Laboratory for Experimental Orthopedics, Department of Orthopedic Surgery, Maastricht University, 6229 HX Maastricht, The Netherlands.
Institute of Life Course and Medical Sciences, University of Liverpool, Liverpool L8 7TX, UK.
Int J Mol Sci. 2023 Aug 8;24(16):12578. doi: 10.3390/ijms241612578.
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 and KO SW1353 cell pools using LentiCRISPRv2/Cas9 and evaluated the ribosome translational capacity by S-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 , but not , 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.
真核核糖体是将 mRNA 中的遗传信息翻译为蛋白质的复杂分子纳米机器。核糖体的组成存在天然异质性。核糖体 RNA(rRNA)的假尿嘧啶化(ψ)是核糖体异质性的关键来源之一。然而，基于 ψ 的核糖体异质性的功能后果及其与人类疾病的相关性尚待理解。我们使用 HydraPsiSeq 和慢性疾病模型（即暴露于骨关节炎性滑液的非骨关节炎原代人关节软骨细胞），证明了疾病微环境能够引发 rRNA ψ 谱的特定位置变化。为了研究鉴定出的差异 rRNA ψ 位点之一（28S-ψ4966），我们使用 LentiCRISPRv2/Cas9 生成了 和 KO SW1353 细胞池，并通过 S-Met/Cys 掺入评估核糖体翻译能力，使用双荧光素酶报告评估翻译起始方式和核糖体保真度，并通过 LC-MS/MS 评估细胞和核糖体蛋白质组。我们发现， 的消耗，但不是 ，降低了 28S-ψ4966 水平。28S-ψ4966 的缺失会影响核糖体蛋白组成和功能，并导致细胞蛋白质组的特异性变化。总体而言，我们的开创性发现表明，细胞通过改变其 rRNA 假尿嘧啶化谱来动态响应其环境中的疾病相关变化，这对核糖体功能和与人类疾病相关的细胞蛋白质组有影响。
