title: Alternative splicing in the fiddler crab cognate ecdysteroid receptor: variation in receptor isoform expression and DNA binding properties in response to hormone.
abstract: RXR cDNA cloning from three Uca species led to the identification of 4 conserved isoforms, indicative of alternative splicing in the hinge and ligand binding domains (LBD). Sequencing of overlapping clones from a Ucapugilator genomic library identified EcR isoforms matching previously identified cDNA variants; in addition, a cryptic exon in the LBD was detected and evidence for expression of this new isoform was obtained from next-generation sequencing. RNA-seq analysis also identified a new amino terminal EcR variant. EcR and RXR transcript abundance increases throughout ovarian maturation in U. pugilator, while cognate receptor transcript abundance remains constant in a related Indo-Pacific species with a different reproductive strategy. To examine if crab RXR LBD isoforms have different physical properties in vitro, electromobility shift assays were performed with different EcR isoforms. The cognate crab and fruit fly receptors differ in their responses to hormone. Ecdysteroids did not increase DNA binding for the crab heterodimers, while ecdysteroids stimulate binding for Drosophilamelanogaster EcR/USP heterodimers. In swapping experiments, UpEcR/USP heterodimers did not show ligand-responsive differences in DNA binding; both crab RXR LBD isoforms, however, conferred ligand-responsive increases in DNA binding with DmEcRs. These data indicate that both UpRXR LBD isoforms can heterodimerize with the heterologous DmEcR receptors and promote ligand and DNA binding. Unresponsiveness of the cognate receptors to ecdysteroid, however, suggest additional factors may be required to mediate endogenous, perhaps isoform-specific, differences in EcR conformation, consistent with previously reported effects of UpRXR isoforms on UpEcR ligand-binding affinities.
authors.affiliation: Department of Biology, University of Oklahoma, Norman, OK 73019, USA. Electronic address: ddurica@ou.edu.
authors.forename: David S
authors.initials: DS
authors.lastname: Durica
authors.affiliation: Department of Biology, University of Oklahoma, Norman, OK 73019, USA.
authors.forename: Sunetra
authors.initials: S
authors.lastname: Das
authors.affiliation: Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019, USA.
authors.forename: Fares
authors.initials: F
authors.lastname: Najar
authors.affiliation: Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019, USA.
authors.forename: Bruce
authors.initials: B
authors.lastname: Roe
authors.affiliation: Department of Biology, University of Oklahoma, Norman, OK 73019, USA.
authors.forename: Barret
authors.initials: B
authors.lastname: Phillips
authors.affiliation: Sree Narayana College, Kannur 670 007, India.
authors.forename: Sudha
authors.initials: S
authors.lastname: Kappalli
authors.affiliation: School of Biotechnology, Chemical and Biomedical Engineering, VIT University, Vellore 632 014, India.
authors.forename: Gopinathan
authors.initials: G
authors.lastname: Anilkumar
mesh_terms: D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003386:Brachyura / Q000502:physiology*; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D026461:Ecdysteroids / Q000494:pharmacology*; D005091:Exons / Q000235:genetics; D007438:Introns / Q000235:genetics; D008024:Ligands; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D011987:Receptors, Steroid / Q000235:genetics* / Q000378:metabolism; D047488:Retinoid X Receptors / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D026461:Ecdysteroids; D008024:Ligands; D020033:Protein Isoforms; D012333:RNA, Messenger; D011987:Receptors, Steroid; D047488:Retinoid X Receptors; C034808:ecdysteroid receptor; D004247:DNA
keywords: EcR; Ecdysteroid receptor; Fiddler crab; Limb regeneration; Oogenesis; RXR