Abstract
The steroid hormone ecdysone coordinates insect growth and development, directing the major postembryonic transition of forms, metamorphosis. The steroid-deficient ecdysoneless1 (ecd1) strain of Drosophila melanogaster has long served to assess the impact of ecdysone on gene regulation, morphogenesis, or reproduction. However, ecd also exerts cell-autonomous effects independently of the hormone, and mammalian Ecd homologs have been implicated in cell cycle regulation and cancer. Why the Drosophila ecd1 mutants lack ecdysone has not been resolved. Here, we show that in Drosophila cells, Ecd directly interacts with core components of the U5 snRNP spliceosomal complex, including the conserved Prp8 protein. In accord with a function in pre-mRNA splicing, Ecd and Prp8 are cell-autonomously required for survival of proliferating cells within the larval imaginal discs. In the steroidogenic prothoracic gland, loss of Ecd or Prp8 prevents splicing of a large intron from CYP307A2/spookier (spok) pre-mRNA, thus eliminating this essential ecdysone-biosynthetic enzyme and blocking the entry to metamorphosis. Human Ecd (hEcd) can substitute for its missing fly ortholog. When expressed in the Ecd-deficient prothoracic gland, hEcd re-establishes spok pre-mRNA splicing and protein expression, restoring ecdysone synthesis and normal development. Our work identifies Ecd as a novel pre-mRNA splicing factor whose function has been conserved in its human counterpart. Whether the role of mammalian Ecd in cancer involves pre-mRNA splicing remains to be discovered.
Highlights
The insect steroid hormones, ecdysteroids, regulate growth, stimulate molting, and orchestrate tissues to undergo complex morphogenetic changes during metamorphosis [1,2,3]
In order to verify the proteome-wide data, we expressed Myc epitope-tagged Ecdysoneless protein (Ecd) in Drosophila S2 cells and using mass spectrometry we examined material that coprecipitated with Myc::Ecd (Figure 1A)
This analysis identified three proteins of the U5 small nuclear ribonucleoprotein particle (snRNP) complex that were previously shown to associate with Ecd [17], namely orthologs of the budding yeast (Saccharomyces cerevisiae) Prp8p, Snu114p, and Brr2p that are encoded by D. melanogaster genes CG8877/prp8, CG4849/eftud2, and CG5931/l(3)72Ab, respectively
Summary
The insect steroid hormones, ecdysteroids, regulate growth, stimulate molting, and orchestrate tissues to undergo complex morphogenetic changes during metamorphosis [1,2,3]. A short-chain dehydrogenase/reductase encoded by shroud (sro) [7] and cytochrome P450 (CYP) enzymes encoded by spook (spo)/ spookier (spok), phantom (phm), disembodied (dib), and shadow (sad) catalyze the subsequent steps to produce ecdysone (E) [8,9]. Once released from the PG, E becomes hydroxylated in peripheral tissues by another CYP, Shade (Shd), to yield the main active hormone, 20-hydroxyecdysone (20E) [10]. Reflecting the necessity of 20E for early cuticle formation, Drosophila melanogaster loss-offunction mutants that are available for sro [7], spo, phm, dib, sad, and shd [8] die as embryos
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