Ectopic miR-975 induces CTP synthase directed cell proliferation and differentiation in Drosophila melanogaster

Abstract

CTP synthase (CTPSyn) is an essential metabolic enzyme, synthesizing precursors required for nucleotides and phospholipids production. Previous studies have also shown that CTPSyn is elevated in various cancers. In many organisms, CTPSyn compartmentalizes into filaments called cytoophidia. In Drosophila melanogaster , only its isoform C (CTPSynIsoC) forms cytoophidia. In the fruit fly’s testis, cytoophidia are normally seen in the transit amplification regions close to its apical tip, where the stem-cell niche is located, and development is at its most rapid. Here, we report that CTPSynIsoC overexpression causes the lengthening of cytoophidia throughout the entirety of the testicular body. A bulging apical tip is found in approximately 34% of males overexpressing CTPSynIsoC . Immunostaining shows that this bulged phenotype is most likely due to increased numbers of both germline cells and spermatocytes. Through a microRNA (miRNA) overexpression screen, we found that ectopic miR-975 concurrently increases both the expression levels of CTPSyn and the length of its cytoophidia. The bulging testes phenotype was also recovered at a penetration of approximately 20%. However, qPCR assays reveal that CTPSynIsoC and miR-975 overexpression each provokes a differential response in expression of a number of cancer-related genes, indicating that the shared CTPSyn upregulation seen in either case is likely the cause of observed testicular overgrowth. This study presents the first instance of consequences of miRNA-asserted regulation upon CTPSyn in D . melanogaster , and further reaffirms the enzyme’s close ties to germline cells overgrowth.

A TRiP RNAi screen to identify molecules necessary for Drosophila photoreceptor differentiation

“…While this domain is associated with DNA binding, it also facilitates protein-protein interactions. CTP synthase is involved in myc -mediated control of cell size ( Aughey et al 2016 ), and regulation of CTPsyn by miR-975 controls cell proliferation and differentiation ( Woo et al 2019 ). While we observed no cell size or proliferation defects, CTP synthase knockdown resulted in aberrant rhabdomere morphology.…”

CTP synthase: the hissing of the cellular serpent

“…Besides, interestingly the overexpression of CTPsyn interacting micro-RNA, miR-975 in Drosophila testis, also showed a similar bulging phenotype in the apical tip, and the cytoophidia were also observed to be lengthened. qPCR assay for both the CTPsynIsoC and miR-975 overexpressions provide evidence for the differential expression of several cancer-related genes, Ack , Brat , IMPDH , Myc , and CTPS , thus reaffirming that CTPsyn upregulation could harness tumor-like testicular overgrowth due to over-proliferation of germline cells (Woo et al 2019 ).

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“…6 ). However, during overexpression studies of D. melanogaster , CTP synthetase isoform C ( CTPsynIsoC ), which is the only isoform that forms cytoophidia (Azzam and Liu 2013 ), causes the elongation of cytoophidia in the entirety of Drosophila testicular body, and around 34% of these males were observed to have testicular apical tip bulging phenotype due to the over-proliferation of germline cells and spermatocytes (Woo et al 2019 ). This suggests that germline cell proliferation, which is a hallmark of a few types of tumorigenesis and cancer formation, could be due to the overexpression of CTPsyn in the rapidly proliferating cells.…”

“…Knocking down CTPsyn in the background of Ras overexpressing flies reduced cell proliferation significantly (Zhou et al 2022 ), supporting a previous finding that suggests blocking CTPsyn activity could be a therapeutic target to treat cancer to inhibit cell proliferation (Sun et al 2022 ). Fascinatingly, blocking the cytoophidia-forming ability of CTPsyn in an active Ras overexpressing environment is sufficient to reduce Ras-dependent cell proliferation (Zhou et al 2022 ), suggesting that cytoophidia formation most likely could be an indication of rapid cell proliferation (Woo et al 2019 ). It is important to note that Ras is also involved in nucleotide synthesis pathway via activation of Myc (Santana-Codina et al 2018 ).…”

A transcriptomic (RNA-seq) analysis of Drosophila melanogaster adult testes overexpressing microRNA-2b-1

“…Approximately 90-100 testes were pooled together for each replicate of RNA extraction using the combination of Trizol reagent (Invitrogen, USA) and RNeasy Mini Kit (Qiagen, Germany) as previously mentioned in Woo et al [3] . Total RNA was used for cDNA library construction following the protocol supplied with the Truseq™ RNA sample prep Kit (Illumina, San Diego, USA).…”