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, MIER1a and b, both of which have been shown to function as transcriptional repressors. Our previous work revealed a dramatic shift in MIER1a subcellular localization from nuclear in normal breast tissue to cytoplasmic in invasive breast carcinoma, suggesting that loss of nuclear MIER1a 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 MIER1a 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 MIER1a to produce the MIER1-3Aa 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 MIER1-3Aa, from 4% to 53%, demonstrating that cytoplasmic localization of this isoform was due to CRM1-dependent nuclear export. Inclusion of exon 3A in MIER1b to produce the N-terminal variant MIER1-3Ab 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 a isoform.