Investigating how alternative splicing processes affect cartilage biology from development to old age

Alternative RNA splicing (AS) and alternative polyadenylation (AP) are two essential mechanisms for the post-transcriptional control of gene expression in eukaryotes. Over 95% of multi-exonic genes are alternatively spliced in humans, allowing a single gene to encode multiple protein isoforms with different (sometimes antagonistic) functions, distinct subcellular localisations and diverse protein-protein interactions. Alternative polyadenylation results in heterogenous transcript pools that share similar protein coding regions but have different length 3'UTRs and transcript stabilities. These two regulatory mechanisms are crucial for normal physiological processes including tissue development, differentiation and homeostasis. Post-transcriptional gene regulation is dysregulated during ageing, and global analysis of mouse tissues revealed that there are an increased number of alternatively spliced genes with age. Alternative splicing is hypothesised to be an important driver in the ageing process in humans as it is involved in regulating genes that control various hallmarks of ageing, including senescence-associated genes. Furthermore, altered expression of splicing factors, as well as abnormal mRNA splicing/polyadenylation, is a common feature of several age-related pathologies including cancer, cardiovascular disease and neurodegeneration.

Targeted studies have highlighted AS and AP as important modulators of cartilage development and differentiation. For example, splicing of several cartilage extracellular matrix genes is developmentally regulated, leading to changes in the protein isoforms that compose the cartilage matrix. However, the role of alternative splicing and polyadenylation in cartilage development, health and age-related dysfunction has not yet been explored at the global transcriptome level.

The aim of this studentship is globally characterise all transcript isoforms expressed in cartilage across the human lifecourse. The students will use a combination of comprehensive bioinformatics analysis and laboratory based cell and molecular biology techniques to;

1. Globally characterise alternative splicing and polyadenylation events in cartilage that occur during embryonic (6wpc, 17wpc) and postnatal development (2-18ys), and during the transition from middle to old age (50-85ys).

2. Identify splicing and polyadenylation changes that take place during the in vitro differentiation of mesenchymal stem cells (MSCs) into cartilage.
3. Examine the effect that specific age-associated mRNA isoforms of key cartilage genes have on cartilage homeostasis and response to physiological stimuli.

Using existing cartilage RNA sequencing datasets, bioinformatics tools will be used to identify cartilage development, differentiation and age-related alternative splice and polyadenylation isoforms. Online tools (e.g. DoChaP and PFAM) will be used to predict the effect of splicing events on the encoded proteins. To mechanistically understanding how splicing changes during development and ageing, mRNA isoforms will be scanned for the binding sites of splicing regulators. Focusing on key cartilage genes, ~100 selected age-related AS and AP events will be validated using capillary gel electrophoresis in a new ageing cohort, with effects on a subset of key proteins tested by western blot. Five AS and five AP events will be manipulated using AONs and the CRISPR-Cas9 system respectively in primary chondrocytes, the SW1353 human chondrosarcoma cells and during chondrogenic differentiation of MSCs, with the effects on chondrocyte differentiation, proliferation, apoptosis, senescence and response to pathological stimuli examined using qRT-PCR, western blot, immunohistochemistry and biochemical assays.