Author Summary Mitosis is the evolutionarily conserved process that enables a dividing cell to equally partition its genetic material between the two daughter cells. The fidelity of mitotic division is crucial for normal development of multicellular organisms and to prevent cancer or birth defects. Understanding the molecular mechanisms of mitosis requires the identification of genes involved in this process. Previous studies have shown that such genes can be readily identified by RNA interference (RNAi) in Drosophila tissue culture cells. Because the inventory of mitotic genes is still incomplete, we have undertaken an RNAi screen using a novel approach. We used a co- expression–based bioinformatic procedure to select a group of 1,000 genes enriched in mitotic functions from a dataset of 13,166 Drosophila genes. This group includes roughly half of the known mitotic genes, implying that it should contain half of all mitotic genes, including those that are currently unknown. We performed RNAi against each of the 1,000 genes in the group. By limiting the number of genes to be examined, we were able to perform a very detailed phenotypic analysis of RNAi cells. This analysis allowed the identification of 70 genes whose mitotic role was previously unknown; 30 are required for proper chromosome segregation and 40 are required to maintain chromosome integrity. Results Construction of a Mitotic-Gene-Enriched Co-Expression List Numerous studies indicate that genes involved in the same biological process tend to be transcriptionally co-expressed (see, for example,[11–13]. We thus exploited extant microarray data [14] to rank the complete set of annotated Drosophila genes according to their co-expression with six well-characterized genes representative of different aspects of mitosis: gluon (glu) encodes a condensin [15]; ida/ APC5, specifies a subunit of the anaphase promoting complex (APC/ C [16]); cid/CenpA is the gene for the centromere-specific histone H3 variant required for kinetochore assembly [17,18]; Eb1 encodes a microtubule (MT)-associated protein required for spindle assembly [19]; zw10 specifies a component of the RZZ complex that helps target cytoplasmic dynein to the kinetochore and that is involved in the spindle checkpoint [20]; and finally sti (Citron kinase) encodes a serine/threonin protein kinase required for the completion of cytokinesis [21–23]. Using the Pearson correlation coefficient, the expression of these prototype genes was correlated with the expression levels of most Drosophila genes across 89 different microrray experiments [14]. The 13,166 probesets contained in this dataset were separately ranked for their co-expression with each prototype gene. We then generated a ranked consensus co- expression list by combining the six gene-specific lists (Table S1). To validate our bioinformatic approach, we determined the rank in the consensus co-expression lists of 164 Drosophila mitotic genes; these genes represent most of the Drosophila mitotic genes so far identified but do not include the genes identified in the recent genome-wide screen performed by Goshima et al. [10]. As shown in Figure 1A and Tables S2 and S3, the first 1000 genes of our consensus co-expression list include 46% of the 164 known mitotic genes. This implies that the first 1000 genes of the same list should contain roughly half of all mitotic genes, including those that are currently unknown. Drosophila Mitotic Genes Identification of Genes Involved in Mitotic Division To identify new mitotic genes, we synthesized a dsRNA for each of the first 1000 genes in our consensus co-expression list. In designing the primers for such RNAs, we minimized gene overlap to avoid off-target effects of dsRNAs [24,25] (Table S4). Each dsRNA was then added to S2 cells grown in 3 milliliters (ml) of culture medium. After a 96 h treatment with dsRNA, the cells were split into two aliquots. 2 ml were fixed with formaldehyde and then stained for both tubulin and DNA. The resulting preparations were then blindly scored by at least two independent observers for abnormalities in spindle morphology and chromo- some segregation. The remaining 1 ml of cell suspension was incubated with colchicine for two hours, hypotonically swollen, fixed with methanol/acetic acid, and stained with DAPI. The metaphase chromosomes obtained in this way were then blindly examined by at least two observers for abnormalities in chromosome structure and/or the presence of chromosome aberrations. We performed two independent RNAi experiments for each gene. In most of these experiments, we examined 50 colchicine-arrested metaphases and 50 tubulin-stained mitotic figures. If the results of the two experiments were significantly different, we performed additional experiments to define the RNAi phenotype. An RNAi phenotype was considered positive only when the frequency of affected cells was significantly different from controls with p,0.001 (using the x2 contingency test; see Methods). Our screen identified 155 genes whose inactivation by RNAi causes a strong mitotic phenotype. Based on phenotypic analysis, these genes can be grouped in seven broad categories that we further subdivided in 18 phenoclusters (PHCs) [26]: 13 genes required for progression through the cell cycle, identified by dsRNAs that result in a complete (or nearly complete) absence of mitotic figures (PHC: NM); 44 genes required for chromosome integrity, identified by dsRNAs that cause chromosome aberra- tions (PHC: CA); 11 genes required for proper mitotic chromo- some condensation (PHCs: CC1–CC3); 41 genes required for regular chromosome segregation (PHCs: CS1–CS5); 33 genes required for spindle assembly (PHCs: SA1–SA4); 7 genes required for cytokinesis (PHCs: CY1 and CY2); and 6 genes required for multiple mitotic functions (PHCs: SC1 and SC2) (Figures 1B, 2, 3, and 4C; Table S5; a synopsis on the functions of these genes can be found in Table S6). Remarkably, the distribution of these mitotic genes in our co-expression list was clearly nonrandom: their frequency decreased with an increase of their rank, further validating our co-expression approach (Figure 1C). Genes Required for Progression through the Cell Cycle We identified 13 dsRNAs that result in the absence of dividing cells at 96 h after treatment initiation (Table S5). Six of these genes (cdc2c, cyclinA, cyclinE, geminin, ran and string) are well-known cell- cycle regulators. Two genes, RpII140 and RpII215, encode the 140 and 215 kDa subunits of RNA polymerase II, respectively. Three genes are involved in RNA metabolism and encode either canonical splicing factors (Prp8 and SF1) or the small DebB ribonucleoprotein, which is also likely to be involved in RNA splicing. Defects in PRPF8, the human homolog of Prp8, are one cause of Retinitis pigmentosa. Of the remaining two genes, CG9273 encodes a protein with similarity to a subunit of DNA replication factor A, and Bx42 specifies a protein involved in Notch signal transduction (Table S6). Genes Required for Chromosome Integrity Although the 1000 genes in our list were selected for their co- expression with mitotic genes, our screen uncovered several functions required for chromosome integrity (Figures 1 and 4; PLoS Genetics | www.plosgenetics.org 2 July 2008 | Volume 4 | Issue 7 | e1000126