A Genome-Wide RNAi Screen to Dissect Centriole Duplication and Centrosome Maturation in Drosophila

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Abstract
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.