Depletion of <i>SNORA33</i> Abolishes ψ of 28S-U4966 and Affects the Ribosome Translational Apparatus

Eukaryotic ribosomes are complex molecular nanomachines translating genetic information from mRNAs into proteins. There is natural heterogeneity in ribosome composition. The pseudouridylation (ψ) of ribosomal RNAs (rRNAs) is one of the key sources of ribosome heterogeneity. Nevertheless, the functional consequences of ψ-based ribosome heterogeneity and its relevance for human disease are yet to be understood. Using HydraPsiSeq and a chronic disease model of non-osteoarthritic primary human articular chondrocytes exposed to osteoarthritic synovial fluid, we demonstrated that the disease microenvironment is capable of instigating site-specific changes in rRNA ψ profiles. To investigate one of the identified differential rRNA ψ sites (28S-ψ4966), we generated SNORA22 and SNORA33 KO SW1353 cell pools using LentiCRISPRv2/Cas9 and evaluated the ribosome translational capacity by 35S-Met/Cys incorporation, assessed the mode of translation initiation and ribosomal fidelity using dual luciferase reporters, and assessed cellular and ribosomal proteomes by LC-MS/MS. We uncovered that the depletion of SNORA33, but not SNORA22, reduced 28S-ψ4966 levels. The resulting loss of 28S-ψ4966 affected ribosomal protein composition and function and led to specific changes in the cellular proteome. Overall, our pioneering findings demonstrate that cells dynamically respond to disease-relevant changes in their environment by altering their rRNA pseudouridylation profiles, with consequences for ribosome function and the cellular proteome relevant to human disease

An improved diagnostic tool to predict cartilage formation in an osteoarthritic joint environment

Osteoarthritis (OA) is a degenerative joint disease with progressive articular cartilage loss. Due to the chondrogenic potential of human mesenchymal stromal cells (MSCs), MSC-based therapies are promising treatment strategies for cartilage loss. However, the local joint microenvironment has a great impact on the success of cartilage formation by MSCs. This local joint environment is different between patients and therefore the outcome of MSC therapies is uncertain. We previously developed gene promoter-based reporter assays as a novel tool to predict the effect of a patient's OA joint microenvironment on the success of MSC-based cartilage formation. Here we describe an improved version of this molecular tool with increased prediction accuracy. For this, we generated fourteen stable cell lines using transcription factor (TF) binding elements (AP1, ARE, CRE, GRE, ISRE, NFAT5, NFκB, PPRE, SBE, SIE, SOX9, SRE, SRF, TCF/LEF) to drive luciferase reporter gene expression, and evaluated the cell lines for their responsiveness to an osteoarthritic microenvironment by stimulation with OA synovium-conditioned medium (OAs-cm; n=31). To study the effect of this OA microenvironment on MSC-based cartilage formation, MSCs were cultured in a three-dimensional pellet culture model while stimulated with OAs-cm. Cartilage formation was assessed histologically and by quantifying sulfated glycosaminoglycan (sGAG) production. Six TF reporters correlated significantly with the effect of OAs-cm on cartilage formation. We validated the predictive value of these TF reporters with an independent cohort of OAs-cm (n=22) and compared the prediction accuracy between our previous and the current new tool. Furthermore, we investigated which combination of reporters could predict the effect of the OA microenvironment on cartilage repair with the highest accuracy. A combination between the TF (NFκB) and the promoter-based (IL6) reporter proved to reach a more accurate prediction compared to the tools separately. These developments are an important step towards a diagnostic tool that can be used for personalized cartilage repair strategies for OA patients

An improved diagnostic tool to predict cartilage formation in an osteoarthritic joint environment

Osteoarthritis (OA) is a degenerative joint disease with progressive articular cartilage loss. Due to the chondrogenic potential of human mesenchymal stromal cells (MSCs), MSC-based therapies are promising treatment strategies for cartilage loss. However, the local joint microenvironment has a great impact on the success of cartilage formation by MSCs. This local joint environment is different between patients and therefore the outcome of MSC therapies is uncertain. We previously developed gene promoter-based reporter assays as a novel tool to predict the effect of a patient's OA joint microenvironment on the success of MSC-based cartilage formation. Here we describe an improved version of this molecular tool with increased prediction accuracy. For this, we generated fourteen stable cell lines using transcription factor (TF) binding elements (AP1, ARE, CRE, GRE, ISRE, NFAT5, NFκB, PPRE, SBE, SIE, SOX9, SRE, SRF, TCF/LEF) to drive luciferase reporter gene expression, and evaluated the cell lines for their responsiveness to an osteoarthritic microenvironment by stimulation with OA synovium-conditioned medium (OAs-cm; n=31). To study the effect of this OA microenvironment on MSC-based cartilage formation, MSCs were cultured in a three-dimensional pellet culture model while stimulated with OAs-cm. Cartilage formation was assessed histologically and by quantifying sulfated glycosaminoglycan (sGAG) production. Six TF reporters correlated significantly with the effect of OAs-cm on cartilage formation. We validated the predictive value of these TF reporters with an independent cohort of OAs-cm (n=22) and compared the prediction accuracy between our previous and the current new tool. Furthermore, we investigated which combination of reporters could predict the effect of the OA microenvironment on cartilage repair with the highest accuracy. A combination between the TF (NFκB) and the promoter-based (IL6) reporter proved to reach a more accurate prediction compared to the tools separately. These developments are an important step towards a diagnostic tool that can be used for personalized cartilage repair strategies for OA patients