Osteoarthritis is a leading cause of disability especially in our ageing society affecting 10% men and 18% women over 60 years old becoming one of the major health problems in the western world. Articular cartilage damage is the dominant hallmark of osteoarthritis, which is age-related and for which there are no disease-modifying treatments. By understanding the processes involved in cartilage ageing we could identify therapeutic targets. We have previously published the mRNA and small non-coding RNA transcriptome signatures in cartilage ageing using RNASeq/small RNASeq and described a dysregulation of matrix, anabolic/catabolic genes as well as small nucleolar RNAs (snoRNAs) and some snoRNA mechanistic roles in chondrocyte biology.
Articular chondrocytes are specialised cells residing in cartilage. Their primary function is maintaining the extracellular matrix requiring continuous mRNA translation by ribosomes. Post-transcriptional modification (PTM;guided by snoRNAs) of ribosomal RNAs fine-tune their translational activity. Ribosome biogenesis and activity is dysfunctional in osteoarthritis and targeting ribosomes is a novel avenue for therapeutics.
With a limited understanding of how translation regulation mechanisms affect pathological processes in age-related diseases such as osteoarthritis we hypothesized that rRNA 2’O-Me and ψ PTM profiles are altered during ageing, affecting chondrocyte translational modus and contributing to osteoarthritis.
Objectives and experimental approaches
1. Detect and quantify global 2’-O-ribose methylation (2’O-Me) and pseudouridylation (ψ) of rRNAs in ageing human chondrocytes; using RiboMethSeq and HydraPsiSeq.
2. Generate snoRNA knockout in human primary chondrocytes; using LentiCRISPRv2/Cas9 and measuring effects on rRNA PTM profiles.
3. Determine types of translation for selected snoRNAs from objective 2 such as translation initiation (cap/IRES – mediated) and translation fidelity: using polysomes combined with RT-qPCR and ribosome profiling.
4. Determine if the ribosome composition and cellular proteome is different in cells lacking selected snoRNAs from objective 2; using mass spectrometry and label-free quantification following ribosome purification.
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