Developing methods for accurate detection of RNA modifications remains a major challenge in epitranscriptomics. Next-generation sequencing-based mapping approaches have recently emerged but, often, they are not quantitative and lack specificity. Pseudouridine (ψ), produced by uridine isomerization, is one of the most abundant RNA modification. ψ mapping classically involves derivatization with soluble carbodiimide (CMCT), which is prone to variation making this approach only semi-quantitative. Here, we developed ‘HydraPsiSeq’, a novel quantitative ψ mapping technique relying on specific protection from hydrazine/aniline cleavage. HydraPsiSeq is quantitative because the obtained signal directly reflects pseudouridine level. Furthermore, normalization to natural unmodified RNA and/or to synthetic in vitro transcripts allows absolute measurements of modification levels. HydraPsiSeq requires minute amounts of RNA (as low as 10–50 ng), making it compatible with high-throughput profiling of diverse biological and clinical samples. Exploring the potential of HydraPsiSeq, we profiled human rRNAs, revealing strong variations in pseudouridylation levels at ∼20–25 positions out of total 104 sites. We also observed the dynamics of rRNA pseudouridylation throughout chondrogenic differentiation of human bone marrow stem cells. In conclusion, HydraPsiSeq is a robust approach for the systematic mapping and accurate quantification of pseudouridines in RNAs with applications in disease, aging, development, differentiation and/or stress response.

BackgroundMesenchymal stem cells (MSCs) are found in synovial fluid (SF) and can easily be harvested during arthrocentesis or arthroscopy. However, SF-MSC characterization and chondrogenicity in collagen sponges have been poorly documented as well as their hypothetical in vivo chondroprotective properties with intra-articular injections during experimental osteoarthritis (OA).MethodsSF-MSCs were isolated from human SF aspirates in patients suffering from advanced OA undergoing total knee joint replacements. SF-MSCs at passage 2 (P2) were characterized by flow cytometry for epitope profiling. SF-MSCs at P2 were subsequently cultured in vitro to assess their multilineage potentials. To assess their chondrogenicity, SF-MSCs at P4 were seeded in collagen sponges for 4 weeks under various oxygen tensions and growth factors combinations to estimate their gene profile and matrix production. Also, SF-MSCs were injected into the joints in a nude rat anterior cruciate ligament transection (ACLT) to macroscopically and histologically assess their possible chondroprotective properties,.ResultsWe characterized the stemness (CD73+, CD90+, CD105+, CD34−, CD45−) and demonstrated the multilineage potency of SF-MSCs in vitro. Furthermore, the chondrogenic induction (TGF-ß1 ± BMP-2) of these SF-MSCs in collagen sponges demonstrated a good capacity of chondrogenic gene induction and extracellular matrix synthesis. Surprisingly, hypoxia did not enhance matrix synthesis, although it boosted chondrogenic gene expression (ACAN, SOX9, COL2A1). Besides, intra-articular injections of xenogenic SF-MSCs did exert neither chondroprotection nor inflammation in ACLT-induced OA in the rat knee.ConclusionsAdvanced OA SF-MSCs seem better candidates for cell-based constructs conceived for cartilage defects rather than intra-articular injections for diffuse OA.