MicroRNAs are abundant in animal genomes, yet little is known about their functions in vivo. Here, we report the production of 80 new Drosophila miRNA mutants by targeted homologous recombination. These mutants remove 104 miRNAs. Together with 15 previously reported mutants, this collection includes 95 mutants deleting 130 miRNAs. Collectively, these genes produce over 99% of all Drosophila miRNAs, measured by miRNA sequence reads. We present a survey of developmental and adult miRNA phenotypes. Over 80% of the mutants showed at least one phenotype using a p < 0.01 significance threshold. We observed a significant correlation between miRNA abundance and phenotypes related to survival and lifespan, but not to most other phenotypes. miRNA cluster mutants were no more likely than single miRNA mutants to produce significant phenotypes. This mutant collection will provide a resource for future analysis of the biological roles of Drosophila miRNAs.
Bicaudal-C (Bic-C) is a multiple KH-domain RNA-binding protein required for Drosophila oogenesis and, maternally, for embryonic patterning. In early oogenesis, Bic-C negatively regulates target mRNAs, including Bic-C, by recruiting the CCR4 deadenylase through a direct association with its NOT3 subunit. Here, we identify a novel function for Bic-C in secretion of the TGF-α homolog Gurken (Grk). In Bic-C mutant egg chambers, Grk is sequestered within actin-coated structures during mid-oogenesis. As a consequence, Egfr signalling is not efficiently activated in the dorsal-anterior follicle cells. This phenotype is strikingly similar to that of trailer hitch (tral) mutants. Consistent with the idea that Bic-C and Tral act together in Grk secretion, Bic-C co-localizes with Tral within cytoplasmic granules, and can be co-purified with multiple protein components of a Tral mRNP complex. Taken together, our results implicate translational regulation by Bic-C and Tral in the secretory pathway.
Dosage-sensitive modifier screening is a powerful tool for linking genes to biological processes. Use of chromosomal deletions permits sampling the effects of removing groups of genes related by position on the chromosome. Here, we explore the use of inducible microRNA transgenes as a complement to deficiency-based modifier screens. miRNAs are predicted to have hundreds of targets. miRNA overexpression provides an efficient means to reduces expression of large gene sets. A collection of transgenes was prepared to allow overexpression of 89 miRNAs or miRNA clusters. These transgenes and a set of genomic deficiencies were screened for their ability to modify the bristle phenotype of the cell-cycle regulator minus. Sixteen miRNAs were identified as dominant suppressors, while the deficiency screen uncovered four genomic regions that contain a dominant suppressor. Comparing the genes uncovered by the deletions with predicted miRNA targets uncovered a small set of candidate suppressors. Two candidates were identified as suppressors of the minus phenotype, Cullin-4 and CG5199/Cut8. Additionally, we show that Cullin-4 acts through its substrate receptor Cdt2 to suppress the minus phenotype. We suggest that inducible microRNA transgenes are a useful complement to deficiency-based modifier screens.
microRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by destabilizing target transcripts and/or inhibiting their translation. miRNAs are thought to have roles in buffering gene expression to confer robustness. miRNAs have been shown to play important roles during tissue development to control cell proliferation, differentiation and morphogenesis. Many miRNAs are expressed in the germ line of Drosophila, and functions have been reported for a few miRNAs in maintenance of stem cell proliferation during oogenesis. Here, we analyse the function of Drosophila miR-989 in oogenesis. miR-989 is abundant in ovaries. Mutants lacking miR-989 did not display gross abnormalities affecting egg chamber formation or maturation. However, the migration of the border cell cluster was severely delayed in miR-989 mutant egg chambers. We demonstrate that miR-989 function is required in the somatic cells in the egg chamber, not in germ line cells for border cell migration. Loss of miR-989 from a fraction of the border cell cluster was sufficient to impair cluster migration as a whole, suggesting a role in border cells. Gene ontology analysis reveals that many predicted miR-989 target mRNAs are implicated in regulating cell migration, cell projection morphogenesis, cell adhesion as well as receptor tyrosine kinase and ecdysone signalling, consistent with an important regulatory role for miR-989 in border cell migration.
BackgroundChanges in cellular metabolism are now recognized as potential drivers of cancer development, rather than as secondary consequences of disease. Here, we explore the mechanism by which metabolic changes dependent on aldehyde dehydrogenase impact cancer development.MethodsALDH7A1 was identified as a potential cancer gene using a Drosophila in vivo metastasis model. The role of the human ortholog was examined using RNA interference in cell-based assays of cell migration and invasion. 1H-NMR metabolite profiling was used to identify metabolic changes in ALDH7A1-depleted cells. Publically available cancer gene expression data was interrogated to identify a gene-expression signature associated with depletion of ALDH7A1. Computational pathway and gene set enrichment analysis was used to identify signaling pathways and cellular processes that were correlated with reduced ALDH7A1 expression in cancer. A variety of statistical tests used to evaluate these analyses are described in detail in the methods section. Immunohistochemistry was used to assess ALDH7A1 expression in tissue samples from cancer patients.ResultsDepletion of ALDH7A1 increased cellular migration and invasiveness in vitro. Depletion of ALDH7A1 led to reduced levels of metabolites identified as ligands for Peroxisome proliferator-activated receptor (PPARα). Analysis of publically available cancer gene expression data revealed that ALDH7A1 mRNA levels were reduced in many human cancers, and that this correlated with poor survival in kidney and liver cancer patients. Using pathway and gene set enrichment analysis, we establish a correlation between low ALDH7A1 levels, reduced PPAR signaling and reduced patient survival. Metabolic profiling showed that endogenous PPARα ligands were reduced in ALDH7A1-depleted cells. ALDH7A1-depletion led to reduced PPAR transcriptional activity. Treatment with a PPARα agonist restored normal cellular behavior. Low ALDH7A1 protein levels correlated with poor clinical outcome in hepatocellular and renal clear cell carcinoma patients.ConclusionsWe provide evidence that low ALDH7A1 expression is a useful prognostic marker of poor clinical outcome for hepatocellular and renal clear cell carcinomas and hypothesize that patients with low ALDH7A1 might benefit from therapeutic approaches addressing PPARα activity.Electronic supplementary materialThe online version of this article (10.1186/s12885-018-5061-7) contains supplementary material, which is available to authorized users.
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