Investigating the Role of Extracellular Vesicle-Mediated Transfer of snoRNAs and Ribosomes in Joint Ageing

About the Project

Ageing causes significant deterioration in musculoskeletal tissues, especially cartilage, contributing to osteoarthritis. Small nucleolar RNAs (snoRNAs) and extracellular vesicles (EVs) change with age and may drive cartilage decline by altering ribosome function and transferring regulatory RNA cargo. We propose EV‑mediated snoRNA and ribosomal transport promotes age‑related joint degeneration.

The deterioration in tissue function associated with increasing age is an emerging clinical challenge. Age-associated cellular and molecular changes in tissues have been reported by numerous studies. However, musculoskeletal tissues are profoundly underrepresented. This is even though age-related musculoskeletal decline and degenerative diseases such as osteoarthritis contribute greatly to the high prevalence of disability in older adults (affecting 30-60% of people over 65 years) and overall healthcare spending.

Cartilage is a crucial component of the articular joint, providing a smooth, lubricated surface for joint movement and acting as a shock absorber. As we age, cartilage undergoes significant changes, including decreased cellularity, altered extracellular matrix composition, and increased stiffness. These changes contribute to the development of osteoarthritis, characterized by the breakdown of cartilage, pain, and reduced joint function.

Small nucleolar RNAs (snoRNAs) are emerging as important regulators of cellular processes in health and disease. SnoRNAs are a class of highly evolutionary conserved small non-coding RNAs. Classically, snoRNAs guide site-specific modifications of ribosomal RNAs (2′-O-methylation and pseudouridylation), thus fine-tuning ribosome function. Furthermore, many snoRNAs have non-canonical functions, guiding modifications of other RNA species and regulating gene expression.

We have reported differential expression of snoRNAs in joint health and disease, human and equine osteoarthritic and ageing cartilage, equine synovial fluid in ageing, murine osteoarthritic joints and serum, human mesenchymal stem cells, and chondrogenic differentiation. These data suggest altered expression of snoRNAs in ageing. Based on our previous findings, I postulate that snoRNAs are crucial drivers of ageing by regulating ribosomes, fine-tuning cellular translation, and through other non-canonical mechanisms.

Extracellular vesicles (EVs) are a heterogeneous group of secreted membranous vesicles whose cargo comprises DNA, RNA, proteins, lipids, and metabolites. They facilitate cell-to-cell communication and exert functions in tissue homeostasis, (patho)physiological processes, and ageing. Studies have shown that the number of EVs secreted by the cell and their content change in ageing. We have demonstrated that joint cells exchange snoRNAs and possibly even ribosomal subunits and fully assembled ribosomes via EVs.

We hypothesise that EVs transport snoRNAs and ribosomal components (possibly even functional ribosomes), contributing to age-related changes in cartilage, promoting osteoarthritis.

Objectives

1. Determine the snoRNA profile in EVs isolated from young and old chondrocytes.
2. Describe the rRNA and ribosomal protein cargo in EVs derived from young and old chondrocytes.
3. Investigate the role of ribosomal components transferred via EVs in chondrocytes and their impact on cellular translation and function.

Methods

Research design

This PhD will employ a mixed-methods approach, combining quantitative and qualitative research methods

Cell isolation and culture

Equine primary chondrocytes will be isolated from young and old horses using established protocols.

EV isolation and characterisation

EVs from young and old chondrocytes will be isolated from cell culture media using size exclusion chromatography and characterized with nanoparticle tracking, electron microscopy and Western blotting for CD9, CD81, and CD63.

EV cargo in ageing chondrocytes

1. RNA Sequencing
   Total RNA will be isolated from EVs from young and old chondrocytes and snoRNA sequencing undertaken.
2. Mass spectrometry proteomics
   Proteins including ribosomal protein changes in EV cargo in ageing chondrocytes will be assessed using mass spectrometry label-free quantification.

Ribosome and polysome profiling

It will be determined whether entire ribosomal subunits (40S and 60S) and fully assembled functional ribosomes (80S) and polysomes are present in EVs using translational competency assessment techniques.

1. In vitro translation assay
2. To assess the translational competency of EV ribosomes, a cell-free in vitro translation assay will be employed. Rabbit reticulocyte lysate will be depleted of endogenous ribosomes and reconstituted with EV-ribosome samples. Translation will be allowed under standard conditions in the presence of heavy arginine and heavy lysine for subsequent SILAC-based proteomics.
3. Protein synthesis detection
    Using SILAC, we will detect de novo synthesis of proteins in the in vitro translation reaction. The focus will be on the identification of de novo synthesised beta-globin protein and other proteins encoded by mRNAs co-isolated with EV-ribosomes.

Outcome

This research will provide valuable insights into the role of EV-mediated transfer of snoRNAs in cartilage ageing and whether ribosomal components are functional in EVs. By achieving the outlined objectives, this study will help bridge the gap in the existing literature and inform future research on the mechanisms of ageing in cartilage.