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The Drosophila Nonspecific Lethal (NSL) complex is a major transcriptional regulator of housekeeping genes. It contains at least seven subunits that are conserved in the human KANSL complex: Nsl1/Wah (KANSL1), Dgt1/Nsl2 (KANSL2), Rcd1/Nsl3 (KANSL3), Rcd5 (MCRS1), MBD-R2 (PHF20), Wds (WDR5) and Mof (MOF/KAT8). Previous studies have shown that Dgt1, Rcd1 and Rcd5 are implicated in centrosome maintenance. Here, we analyzed the mitotic phenotypes caused by RNAi-mediated depletion of Rcd1, Rcd5, MBD-R2 or Wds in greater detail. Depletion of any of these proteins in Drosophila S2 cells led to defects in chromosome segregation. Consistent with these findings, Rcd1, Rcd5 and MBD-R2 RNAi cells showed reduced levels of both Cid/CENP-A and the kinetochore component Ndc80. In addition, RNAi against any of the four genes negatively affected centriole duplication. In Wds-depleted cells, the mitotic phenotypes were similar but milder than those observed in Rcd1-, Rcd5- or MBD-R2-deficient cells. RT-qPCR experiments and interrogation of published datasets revealed that transcription of many genes encoding centromere/kinetochore proteins (e.g., cid, Mis12 and Nnf1b), or involved in centriole duplication (e.g., Sas-6, Sas-4 and asl) is substantially reduced in Rcd1, Rcd5 and MBD-R2 RNAi cells, and to a lesser extent in wds RNAi cells. During mitosis, both Rcd1-GFP and Rcd5-GFP accumulate at the centrosomes and the telophase midbody, MBD-R2-GFP is enriched only at the chromosomes, while Wds-GFP accumulates at the centrosomes, the kinetochores, the midbody, and on a specific chromosome region. Collectively, our results suggest that the mitotic phenotypes caused by Rcd1, Rcd5, MBD-R2 or Wds depletion are primarily due to reduced transcription of genes involved in kinetochore assembly and centriole duplication. The differences in the subcellular localizations of the NSL components may reflect direct mitotic functions that are difficult to detect at the phenotypic level, because they are masked by the transcription-dependent deficiency of kinetochore and centriolar proteins., Competing Interests: The authors have declared that no competing interests exist.