Alternative splicing in the fiddler crab cognate ecdysteroid receptor: Variation in receptor isoform expression and DNA binding properties in response to hormone

Jul 16, 2014General and comparative endocrinology

Different versions of the fiddler crab hormone receptor vary in how they respond to the hormone

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Abstract

Four conserved isoforms of RXR were identified through cDNA cloning from three Uca species.

Alternative splicing occurs in the hinge and ligand binding domains of RXR.
A new cryptic exon and an amino terminal variant of EcR were detected through genomic sequencing and RNA-seq analysis.
Transcript abundance of EcR and RXR increases during ovarian maturation in U. pugilator, while it remains constant in a related species with a different reproductive strategy.
Electromobility shift assays showed that crab RXR LBD isoforms have different physical properties compared to fruit fly receptors.
Crab RXR LBD isoforms can heterodimerize with Drosophila EcR receptors, enhancing ligand and DNA binding.
Ecdysteroids do not increase DNA binding for crab heterodimers, suggesting that additional factors may influence receptor conformation.

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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.

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Alternative splicing in the fiddler crab cognate ecdysteroid receptor: Variation in receptor isoform expression and DNA binding properties in response to hormone

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