A genome-wide RNAi screen to dissect centriole duplication and centrosome maturation in Drosophila.
A genome-wide RNAi screen to dissect centriole duplication and centrosome maturation in Drosophila.
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全基因组RNAi筛选，用于剖析果蝇中的中心重复和中心体成熟。
DOI：
10.1371/journal.pbio.0060224
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发表时间：
2008-09-16
期刊：
影响因子：
9.8
通讯作者：
Raff, Jordan
中科院分区：
文献类型：
--
作者：
Dobbelaere, Jeroen;Josue, Filipe;Suijkerbuijk, Saskia;Baum, Buzz;Tapon, Nicolas;Raff, Jordan
中心体由一对中心粒组成，中心粒被无定形的中心粒周围物质（PCM）包围。在这里，我们在果蝇细胞中进行了基于显微镜的全基因组RNA干扰（RNAi）筛选，以鉴定中心粒复制和有丝分裂PCM募集所需的蛋白质。我们分析了92%的果蝇基因组（13059个基因），确定了32个参与中心体功能的基因。一系列广泛的二次筛选将这些基因分为四类：(1)中心粒复制需要9个基因，(2)中心体成熟需要11个基因，(3)两种功能都需要9个基因，(4)3个基因调节中心体分离。这32个点包括几个新的中心体成分，其中一些具有人类同源物。此外，我们发现只有Polo和Centrosomin （Cnn）两种蛋白的个体缺失就可以完全阻断中心体的成熟。在有丝分裂过程中，Cnn以polo依赖的方式被磷酸化，这表明在果蝇中，Cnn的polo依赖磷酸化启动了中心体的成熟。细胞周期的一个主要目标是准确地分离两个子细胞之间的复制染色体。为了实现这一点，一对中心体组织一个由微管组成的双极纺锤体；染色体在纺锤体上排列，然后分离到两个纺锤极。中心体也是形成纤毛和鞭毛所必需的，它们存在于许多真核细胞中；中心体功能障碍是许多人类癌症和几种神经系统疾病的共同特征，而影响纤毛功能的基因突变引起了几种人类疾病。在这里，我们使用RNA干扰进行全基因组筛选，试图鉴定模式生物黑腹果蝇中心体功能所需的所有蛋白质。我们鉴定了所有已知的果蝇中心体功能所需的16种中心体蛋白，以及16种新的中心体成分或调节因子。我们确认了几个成分的中心体位置，并对它们的功能进行了一些分析。我们相信，我们正在接近果蝇中心体功能所需蛋白质的完整清单。RNAi筛选鉴定了果蝇体内16种新的中心体成分或调节因子，并对其功能进行了分子解剖，阐明了Polo激酶在中心周围物质成熟中的作用。
Centrosomes comprise a pair of centrioles surrounded by an amorphous pericentriolar material (PCM). Here, we have performed a microscopy-based genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins required for centriole duplication and mitotic PCM recruitment. We analysed 92% of the Drosophila genome (13,059 genes) and identified 32 genes involved in centrosome function. An extensive series of secondary screens classified these genes into four categories: (1) nine are required for centriole duplication, (2) 11 are required for centrosome maturation, (3) nine are required for both functions, and (4) three genes regulate centrosome separation. These 32 hits include several new centrosomal components, some of which have human homologs. In addition, we find that the individual depletion of only two proteins, Polo and Centrosomin (Cnn) can completely block centrosome maturation. Cnn is phosphorylated during mitosis in a Polo-dependent manner, suggesting that the Polo-dependent phosphorylation of Cnn initiates centrosome maturation in flies. A major goal of the cell cycle is to accurately separate the duplicated chromosomes between two daughter cells. To achieve this, a pair of centrosomes organise a bipolar spindle made of microtubules; the chromosomes line up on the spindle and are then separated to the two spindle poles. Centrosomes are also required for the formation of cilia and flagella, which are present in many eukaryotic cells; centrosome dysfunction is a common feature of many human cancers and several neurological disorders, whereas mutations in genes that affect cilia function give rise to several human diseases. Here, we perform a genome-wide screen using RNA interference to try to identify all of the proteins required for centrosome function in the model organism Drosophila melanogaster (a fruitfly). We identified all 16 of the centrosomal proteins that were already known to be required for centrosome function in Drosophila, as well as 16 new centrosomal components or regulators. We confirmed the centrosomal location of several of the components and performed some analysis of their functions. We believe we are approaching a complete inventory of the proteins required for centrosome function in flies. An RNAi screen identifies 16 new centrosomal components or regulators inDrosophila, and molecular dissection of their function addresses the role of Polo kinase in the maturation of pericentriolar material.
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DOI：
10.1083/jcb.129.6.1617
发表时间：
1995-06
期刊：
The Journal of cell biology
影响因子：
--
作者：
Golsteyn RM;Mundt KE;Fry AM;Nigg EA
通讯作者：
Nigg EA
影响因子：
12.3
作者：
Boutros M;Brás LP;Huber W
通讯作者：
Huber W
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
影响因子：
64.8
作者：
Cowan, CR;Hyman, AA
通讯作者：
Hyman, AA
影响因子：
4
作者：
Eley, L;Yates, LM;Goodship, JA
通讯作者：
Goodship, JA