A disease-linked lncRNA mutation in RNase MRP inhibits ribosome synthesis [RMRP_SHAPE]. A disease-linked lncRNA mutation in RNase MRP inhibits ribosome synthesis [RMRP_SHAPE]

Mutations in the human RMRP gene cause Cartilage Hair Hypoplasia (CHH), an autosomal recessive disorder characterized by skeletal abnormalities and impaired T-cell activation. RMRP encodes a non-coding RNA, which forms the core of the RNase MRP ribonucleoprotein complex. In budding yeast, RMRP cleaves a specific site in the pre-ribosomal RNA (pre-rRNA) during ribosome synthesis. CRISPR-mediated disruption of RMRP in human cells lines caused growth arrest, with pre-rRNA accumulation. Here, we analyzed disease-relevant primary cells, showing that mutations in RMRP impair mouse T cell activation and delay pre-rRNA processing. Analysis of pre-rRNA processing in patient-derived human fibroblasts with CHH-linked mutations showed a similar pattern of processing delay. Human cells engineered with the most common CHH mutation (70AG in RMRP) show specifically impaired pre-rRNA processing, resulting in reduced mature rRNA and a reduced ratio of cytosolic to mitochondrial ribosomes. Moreover, the 70AG mutation caused a reduction in intact RNase MRP complexes. Together, these results indicate that CHH is a ribosomopathy, and the first human disorder of rRNA processing to be described. Overall design: Wildtype or RMRP 70A->G mutant cells were grown to log phase in RPMI. The SHAPE reagent 1M7 (or DMSO for untreated samples) was added to a final concentration of 10 mM and in-cell acylation reaction left to proceed for 15 minutes at 37 degrees. RNA was extracted and reverse transcribed using an RMRP-specific primer. Sites of RNA modification are represented as mutations or deletions in the sequencing, and were analysed with the ShapeMapper 2 tool.

人类RMRP基因的突变可引发软骨毛发发育不全（Cartilage Hair Hypoplasia, CHH），这是一类以骨骼畸形与T细胞活化受损为典型表型的常染色体隐性遗传病。RMRP编码一种非编码RNA，该RNA是核糖核酸酶MRP（RNase MRP）核糖核蛋白复合物的核心组分。在出芽酵母中，RMRP可在核糖体合成过程中，剪切前体核糖体RNA（pre-ribosomal RNA, pre-rRNA）的特定位点。在人类细胞系中经CRISPR介导的RMRP敲除会引发细胞生长停滞，并伴随pre-rRNA的积累。本研究针对疾病相关性原代细胞展开分析，结果显示RMRP的突变会损伤小鼠T细胞活化，并延缓pre-rRNA的加工过程。对携带CHH相关突变的患者来源人类成纤维细胞开展的pre-rRNA加工分析，同样观察到了加工延迟的现象。携带CHH最常见突变（RMRP基因70AG位点突变）的工程改造人类细胞，其pre-rRNA加工过程出现特异性受损，最终造成成熟rRNA水平下调，以及胞质核糖体与线粒体核糖体的比值降低。此外，70AG位点突变还会导致完整核糖核酸酶MRP（RNase MRP）复合物的数量减少。综合上述结果，本研究表明CHH属于核糖体病，亦是目前已报道的首例以rRNA加工异常为致病机制的人类遗传病。
实验整体设计：将野生型或RMRP 70A→G突变体细胞在RPMI培养基中培养至对数生长期。向培养基中加入SHAPE试剂1M7（未处理组加入等体积二甲基亚砜（dimethyl sulfoxide, DMSO）），使其终浓度达到10 mM，随后于37℃下进行15分钟的细胞内酰化反应。提取RNA并使用RMRP特异性引物进行反转录。测序数据中以突变或缺失信号表征RNA修饰位点，并通过ShapeMapper 2工具完成数据分析。