Unexpected Role of the Steroid-Deficiency Protein Ecdysoneless in Pre-mRNA Splicing Ann-Katrin Claudius 1 , Patrizia Romani 2 , Tobias Lamkemeyer 3 , Marek Jindra 4" *, Mirka Uhlirova 1" * 1 Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany, 2 Dipartimento di Biologia Evoluzionistica Sperimentale, Universita ` di Bologna, Bologna, Italy, 3 Proteomics Facility, CECAD, University of Cologne, Cologne, Germany, 4 Biology Center, Academy of Sciences of the Czech Republic, Ceske Budejovice, Czech Republic Abstract The steroid hormone ecdysone coordinates insect growth and development, directing the major postembryonic transition of forms, metamorphosis. The steroid-deficient ecdysoneless 1 (ecd 1 ) 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 ecd 1 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. Citation: Claudius A-K, Romani P, Lamkemeyer T, Jindra M, Uhlirova M (2014) Unexpected Role of the Steroid-Deficiency Protein Ecdysoneless in Pre-mRNA Splicing. PLoS Genet 10(4): e1004287. doi:10.1371/journal.pgen.1004287 Editor: Kirst King-Jones, University of Alberta, Canada Received October 24, 2013; Accepted February 20, 2014; Published April 10, 2014 Copyright: ß 2014 Claudius et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Sofja Kovalevskaja Award from the AvH and CECAD funds from the DFG (Germany) to MU. MJ was supported by Marie Curie Fellowship Award 276569 from the European Union. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jindra@entu.cas.cz (MJ); mirka.uhlirova@uni-koeln.de (MU) " MJ and MU are joint senior authors on this work. Introduction The insect steroid hormones, ecdysteroids, regulate growth, stimulate molting, and orchestrate tissues to undergo complex morphogenetic changes during metamorphosis [1–3]. Temporal control of ecdysteroid synthesis by the larval prothoracic gland (PG) is therefore critical (for recent reviews see [4,5]). The biosynthetic pathway commences by converting cholesterol to 7- dehydrocholesterol by a Rieske oxygenase Neverland (Nvd) [6]. 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) then 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-of- function mutants that are available for sro [7], spo, phm, dib, sad, and shd [8] die as embryos. Understandably, Drosophila mutants that display reduced steroid titers during larval development provide invaluable experimental tools. Among these, the ecdysoneless 1 (ecd 1 ) mutants are homozygous viable at 22uC, but exposure to 29uC reduces their ecdysteroid titer and causes a developmental arrest [11,12]. The ecd 1 allele has been widely used since its discovery in 1977 [11] to test effects of ecdysteroid signaling on a number of processes from morphogen- esis to reproduction to behavior. Yet why these mutants lack the hormone has not been determined. Our original identification of the ecd gene [13] has revealed homology from fission yeast to humans but none that would illuminate the mode of Ecd action. Mammalian Ecd (also known as SGT1 and hEcd in humans) has been shown to stimulate cell proliferation by interacting with the Retinoblastoma (Rb) proteins [14]. Conditional deletion of the Ecd gene from mouse embryonic fibroblasts stalls these cells at the G 1 -S phase transition, suggesting that Ecd normally lifts the inhibitory effect of Rb on E2F-dependent cell cycle progression [14]. High hEcd expression has been correlated with malignancy of human breast [15] and pancreatic [16] tumors. In a mouse model, Ecd has been suggested to promote tumorigenesis via enhancing glucose import and glycolysis in the pancreatic tumor cells [16]. These observations illustrate the emerging importance of Ecd, but an underlying mechanism for Ecd action is still lacking. A systematic mapping of Drosophila protein-protein interactions [17] has uncovered contacts between Ecd and proteins responsible for pre-mRNA splicing. Among these are members of the U5 small nuclear ribonucleoprotein particle (snRNP) complex, PLOS Genetics | www.plosgenetics.org 1 April 2014 | Volume 10 | Issue 4 | e1004287