SNORA33 Knockdown in chondrocytes

Background: In the experiment where we exposed chondrocytes of 4 healthy donors to OA-SF for 14 days we found several 2’O-methylation and pseudouridylation sites to be differentially modified in OA-SF condition compared to control (regular medium condition). We linked these PTMs to box C/D and H/ACA snoRNAs and selected 7 sites for validation. We did a CRISR/Cas9 mediated knock-down (KD) (not a complete knock-out (KO), as we didn’t pick clones) of these seven selected snoRNAs. Out of these seven snoRNAs, snoRNA33 knock-down stood out as the pseudouridylation at the corresposing site basically disappeared. We also saw differences in translation initiation and fidelity in snoRNA33 depleted cells. Therefore we decided to explore whether the ribosome composition (core and associated ribosomal proteins) is different in cells lacking snoRNA33 = RiboProteome samples (RP) and whether the proteome of these cells is different = Proteome samples (P). In both cases we used a GFP targeting CRISPR/Cas9 cell line as a control.