The Analysis Of Interaction Between ERF3and Survivin From Homo Sapiens

Translation termination in eukaryotes requires two release factors, eukaryotic release factor1(eRF1) and eRF3, to complete protein synthesis. eRF3is a GTPase associated with eRF1in a complex to ensure efficient stop codon recognition and fast polypeptide release. Studies have related eRF3with cell cycle regulation, cytoskeleton organization, and tumorigenesis.In mammals, two genes encode two distinct forms of eRF3, eRF3a and eRF3b, which differ in their N-terminal domains. Three transcript variants of eRF3a have been found that the variant2is one amino acid shorter than variant1and the variant3lacks about140amino acids at the N-terminal, compared to variant1.Survivin is the smallest and structural unique member of inhibitor of apoptosis (IAP) protein family, which deserves growing attention due to its universal over-expression in human tumors, and its prominent role in disparate networks of cellular division, intracellular signaling and apoptosis.Both eRF3and survivin are involved in cell cycle regulation and tumorigenesis, respectively. Whether there exists coordination between eRF3and survivin in these processes remains unknown. This study confirmed the interaction between eRF3a-F3, eRF3b and survivin by using yeast two hybrid and pull down assays, and mapped the interacting domains on eRF3and survivin. Furthermore, we analyzed the subcellular localization of eRF3and survivin, which provide a major clue for investigation of regulation during cell division. The results obtained are as follows.The full-length cDNA of human survivin was cloned from HeLa cells and a set of recombinant plasmids were constructed for yeast two hybrid(Y2H). The plasmids were co-transformed into the yeast strain Saccharomyces cerevisiae AH109and the clones were strictly selected by using medium of SD-Leu-Trp-His-Ade. Finally, the clones were analyzed by β-galactosidase filter-lift assays. The results displayed that yeast strain expressing both eRF3a-F3/survivin genes and eRF3b/survivin genes were observed to turn blue, similar to that of positive control expressing eRF1/eRF3a-F3genes and eRF1/eRF3b genes, whereas no blue color were observed on filter of negative control only expressing survivin, eRF3a-F3or eRF3b, suggesting the interaction between eRF3a-F3, eRF3b and survivin.To further affirm the interaction of both molecules, a series recombinant expression plasmids were conducted for pull down assays and transformed into Escherichia coli BL21(DE3) strain to express fusion protein. GST and His fusion proteins were purified by affinity chromatography on glutathione-Sepharose TM4B beads or Ni-NTA beads, respectively. Supernatant containing recombinant His-eRF3a-F3was loaded into the GS4B beads that contain immobilized GST-survivin, while supernatant containing recombinant GST-eRF3b was loaded into the Ni-NTA beads that contain immobilized His-survivin. After incubation at4℃, beads were washed to remove non-specific proteins. Bound proteins were analyzed by SDS-PAGE and Western blotting using anti-His antibody or anti-GST antibody, respectively. Both results indicated that eRF3and survivin in Homo sapiens could interact in vitro.To identify the minimal interacting domains on eRF3and survivin, the eRF3a-F3, eRF3b and survivin were mutated and seven recombinant plasmids were obtained. The Y2H results suggested that survivin binding domain on eRF3should be located1-72aa of eRF3a-F3or131-200aa of eRF3b. The1-72aa of eRF3a-F3was correspond to139-210aa of eRF3a-F1and130-201aa of eRF3b, suggesting this region might be essential for interaction between eRF3and survivin. The results of assays also revealed that65-76aa region on BIR domain of survivin was an essential motif for binding to eRF3, where amino acids (68EPDDD72) formed a beta fold with negative charge.Furthermore, in order to investigate the function of both factors in intracellular processes, the coding regions of cDNA for eRF3a-F3, heRF3b and survivin were fused in frame to the green fluorescent protein (GFP) and red fluorescent protein (RFP). Over expression of GFP-heRF3a-F3and GFP-heRF3b lead to the extensive nuclear accumulation of the GFP fusion protein in HeLa cell, and RFP-survivin expressed the red florescence in nucleus. Both factors were co-localized in nucleus of HeLa cell, suggesting they might function in nuclear processes.