Small nucleolar RNAS in OA pathogenesis and cartilage biology

Abstract

Purpose: Small nucleolar RNAs (snoRNAs) are non-coding RNAs of 50-150 nucleotides long with a main task in post-transcriptional modification of ribosomal RNAs (rRNAs). SnoRNAs can be classified into box C/D and box H/ACA snoRNAs. Guided by sequence complementarity between dedicated snoRNAs and positions on the rRNA sequence, the rRNA is site-specifically modified. Box C/D snoRNAs are involved in 2’O-ribose methylation, while box H/ACA in pseudouridylation. A small subset of snoRNAs (e.g., U3, U8 and RMRP), have essential functions in endoribonucleolytic processing of 47S pre-rRNA. Although hundreds of snoRNAs have been identified, their significance is unknown for the cartilage/OA field. We are currently undertaking a number of targeted and discovery studies to investigate the relevance of snoRNAs for chondrocyte differentiation, OA disease progression and OA biomarker identification. Methods: General chondrocyte molecular biology methods were used to isolate, culture and analyse chondrocytes, cartilage and serum/synovial fluid. Transcriptomics was performed using micro-array and RNAseq approaches. Intervention with snoRNA expression was performed by transfecting anti-sense oligonucleotides. Results: Expression of RMRP snoRNA is regulated during chondrogenic differentiation and under control of PTHrP, TGF-beta and BMP-2. RMRP snoRNA function was found to be associated with 47S pre-rRNA processing and necessary for chondrogenic differentiation. Expression of rRNAs and ribosome translation capacity was found to adapt as function of chondrogenic differentiation and RNAseq expression profiling of ATDC5 chondrogenic differentiation uncovered a variety of snoRNAs with differentiation phase-dependent expression profiles. We investigated snoRNA expression in mouse knee joint ageing and post-traumatic OA (DMM). Ageing and OA development led to alterations of specific snoRNA expression in whole mouse joints. Micro-array expression profiling of young, old-protected and old-OA human articular knee cartilage demonstrated age-specific and OA-specific expression of snoRNAs. We are now interrogating whether age- and OA-specific snoRNAs functionally interact with the chondrocyte phenotype and may relate to OA-associated changes in chondrocyte biology. Indeed we have found evidence that alterations in U3 snoRNA expression leads to OA-like changes in the chondrocyte phenotype and that expression of U3 snoRNA can be influenced by morphogens and cytokines. This is associated with changes in 47S pre-rRNA maturation and mature rRNA levels. Also knock-down of OA-specific snoRNAs SNORD78, SNORD26 or SNORD96 induces changes in chondrocyte differentiation status and rRNA levels. Our snoRNA biomarker studies have identified ageing- and OA-specific expression of snoRNAs. In a mouse ageing model we discovered that in the serum of aged mice higher expression of SNORA31 and SNORD23 can be detected and that SNORD116 is dramatically elevated in the serum of mouse and equine OA subjects. We are now exploring the snoRNA content of healthy and OA synovial fluid to identify snoRNA biomarkers for early OA development and investigate whether synovial fluid-mediated inter-tissue communication within the joint involves snoRNAs. Conclusions: Collectively our data show for the first time that specific snoRNAs are differentially expressed due to chondrocyte differentiation and in ageing- and OA cartilage. Expression of specific snoRNAs responds to morphogens and cytokines and our identified snoRNAs influence the phenotypic behaviour of the chondrocyte, potentially via alterations in processing and post-trancriptional modification of rRNAs. SnoRNAs provide an unexplored class of non-coding RNAs relevant for chondrocyte biology. We are now studying further in-depth the variety of identified snoRNAs in chondrocyte differentiation, -homeostasis and -disease processes and identifying snoRNA candidates that may be used as potential biomarkers for OA.