Abstract
BackgroundAlternative splicing is critical for generating complex proteomes in response to extracellular signals. Nuclear receptors including estrogen receptor alpha (ERα) and their ligands promote alternative splicing. The endogenous targets of ERα:estradiol (E2)-mediated alternative splicing and the influence of extracellular kinases that phosphorylate ERα on E2-induced splicing are unknown.MethodsMCF-7 and its anti-estrogen derivatives were used for the majority of the assays. CD44 mini gene was used to measure the effect of E2 and AKT on alternative splicing. ExonHit array analysis was performed to identify E2 and AKT-regulated endogenous alternatively spliced apoptosis-related genes. Quantitative reverse transcription polymerase chain reaction was performed to verify alternative splicing. ERα binding to alternatively spliced genes was verified by chromatin immunoprecipitation assay. Bromodeoxyuridine incorporation-ELISA and Annexin V labeling assays were done to measure cell proliferation and apoptosis, respectively.ResultsWe identified the targets of E2-induced alternative splicing and deconstructed some of the mechanisms surrounding E2-induced splicing by combining splice array with ERα cistrome and gene expression array. E2-induced alternatively spliced genes fall into at least two subgroups: coupled to E2-regulated transcription and ERα binding to the gene without an effect on rate of transcription. Further, AKT, which phosphorylates both ERα and splicing factors, influenced ERα:E2 dependent splicing in a gene-specific manner. Genes that are alternatively spliced include FAS/CD95, FGFR2, and AXIN-1. E2 increased the expression of FGFR2 C1 isoform but reduced C3 isoform at mRNA level. E2-induced alternative splicing of FAS and FGFR2 in MCF-7 cells correlated with resistance to FAS activation-induced apoptosis and response to keratinocyte growth factor (KGF), respectively. Resistance of MCF-7 breast cancer cells to the anti-estrogen tamoxifen was associated with ERα-dependent overexpression of FGFR2, whereas resistance to fulvestrant was associated with ERα-dependent isoform switching, which correlated with altered response to KGF.ConclusionE2 may partly alter cellular proteome through alternative splicing uncoupled to its effects on transcription initiation and aberration in E2-induced alternative splicing events may influence response to anti-estrogens.
Highlights
Alternative splicing is critical for generating complex proteomes in response to extracellular signals
Two cell systems were used: 1) 293 cells requiring transfection of both the CD44 minigene and ERα or its mutants; 2) MCF-7p and MCF-7AKT cells transfected with the CD44 minigene
This study identified E2 regulated alternative splicing of genes involved in apoptosis and breast epithelial cell transformation
Summary
Alternative splicing is critical for generating complex proteomes in response to extracellular signals. Nuclear receptors including estrogen receptor alpha (ERα) and their ligands promote alternative splicing. The endogenous targets of ERα:estradiol (E2)-mediated alternative splicing and the influence of extracellular kinases that phosphorylate ERα on E2-induced splicing are unknown. Estradiol (E2) signaling primarily involves activation of nuclear receptors, estrogen receptors alpha (ERα) and beta (ERβ), which function as transcription factors that regulate gene expression through either DNA binding or through protein-protein interaction [1,2]. E2 signaling in Alternative splicing is an important post-transcriptional mechanism that permits the generation of multiple protein products from a single gene. RS domains of SR proteins contain multiple copies of consensus AKT phosphorylation site RXRXXS and AKT controls their activity in the nucleus and cytoplasm [11]
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