylation sites. There is currently a limited understanding about how this process is influenced in cartilage, particularly in response to relevant physiological factors such as ageing. This proposal will address this by determining how age affects post-transcriptional gene regulation in articular chondrocytes. Using an equine model, which we have shown to have strong parallels with human tissue ageing, we will measure the rate of mRNA decay across the articular chondrocyte transcriptome, using cutting-edge next generation sequencing methodology. To identify the mechanistic processes that govern age and differentiation-dependent regulation of mRNA decay we will also perform transcriptome-wide analysis of polyadenylation events using the same conditions, identifying groups of transcripts whose decay rates are significantly affected by age and that also exhibit alternative polyadenylation. We will establish qPCR assays to determine whether age-specific changes in mRNA decay and alternative polyadenylation also influences mRNA translation using a polysome profiling approach. Follow on studies will use RNA-Seq and qPCR assays to determine that similar polyadenylation site usage is observed with age in human articular chondrocytes. From there we intend to perform proof of concept studies, initially using CRISPR/Cas9 gene-editing in a model human cell line to mutate age dependent polyadenylation sites, determining locations that are able to specifically regulate gene expression. Further development of this approach will examine how efficiently mRNA-specific oligonucleotide antagonists can reproduce the effects of the mutations, and whether this is able to work in primary cells. This work will provide a platform for future translational studies aimed at generating reagents that can target the post-transcriptional regulation of age-associated genes for potential therapeutic gain.

Lay abstract: Osteoarthritis is an age-related disease, characterised by the destruction of the articular cartilage that lines the ends of the bones within our joints. It is a leading cause of disability among older people throughout the world with around 10-12% of the adult population suffering from the disease. This project aims to determine how a specific form of genetic control is affected by old age in cells from articular cartilage. When genes are turned on, the information in their DNA is copied into an intermediate molecule called RNA, which then acts as a blueprint for the production of a particular protein. The number of RNA blueprints is controlled through a balance between how quickly they are made and how quickly they are destroyed. Cells actively destroy the blueprints of some genes at a high rate, which allows them to switch off the gene quickly if required. We have previously shown that the rate of destruction of some RNAs is altered in articular cartilage cells in elderly people who have osteoarthritis. However, we have not yet been able to definitively identify to what degree age itself is a driving factor in this process. This project aims to determine the effect of age on the destruction of the RNA of all of our genes in articular cartilage cells using techniques that allow us to measure all of the genes simultaneously. To identify the mechanisms that lead to age-associated changes, we will also investigate specific structural aspects of RNAs that may affect their longevity whilst also determining whether the RNA of genes affected by age functions differently. Finally, we will perform proof of principle experiments to determine how we can manipulate the structural properties of RNAs that promotes a reversal of the effects of ageing. Overall, this project will answer outstanding questions regarding the healthy ageing of articular cartilage, with great relevance to our understanding of how osteoarthritis develops. Furthermore, it will develop interventional approaches that have the potential to be used to promote healthy ageing of our joints.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;MUSCULOSKELETAL