title: Isoform-specific regulation of a steroid hormone nuclear receptor by an E3 ubiquitin ligase in Drosophila melanogaster.
abstract: The steroid hormone 20-hydroxyecdysone (20E) regulates gene transcription through the heterodimeric nuclear receptor composed of ecdysone receptor (EcR) and Ultraspiracle (USP). The EcR gene encodes three protein isoforms--A, B1, and B2--with variant N-terminal domains that mediate tissue and developmental stage-specific responses to 20E. Ariadne-1a is a conserved member of the RING finger family of ubiquitin ligases first identified in Drosophila melanogaster. Loss-of-function mutations at key cysteines in either of the two RING finger motifs, as well as general overexpression of this enzyme, cause lethality in pupae, which suggests a requirement in metamorphosis. Here, we show that Ariadne-1a binds specifically the isoform A of EcR and ubiquitylates it. Co-immunoprecipitation experiments indicate that the full sequence of EcRA is required for this binding. Protein levels of EcRA and USP change in opposite directions when those of ARI-1a are genetically altered. This is an isoform-specific, E3-dependent regulatory mechanism for a steroid nuclear receptor. Further, qRT-PCR experiments show that the ARI-1a levels lead to the transcriptional regulation of Eip78C, Eip74EF, Eip75B, and Br-C, as well as that of EcR and usp genes. Thus, the activity of this enzyme results in the regulation of dimerizing receptors at the protein and gene transcription levels. This fine-tuned orchestration by a conserved ubiquitin ligase is required during insect metamorphosis and, likely, in other steroid hormone-controlled processes across species.
authors: Instituto Cajal, Consejo Superior de Investigaciones Científicas, Madrid 28002, Spain.
authors: Ana-Citlali
authors: AC
authors: Gradilla
authors: Alicia
authors: A
authors: Mansilla
authors: Alberto
authors: A
authors: Ferrús
mesh_terms: D021581:Active Transport, Cell Nucleus; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D029721:Drosophila Proteins / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000166:cytology / Q000201:enzymology / Q000254:growth & development / Q000378:metabolism*; D004440:Ecdysone / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D008297:Male; D008675:Metamorphosis, Biological; D010641:Phenotype; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011987:Receptors, Steroid / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D059010:Single-Cell Analysis; D013379:Substrate Specificity; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: C412166:Ari-1 protein, Drosophila; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D020033:Protein Isoforms; D011987:Receptors, Steroid; D014157:Transcription Factors; C066030:USP protein, Drosophila; C055469:ecdysone receptor; D004440:Ecdysone; D044767:Ubiquitin-Protein Ligases