A protein inventory of human ribosome biogenesis reveals an essential function of exportin 5 in 60S subunit export.
A protein inventory of human ribosome biogenesis reveals an essential function of exportin 5 in 60S subunit export.
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DOI：
10.1371/journal.pbio.1000522
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发表时间：
2010-10-26
期刊：
影响因子：
9.8
通讯作者：
Kutay U
中科院分区：
文献类型：
--
作者：
Wild T;Horvath P;Wyler E;Widmann B;Badertscher L;Zemp I;Kozak K;Csucs G;Lund E;Kutay U
对人核糖体生物发生因子的系统研究表明，真核细胞核糖体合成的许多方面与酵母中研究得很好的过程是保守的，并确定了高等真核生物特有的60S亚基的输出途径。真核生物核糖体亚基的组装是一个复杂的、多步骤的过程，到目前为止主要在酵母中进行研究。在酿酒酵母中，40S和60S核糖体亚基的有序组装需要200多个因子，包括核糖体蛋白和反式作用因子。到目前为止，这些酵母核糖体合成因子的人类同源物还很少被鉴定。在这里，我们使用系统的RNA干扰(RNAi)方法来分析464个候选因素对人类细胞核糖体亚单位生物发生的贡献。该屏幕是基于视觉读数，使用可诱导的荧光核糖体蛋白作为记者。通过使用有监督的机器学习技术进行基于计算机的图像分析，我们获得了153种人类蛋白质与核糖体合成功能联系的证据。我们的数据表明，核糖体组装的核心特征在酵母和人类之间是保守的，但存在差异，例如在60S亚基输出方面。出乎意料的是，我们的RNAi筛查发现，在人类细胞60S亚单位的核出口中，需要出口受体Exportin 5(Exp5)。我们发现Exp5，就像已知的CRM1中的60S Exportion一样，以RanGTP依赖的方式与60S前的颗粒结合。对Exp5或CRM1功能的干扰阻止了人类细胞和青蛙卵母细胞中60s的输出，而40s的输出只有在抑制CRM1时才受到影响。因此，60S亚基的输出依赖于脊椎动物细胞中至少两个RanGTP结合的输出蛋白。核糖体是合成蛋白质的分子机器，存在于每个细胞中。每个核糖体都有一个小的和一个大的亚基，每个亚基依次由核糖体RNA和许多核糖体蛋白组成。在真核细胞中，这些核糖体亚基的产生是一个复杂的过程，需要数百个因素的参与。未成熟的核糖体亚基源自核仁(专门合成核糖体的核区)，穿过核内部并输出到细胞质，在那里它们的组装完成。近年来，核糖体生产中的缺陷与人类疾病有关已经变得明显，但我们对这一基本过程的了解主要是基于酵母这一单细胞真核生物的知识。我们开始通过单独消耗大约500种不同的细胞蛋白来系统地识别参与在人类细胞中制造核糖体的因素。利用显微分析，我们确定了大约150个人类核糖体合成因子，从而极大地扩展了我们对人类核糖体生物发生的了解。我们的数据集不仅揭示了这一基本细胞过程在进化上保守的许多方面，而且还使我们描述了高等真核细胞所特有的大核糖体亚基的输出途径。
A systematic search for human ribosome biogenesis factors shows conservation of many aspects of eukaryotic ribosome synthesis with the well-studied process in yeast and identifies an export route of 60S subunits that is specific for higher eukaryotes. The assembly of ribosomal subunits in eukaryotes is a complex, multistep process so far mostly studied in yeast. In S. cerevisiae, more than 200 factors including ribosomal proteins and trans-acting factors are required for the ordered assembly of 40S and 60S ribosomal subunits. To date, only few human homologs of these yeast ribosome synthesis factors have been characterized. Here, we used a systematic RNA interference (RNAi) approach to analyze the contribution of 464 candidate factors to ribosomal subunit biogenesis in human cells. The screen was based on visual readouts, using inducible, fluorescent ribosomal proteins as reporters. By performing computer-based image analysis utilizing supervised machine-learning techniques, we obtained evidence for a functional link of 153 human proteins to ribosome synthesis. Our data show that core features of ribosome assembly are conserved from yeast to human, but differences exist for instance with respect to 60S subunit export. Unexpectedly, our RNAi screen uncovered a requirement for the export receptor Exportin 5 (Exp5) in nuclear export of 60S subunits in human cells. We show that Exp5, like the known 60S exportin Crm1, binds to pre-60S particles in a RanGTP-dependent manner. Interference with either Exp5 or Crm1 function blocks 60S export in both human cells and frog oocytes, whereas 40S export is compromised only upon inhibition of Crm1. Thus, 60S subunit export is dependent on at least two RanGTP-binding exportins in vertebrate cells. Ribosomes are molecular machines that synthesize proteins and are found in every cell. Each ribosome has a small and a large subunit, each of which is in turn composed of ribosomal RNA and many ribosomal proteins. In eukaryotic cells, the generation of these ribosomal subunits is a complex process requiring the participation of hundreds of factors. Originating from the nucleolus (a nuclear region specialized in ribosome synthesis) immature ribosomal subunits pass through the nuclear interior and are exported to the cytoplasm, where their assembly is finalized. In recent years, it has become apparent that defects in ribosome production are associated with human diseases, but our knowledge about this fundamental process is largely based on knowledge derived from yeast, a unicellular eukaryote. We set out to systematically identify factors involved in making ribosomes in human cells by individually depleting around 500 different cellular proteins. Using microscopic analysis, we identified approximately 150 human ribosome synthesis factors, thereby significantly extending our knowledge about human ribosome biogenesis. Our dataset not only revealed many evolutionarily conserved aspects of this essential cellular process but also led us to characterize an export route for the large ribosomal subunit that is specific for higher eukaryotic cells.
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DOI：
10.1083/jcb.200501141
发表时间：
2005-08-01
期刊：
The Journal of cell biology
影响因子：
--
作者：
Hölzel M;Rohrmoser M;Schlee M;Grimm T;Harasim T;Malamoussi A;Gruber-Eber A;Kremmer E;Hiddemann W;Bornkamm GW;Eick D
通讯作者：
Eick D
影响因子：
12.3
作者：
Carpenter AE;Jones TR;Lamprecht MR;Clarke C;Kang IH;Friman O;Guertin DA;Chang JH;Lindquist RA;Moffat J;Golland P;Sabatini DM
通讯作者：
Sabatini DM
DOI：
10.1073/pnas.84.3.629
发表时间：
1987-02-01
影响因子：
11.1
作者：
CRAIG, N;KASS, S;SOLLNERWEBB, B
通讯作者：
SOLLNERWEBB, B
影响因子：
16
作者：
Grandi, P;Rybin, V;Hurt, E
通讯作者：
Hurt, E
影响因子：
--
作者：
Freed EF;Bleichert F;Dutca LM;Baserga SJ
通讯作者：
Baserga SJ