Nuclear Distribution Of Eukaryotic Initiation Factor3g And Its Interacting Nuclear Proteins

Eukaryotic translation initiation factors (eIFs) are a group of important functional cellular proteins that form protein translation initiation complex, which initiates protein synthesis. Six eIFs (eIFl-eIF6) have been identified up to date, each of them is consisted of several subunits. Aberrant expressions of some subunits of eIFs in various types of cancer have been reported to be associated with malignant behavior of cancer cells. For instance, eIF4E is overexpressed in cancer cells and can be used as an effective therapeutic target. eIF3is the largest and most complicate translation initiation factor, and one key factor that links other eIFs in the translation initiation complex. eIF3is comprised of13subunits with a molecular weight of approximately700～800kDa. Its subunits are named eIF3a-eIF3m according to their molecular weight. eIF3g is a core subunit of eIF3and known to be involved in the translation reinitiation process. Recent studies showed that eIF3g contains binding sites for cytoskeleton protein4.1R and apoptosis inducing factor. We previously found that eIF3g was overexpressed in an adriamycin (ADR)-resistant human erythroleukemia cell line, downregulation of eIF3g could inhibit drug resistance and growth of leukemia cells. We also observed a positive correlation between overexpression of eIF3g and lymph node metastasis in a pilot study to evaluate the clinical significance of eIF3g overexpression in breast cancer. These results provided us the basis for further investigation on the roles and mechanism of eIF3g plays in cancer. We hypothesize that eIF3g may be involved in the development and/or in the progression of cancer by mechanisms other than that in translation regulatory.In this subject, we firstly predicted that eIF3g may have a nuclear location through online bioinformatics tools. The nuclear location of eIF3g was confirmed by experiments; its interacting nuclear proteins were identified and confirmed for further research. We confirmed the nuclear distribution of eIF3g by performing subcellular fractionation in combination with Western blotting and immunofluorescent stains. By co-immunoprecipitation (co-IP) in combination with mass spectrometry, candidate eIF3g-interacting proteins in nucleus were identified, including hnRNP U, HSZFP36and β-acthi. The protein-protein interaction was further confirmed by co-IP, cross-linking and GST-pulldown followed by Western blotting, and the co-localization of these proteins by confocal microscopy. Specific contents are listed as follows:1. Prediction and confirmation of the nuclear location of eIF3gBioinformatics with state-of-art online tools, including WoLF PSORT (http://wolfpsort.org), PROST II prediction (http://psort.hgc.jp/form2.html), Subnuclear Compartments Prediction System (http://array.bioengr.uic.edu/subnuclear.htm) and PredictProtein (https://www.predictprotein.org) were used to predict eIF3g distribution and potential function(s). Then subcellular fractionation followed by Western blotting and immunofluorescent stains followed by con-focal were used to verify the subcelluar location of eIF3g. Those four bioinformatics tools predicted a nuclear location of eIF3g; and the PredictProtein predicted that eIF3g may combine to the DNA or RNA in the nucleus. The nuclear location was confirmed by Western blotting and con-focal.2. Identification of nuclear proteins that may interact with eIF3gTo further study the nuclear proteins that may interact with eIF3g, we constructed a human breast cancer cell line (Bcap37/Tet-on-eIF3g) with doxycycline-inducible overexpression of eIF3g. We isolated the nuclear fraction of eIF3g-expressing cells, and performed co-IP with anti-eIF3g antibody, followed by SDS-PAGE and visualized by Coomassie blue staining. Compared with control group, four bands were selected for subsequent mass spectrometry analysis for protein characterization. Those proteins are hnRNP U, HSZFP36, P-actin and eIF3A respectively. Their nuclear locations were confirmed through Western blotting and con-focal.3. Confirmation of interaction between eIF3g and hnRNP U, HSZFP36and β-actinThe nuclear fraction of Bcap37/Tet-on-eIF3g cells was harvested for co-and reverse-co-IP. The co-IP results demonstrated that eIF3g antibody pulled down hnRNP U, HSZFP36and β-actin. The reverse-co-IP results demonstrated that hnRNP U, HSZFP36and β-actin antibodies pulled down eIF3g. We then performed in vitro crosslinking in combination with Western blotting in nucleus, demonstrated that eIF3g forms complex with hnRNP U, HSZFP36and β-actin. Further, GST-eIF3g fusion protein vector was constructed for GST-pulldown experiment. The following Western blotting detected the interaction between eIF3g and hnRNP U, HSZFP36, and P-actin. At last the co-localization of eIF3g and HSZFP36, P-actin was observed through con-focal. Thus, the physical interactions between eIF3g and hnRNP U, HSZFP36, and β-actin were confirmed by performing co-IP, in vitro crosslinking, GST-pulldown and con-focal.Conclusion:Our data reveal the novel finding that eIF3g locates in the nucleus and interacts with hnRNP U, HSZFP36and β-actin.