IDENTIFICATION OF A NOVEL IMPORTIN ALPHA PREDOMINANTLY EXPRESSED IN BOVINE OOCYTES AND EARLY EMBRYOS

Abstract

Early embryonic development is a complex process that involves timely and quantitative control of gene transcription. Nuclear proteins, including transcription factors and chromatin remodeling proteins, are required for initiation of transcription in early embryos prior to embryonic genome activation. The nuclear transport of these proteins is mediated by transport factors such as importins. Through analysis of EST sequences from a bovine oocyte cDNA library and the database, we identified a novel transcript which shows sequence similarity to importin alpha subunits. The novel transcript (contig) is represented by multiple ESTs that are only present in the oocyte library and a 2-cell embryo library. The largest cDNA clone obtained from the oocyte library is 720 bp in length containing a poly (A) tail. Additional 5′ end sequence was obtained by RT-PCR amplification of cDNA from a fetal ovary using primers designed based on a bovine genomic sequence. The final assembled cDNA is 1680 bp (missing 5′-UTR). Sequence analysis revealed that the cDNA has an open reading frame encoding a protein of 522 amino acids and the predicted protein shares 80%, 79%, 67%, and 65% sequence identity with bovine importin alpha 1, alpha 2, alpha 3 and alpha 7, respectively. The protein contains a conserved importin beta binding domain (IBB) at its N-terminus and 2 ARM (Armadillo/beta-catenin-like repeats) motifs in the central region. Analysis of tissue distribution of this novel transcript by reverse transcription polymerase chain reaction revealed that this gene is predominantly expressed in ovarian samples. Quantitative real time PCR analysis demonstrated that the expression of this novel importin alpha is significantly higher in GV stage oocytes as compared to importin alpha 1, alpha 2, alpha 3 and alpha 7. Analysis of temporal expression of the novel transcript during early embryonic development showed that the gene is highly expressed in GV and MII stage oocytes and remains at high levels of expression in embryos at 2-cell, 4-cell, and 8-cell stages but the transcript was barely detectable in morula and blastocyst stage embryos. These results suggest that this novel importin is responsible for nuclear transport of certain key transcription factors and/or other essential nuclear proteins required for activation of the embryonic genome during early embryogenesis. (platform)