Deficient RPS19 protein production induces cell cycle arrest in erythroid progenitor cells
Deficient RPS19 protein production induces cell cycle arrest in erythroid progenitor cells
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DOI：
10.1111/j.1365-2141.2007.06930.x
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发表时间：
2008-02-01
影响因子：
6.5
通讯作者：
Yamaguchi, Kazunari
中科院分区：
文献类型：
--
作者：
Kuramitsu, Madoka;Hamaguchi, Isao;Yamaguchi, Kazunari
核糖体蛋白S19（RPS 19）基因是引起先天性成红细胞减少症（Diamond-Blackfan anaemia，DBA）的基因之一。虽然RPS 19单倍不足已被认为是DBA发病机制的发病机制，但其顺序机制尚未阐明。为了分析DBA患者特异性RPS 19错义突变的后果，我们制备了突变的RPS 19表达载体。12个C-末端Flag标记的错义突变体从逆转录病毒载体中外源表达，并通过Western印迹分析和流式细胞术进行分析。当这12个突变体在红白血病细胞系K562和人骨髓CD 34（+）细胞中表达时，几乎所有的突变蛋白（除了G120 R）都是不稳定的，并且突变的RPS 19蛋白的水平显著低。为了解决RPS 19表达缺陷对细胞增殖的影响，通过siRNA下调RPS 19。人CD 34（+）细胞中RPS 19的抑制性表达在GO时产生了升高的细胞数量，并诱导了BM细胞中红系祖细胞特异性缺陷。这些结果表明，异常的核糖体生物合成导致造血祖细胞的细胞周期停滞不足，并且随后，红系祖细胞受到特别的阻碍。这些体外表型的遗传操作的CD 34（+）细胞模拟DBA发病机制。
The gene encoding ribosomal protein S19 (RPS19) is one of the responsible genes for Diamond-Blackfan anaemia (DBA), a congenital erythroblastopenia. Although haplo-insufficiency of RPS19 has been suggested to be the onset mechanism underlying the pathogenesis of DBA, the sequential mechanism has not been elucidated. In order to analyse the consequences of the missense mutation of RPS19 specific for DBA patients, we made mutated RPS19 expression vectors. Twelve C-terminally Flag-tagged missense mutants were exogenously expressed from retroviral vectors and analysed by Western blot analysis and flow cytometry. When these 12 mutants were expressed in the erythro-leukaemic cell lines K562 and human bone marrow CD34(+) cells, almost all of the mutant proteins (except for G120R) were unstable, and the levels of mutated RPS19 protein were significantly low. To address the effect of deficient RPS19 expression on cell proliferation, RPS19 was downregulated by siRNA. Repressive expression of RPS19 in human CD34(+) cells produced an elevated number of cells at GO and induced erythroid progenitor-specific defects in BM cells. These results suggest that abnormal ribosomal biogenesis causes inadequate cell cycle arrest in haematopoietic progenitors, and that, subsequently, erythroid progenitors are specifically hampered. These in vitro phenotypes of genetically manipulated CD34(+) cells mimic DBA pathogenesis.