A toolset to study functions of Cytosolic non-specific dipeptidase 2 (CNDP2) using Drosophila as a model organism

Abstract Background Expression of the CNDP2 gene is frequently up- or down-regulated in different types of human cancers. However, how the product of this gene is involved in cell growth and proliferation is poorly understood. Moreover, our knowledge of the functions of the CNDP2 orthologs in well-established model organisms is scarce. In particular, the function of the D. melanogaster ortholog of CNDP2, encoded by the CG17337 gene (hereafter referred to as dCNDP2), is still unknown. Results This study was aimed at developing a set of genetic and molecular tools to study the roles of dCNDP2. We generated a dCNDP2 null mutation (hereafter ∆dCNDP2) using CRISPR/Cas9-mediated homologous recombination (HR) and found that the ∆dCNDP2 mutants are homozygous viable, morphologically normal and fertile. We also generated transgenic fly lines expressing eGFP-tagged and non-tagged dCNDP2 protein, all under the control of the UAS promoter, as well as polyclonal antibodies specific to dCNDP2. Using these tools, we demonstrate that only one of the two predicted dCNDP2 isoforms is expressed throughout the different tissues tested. dCNDP2 was detected in both the cytoplasm and the nucleus, and was found to be associated with multiple sites in the salivary gland polytene chromosomes. Conclusions The dCNDP2 gene is not essential for fly viability under standard laboratory conditions. The subcellular localization pattern of dCNDP2 suggests that this protein might have roles in both the cytoplasm and the nucleus. The genetic and molecular tools developed in this study will allow further functional characterization of the conserved CNDP2 protein using D. melanogaster as a model system

RNAi-mediated depletion of the NSL complex subunits leads to abnormal chromosome segregation and defective centrosome duplication in Drosophila mitosis.

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