Mandy Meekels Steinbusch

The cellular processes in which snoRNAs participate are fundamental processes needed for proper cell function, yet their roles in cell differentiation, homeostasis and disease in general have been poorly investigated. Moreover, in the context of chondrogenic differentiation, cartilage homeostasis and disease, snoRNAs have not been investigated so far. In the work presented in this thesis we therefore investigated the involvement of snoRNAs in several chondrocyte and cartilage facets.

Chapter 2 Expression of RMRP RNA is regulated in chondrocyte hypertrophy and determines chondrogenic differentiation
Mutations in the RMRP-gene, encoding the snoRNA component of the RNase MRP complex, are the cause of cartilage-hair hypoplasia (CHH). CHH is associated with severe dwarfism caused by impaired skeletal development. However, it is not clear why mutations in RMRP snoRNA lead to skeletal dysplasia. Since chondrogenic differentiation of the growth plate is required for development of the long bones, we hypothesized that the RMRP snoRNA plays a pivotal role in chondrogenic differentiation. Expression of Rmrp RNA and RNase MRP protein subunits was detected in the murine growth plate and during the course of chondrogenic differentiation of ATDC5 cells, where Rmrp RNA expression was found to be correlated with chondrocyte hypertrophy. Genetic interference with Rmrp RNA expression in ATDC5 cells caused a deregulation of chondrogenic differentiation, with a prominent impact on hypertrophy and changes in pre-rRNA processing and rRNA levels (reduced levels of 18S and 5.8S rRNA). Promoter reporter studies showed that Rmrp RNA expression responds to chondrogenic morphogens such as PTHrP and bFGF (reduced promoter activity) and TGFβ3, BMP-2, WNT-3A and WNT-5A (increased promoter activity). Chondrogenic trans-differentiation of CHH fibroblasts was impaired with a pronounced impact on hypertrophic differentiation, increased levels of PTHrP and accumulation of the ITS-1 pre-rRNA processing intermediate. Together, our data show that RMRP RNA expression is regulated during different stages of chondrogenic differentiation and indicate that RMRP RNA may play a pivotal role in chondrocyte hypertrophy, with potential consequences for CHH pathobiology.

Chapter 3 The anti-viral protein viperin regulates chondrogenic differentiation via CXCL10 protein secretion
RNase MRP has a number of substrate RNAs and from most it is not clear how these substrate RNAs may influence cell biological processes, nor is it known how these substrates may be involved in the development of CHH or chondrogenic differentiation. One of these substrate RNAs is the mRNA of viperin. Viperin, an abbreviation for Virus Inhibitory Protein, Endoplasmic Recticulum-associated, Interferon (IFN)-inducible, is a protein located in the endoplasmic reticulum and it is well described for its role as an antiviral protein. Viperin expression has been shown to be increased in CHH leukocytes and following knockdown of RNase MRP subunits. We discovered that viperin is expressed in differentiating chondrocytic cells and regulates their protein secretion and the outcome of chondrogenic differentiation by influencing TGF-β/SMAD2/3 activity via CXCL10, where CXCL10 inhibits chondrogenic differentiation. Moreover, we observed disturbances in this viperin–CXCL10–TGF-β/SMAD2/3 axis in CHH chondrocytic cells. Our results indicate that the anti-viral protein viperin controls chondrogenic differentiation by influencing secretion of soluble proteins and we identified a molecular route that may explain impaired chondrogenic differentiation of cells from individuals with CHH.

Chapter 4 Adaptation of protein translational apparatus during ATDC5 chondrogenic differentiation
The RMRP snoRNA is one highly specific snoRNA belonging to a small group of non-canonical snoRNAs. The majority of the cell’s snoRNAs however belong to a group of canonical snoRNAs involved in the post-transcriptional modification of rRNAs. Fine-tuning of the cell’s rRNA pool by snoRNA-mediated post-transcriptional modifications is believed to determine ribosome activity and control ribosome translation fidelity. It is expected that this is particularly important in translationally active cells, like growth plate chondrocytes, in order to accurately and efficiently synthesize the proteins required for building the cartilaginous growth plate extracellular matrix and support their high speed of proliferation. We therefore charted the full spectrum of snoRNAs expressed during different phases of chondrogenic differentiation. snoRNAs were found to be differentially expressed during ATDC5 chondrogenic differentiation. In addition, rRNA post-transcriptional modifiers, the 2’O-ribose methylase fibrillarin and the pseudouridylase dyskerin, as well as UBF-1 (involved in rDNA transcription) expression and 18S, 5.8S and 28S rRNA content per cell adapted to the differentiation status of ATDC5 cells. Overall an impact on the translational capacity of the cell, depending on the differentiation status of the ATDC5 cells, was demonstrated. Thus, our data showed that chondrogenic differentiation is associated with significant regulation of mechanisms involving ribosome biogenesis and translation activity. Differentiation-phase specific expression of snoRNAs suggests that specific snoRNAs may modulate the chondrocyte’s developing phenotype via an rRNA post-transcriptional modification-based ribosome heterogeneity mechanism, thereby potentially facilitating the observed dynamics in translational activity impacting the course of chondrogenic differentiation. Future work is expected to uncover the extent of ribosome heterogeneity and regulation in cellular differentiation and its potential implications for human disease.

Chapter 5 Serum snoRNAs as biomarkers for joint ageing and post-traumatic osteoarthritis
In osteoarthritis, like in chondrogenic differentiation, the chondrogenic phenotype is actively changing. The development of effective treatments for the age-related disease osteoarthritis and the ability to predict disease progression has been hampered by the lack of biomarkers able to demonstrate the course of the disease. Profiling the expression patterns of snoRNAs in murine joint ageing and osteoarthritis may provide insight in their contribution to joint pathology, their use as diagnostic biomarkers and potential as therapeutic targets. SnoRNASeq identified differential expression of 6 snoRNAs in young versus old joints and 5 snoRNAs in old sham versus old experimental osteoarthritic joints. In serum we found differential presence of 27 snoRNAs in young versus old serum and 18 snoRNAs in old sham versus old experimental osteoarthritic serum. Profiling the expression patterns of snoRNAs is the initial step in determining their functional significance in ageing and osteoarthritis, and provides potential diagnostic biomarkers and therapeutic targets. Our results established snoRNAs as novel markers of musculoskeletal ageing and osteoarthritis and implicate specific changes in snoRNA abundance in joint ageing (SNORD88 and SNORD38 were respectively decreased and increased) and OA, suggesting the potential use of snoRNAs such as SNORA73 and SNORD23 as a novel biomarker for joint ageing, SNORA64, SNORD46 and SNORD116 for OA, SNORD18 for ageing and OA.

Conclusions
In the general discussion the data described in this thesis were discussed with respect to each other and with outsight to future directions for snoRNA research in cartilaginous tissues. In addition, we provided an overview of the molecular interactions identified from this thesis. Overall, the work presented in this thesis provides directions for pathways in which snoRNAs function in chondrocyte development and disease.