Drosophila Mitotic Genes Figure 12. Mitotic phenotypes observed in the SA3 phenocluster. In merges, DNA is blue and tubulin green. Spindles with broad, anastral poles in NiPp1 (A) and CG6937 (B) RNAi cells. Note that these spindles exhibit a normal microtubule density. Scale bar, 5 mm. doi:10.1371/journal.pgen.1000126.g012 validate our co-expression-based method for the identification of mitotic genes by RNAi. We believe that elaboration of consensus co-expression lists using genes in the same phenocluster will provide many candidate genes for small-scale RNAi screens aimed at completing the inventory of proteins involved in specific mitotic processes. One striking and unanticipated finding among our results merits special attention. We identified 17 highly conserved splicing factors that are required for sister chromatid separation at anaphase. RNAi for the genes encoding these factors resulted in two types of aberrant mitotic figures. Downregulation of the genes in the CS3 phenocluster resulted in mitotic cells showing scattered chromosomes without apparent kinetochore-spindle connections. This phenotype was identical to that caused by downregulation of genes encoding well- known kinetochore proteins such as cid, CG9938/Ndc80/Hec1, CG8902/Nuf2 or l(1)G023/Nsl1. In RNAi cells for genes in the CS4 phenocluster, most chromosomes showed regular connections with the spindle fibers and remained at the center of the cell, a phenotype similar to that produced by downregulation of Separase. However, RNAi for all of the CS4 genes and some of the CS3 genes produced a fraction of cells with an intermediate CS3/CS4 phenotype. These observations raise the possibility that inactivation of the splicing factor genes of both phenoclusters causes the same primary defect in centromere/kinetochore organization. One can envisage that when this defect is strong, both sister chromatid separation and kinetechore MT-interaction are affected; when the defect is weak, sister chromatid separation would be disrupted with little effect on kinetochore function. Splicing factors have previously been implicated in mitosis in fission yeast, Drosophila and human cells [10,53–55]. However, the precise mitotic function of these splicing factors has never been described. We have clearly shown here that splicing factors are required for sister chromatid separation. However, the mecha- nisms by which splicing factors regulate centromere/kinetochore function remain unclear. It is possible that these factors mediate the splicing of one or more pre-mRNAs, whose protein products play crucial roles for proper centromere or spindle function. Alternatively, the splicing factors may be involved in the production and/or stabilization of spindle- or centromere- associated structural RNAs. Recent studies have shown that RNA associates with the mitotic spindle and plays a translation- independent role in spindle assembly [56]. Moreover, there is evidence that maize and human kinetochores are enriched in single-stranded RNAs encoded by centromeric DNA sequences. It has been suggested that these RNAs may facilitate proper assembly of centromere-specific nucleoprotein complexes [57,58]. Deciphering the precise role of splicing factors in centromere/kinetochore assembly and functioning will be a challenging task for future studies. Methods Generation of Co-Expression Lists Co-expression analysis was perfomed on a previously described gene expression dataset [14]. This comprised 267 GeneChip Drosophila Genome Arrays (Affymetrix, Santa Clara, CA, USA) PLoS Genetics | www.plosgenetics.org 14 July 2008 | Volume 4 | Issue 7 | e1000126