title: Differential splicing alters subcellular localization of the alpha but not beta isoform of the MIER1 transcriptional regulator in breast cancer cells.
abstract: MIER1 was originally identified in a screen for novel fibroblast growth factor activated early response genes. The mier1 gene gives rise to multiple transcripts encoding protein isoforms that differ in their amino (N-) and carboxy (C-) termini. Much of the work to date has focused on the two C-terminal variants, MIER1α and β, both of which have been shown to function as transcriptional repressors. Our previous work revealed a dramatic shift in MIER1α subcellular localization from nuclear in normal breast tissue to cytoplasmic in invasive breast carcinoma, suggesting that loss of nuclear MIER1α may play a role in breast cancer development. In the present study, we investigated whether alternative splicing to include a cassette exon and produce an N-terminal variant of MIER1α affects its subcellular localization in MCF7 breast carcinoma cells. We demonstrate that this cassette exon, exon 3A, encodes a consensus leucine-rich nuclear export signal (NES). Inclusion of this exon in MIER1α to produce the MIER1-3Aα isoform altered its subcellular distribution in MCF7 cells from 81% nuclear to 2% nuclear and this change in localization was abrogated by mutation of critical leucines within the NES. Treatment with leptomycin B (LMB), an inhibitor of the nuclear export receptor CRM1, resulted in a significant increase in the percentage of cells with nuclear MIER1-3Aα, from 4% to 53%, demonstrating that cytoplasmic localization of this isoform was due to CRM1-dependent nuclear export. Inclusion of exon 3A in MIER1β to produce the N-terminal variant MIER1-3Aβ however had little effect on the nuclear targeting of this isoform. Our results demonstrate that alternative splicing to include exon 3A specifically affects the localization pattern of the α isoform.
authors.affiliation: Terry Fox Cancer Research Laboratories, Division of BioMedical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St John's, Newfoundland, Canada.
authors.forename: Jaclyn A
authors.initials: JA
authors.lastname: Clements
authors.forename: F Corinne
authors.initials: FC
authors.lastname: Mercer
authors.forename: Gary D
authors.initials: GD
authors.lastname: Paterno
authors.forename: Laura L
authors.initials: LL
authors.lastname: Gillespie
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000235:genetics; D017398:Alternative Splicing; D001943:Breast Neoplasms / Q000378:metabolism*; D045744:Cell Line, Tumor; D003062:Codon; D004268:DNA-Binding Proteins; D005091:Exons; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D010957:Plasmids / Q000378:metabolism; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D012326:RNA Splicing; D014157:Transcription Factors / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*
chemical_list: D003062:Codon; D004268:DNA-Binding Proteins; C470480:MIER1 protein, human; D009687:Nuclear Proteins; D020033:Protein Isoforms; D014157:Transcription Factors