Identification of the role of small nucleolar RNAs in cartilage ageing

Description

To reduce health inequities and improve the lives of older people we need to understand the ageing process at the molecular level. This will promote ‘healthy’ ageing. Alterations in small nucleolar RNA (snoRNA) levels represents a novel consequence of ageing, enabling future therapies for age-related diseases.

The majority of snoRNAs direct post/co-transcriptional chemical modifications of RNA substrates, including ribosomal RNAs. Site-directed 2’-O-ribose-methylation (2′-O-Me) or pseudouridylation (Ψ) of target RNAs by specific snoRNAs functionally fine-tunes translation. Whilst their importance in cell protein synthesis machinery is documented, it is unknown if age-related rRNA chemical modifications changes ribosomal translational capacity.

We mapped the rRNA post-transcriptional modification (PTM) landscape using an in vitro model and identified osteoarthritis (an age-related joint disease) dependent changes in rRNA PTMs; demonstrating for the first time in mammalian cells that CRISPR/Cas9 functional impairment of SNORA22 and SNORA33 mediated Ψ U5001, leading to putative pathogenic ribosome functional changes. 

We identified differential expression of snoRNAs in ageing human/equine cartilage, and murine joints. A metanalysis of these datasets identified putative age-related candidates conserved across species.

Hypothesis and aims

We hypothesise that snoRNAs are drivers of cartilage biological ageing through both changes in post-transcriptional modification characteristics of the ribosome’s rRNA due to canonical snoRNAs; affecting ribosome biogenesis and protein translation. We postulate that snoRNAs are important age-related molecules whose dysregulation leads to increased vulnerability to age-related degenerative diseases due to altered anabolism/catabolism, or ribosome biogenesis effecting pre-rRNA processing and p53 signalling, important for ribosome biogenesis and protein translation.

We will test our hypothesis with the following methods:

• Using our proven horse model cartilage derived from healthy young and old horses will be subject to snoRNA sequencing and differential expression analysis. RiboMethSeq and HydraPsiSeq will be undertaken to detect and quantify 2’O-Me and ψ of rRNAs. Results will be mapped back to differentially expressed snoRNAs identifying dysregulated snoRNAs that concomitantly changing their specific PTMs.
• We will demonstrate how selected snoRNAs inhibition (with our optimised CRISPR/CAS9 methods) in human chondrocytes/chondrocyte cell lines impacts ribosome biogenesis apoptosis and p53 signalling.
• The cell’s responses following CRISPR to oxidative stress, inflammation, senescence, ER stress will be measured as important mechanisms of joint ageing using qRT-PCR, immunoblotting, IHC and Northern Blotting (rRNA processing).
• We will demonstrate how selected snoRNAs inhibition (with our optimised CRISPR/CAS9 methods) in human chondrocytes/chondrocyte cell lines impacts ribosome biogenesis apoptosis and p53 signalling.
• The cell’s responses following CRISPR to oxidative stress, inflammation, senescence, ER stress will be measured as important mechanisms of joint ageing using qRT-PCR, immunoblotting, IHC and Northern Blotting (rRNA processing).