Identification of Drosophila Mitotic Genes by Combining Co-Expression Analysis and RNA Interference Maria Patrizia Somma1, Francesca Ceprani1, Elisabetta Bucciarelli1, Valeria Naim1¤a, Valeria De Arcangelis1, Roberto Piergentili1, Antonella Palena1, Laura Ciapponi1, Maria Grazia Giansanti1, Claudia Pellacani1, Romano Petrucci1, Giovanni Cenci2, Fiammetta Vernı`1, Barbara Fasulo1¤b, Michael L. Goldberg3, Ferdinando Di Cunto4, Maurizio Gatti1* 1 Dipartimento di Genetica e Biologia Molecolare, Istituto di Biologia e Patologia Molecolari del CNR, Rome, Italy, 2 Dipartimento di Biologia di Base ed Applicata, Universita` dell’Aquila, L’Aquila, Italy, 3 Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, United States of America, 4 Molecular Biotechnology Center, Universita` di Torino, Torino, Italy Abstract RNAi screens have, to date, identified many genes required for mitotic divisions of Drosophila tissue culture cells. However, the inventory of such genes remains incomplete. We have combined the powers of bioinformatics and RNAi technology to detect novel mitotic genes. We found that Drosophila genes involved in mitosis tend to be transcriptionally co-expressed. We thus constructed a co-expression–based list of 1,000 genes that are highly enriched in mitotic functions, and we performed RNAi for each of these genes. By limiting the number of genes to be examined, we were able to perform a very detailed phenotypic analysis of RNAi cells. We examined dsRNA-treated cells for possible abnormalities in both chromosome structure and spindle organization. This analysis allowed the identification of 142 mitotic genes, which were subdivided into 18 phenoclusters. Seventy of these genes have not previously been associated with mitotic defects; 30 of them are required for spindle assembly and/or chromosome segregation, and 40 are required to prevent spontaneous chromosome breakage. We note that the latter type of genes has never been detected in previous RNAi screens in any system. Finally, we found that RNAi against genes encoding kinetochore components or highly conserved splicing factors results in identical defects in chromosome segregation, highlighting an unanticipated role of splicing factors in centromere function. These findings indicate that our co-expression–based method for the detection of mitotic functions works remarkably well. We can foresee that elaboration of 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 mitotic proteins. Citation: Somma MP, Ceprani F, Bucciarelli E, Naim V, De Arcangelis V, et al. (2008) Identification of Drosophila Mitotic Genes by Combining Co-Expression Analysis and RNA Interference. PLoS Genet 4(7): e1000126. doi:10.1371/journal.pgen.1000126 Editor: R. Scott Hawley, Stowers Institute for Medical Research, United States of America Received January 22, 2008; Accepted June 16, 2008; Published July 18, 2008 Copyright: 2008 Somma et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by grants from AIRC (Italian Association for Cancer Research) and Italian Telethon to MG, and by a PRIN grant from MUR (Ministero dell’Universita` e della Ricerca) to MG and FD. Competing Interests: The authors have declared that no competing interests exist. * E-mail: maurizio.gatti@uniroma1.it ¤a Current address: Institut Gustave Roussy, Villejuif, France ¤b Current address: MCDB Department, University of California Santa Cruz, Santa Cruz, California, United States of America Introduction RNA interference (RNAi) in Drosophila cell cultures is a powerful tool for the identification of proteins involved in mitotic cell division. The addition of a double stranded RNA (dsRNA) to the cell medium leads to rapid downregulation of the corresponding mitotic protein, resulting in a specific and penetrant phenotype [1– 10]. Identification of mitotic genes/proteins by RNAi has thus far relied on two general approaches. The first involved genome-wide screens to detect gross changes in cell and nuclear morphology [3,6,8], defects in cytokinesis [7,8] or in spindle and centrosome structure [10]. Most of these screens were performed using automated microscopy [3,6,8] or the visual analysis of a very simple phenotype [7]. In a second approach, RNAi experiments were performed on selected gene groups, such as those encoding kinesins, actin-binding proteins, kinases or phosphatases [2,4,5,9]. Cells depleted for these proteins were examined by standard fluorescence microscopy that allowed detection of a wide spectrum of mitotic abnormalities. Although genome-wide and gene-specific approaches have identified many mitotic functions, the inventory of such proteins is likely to be largely incomplete. For example, RNAi has never been used to detect genes involved in establishing proper mitotic chromosome morphology or required to maintain mitotic chromo- some integrity. We present here a novel approach for the identification of mitotic proteins by RNAi. Using a co-expression- based bioinformatic procedure, we generated a list of 1000 Drosophila genes highly enriched in mitotic functions. We then performed RNAi experiments for each of these 1000 genes, and examined both mitotic chromosome structure and spindle morphology in the treated cells. This screen has led to the identification of 142 mitotic genes, 70 of which have not been previously implicated in mitosis. PLoS Genetics | www.plosgenetics.org 1 July 2008 | Volume 4 | Issue 7 | e1000126