Eukaryotic initiation factor 4G (eIF4G) promotes mRNA recruitment to the ribosome by binding to the mRNA cap-and poly(A) tail-binding proteins eIF4E and Pap1p. eIF4G also binds eIF4A at a distinct HEAT domain composed of five stacks of antiparallel ␣-helices. The role of eIF4G in the later steps of initiation, such as scanning and AUG recognition, has not been defined. Here we show that the entire HEAT domain and flanking residues of Saccharomyces cerevisiae eIF4G2 are required for the optimal interaction with the AUG recognition factors eIF5 and eIF1. eIF1 binds simultaneously to eIF4G and eIF3c in vitro, as shown previously for the C-terminal domain of eIF5. In vivo, cooverexpression of eIF1 or eIF5 reverses the genetic suppression of an eIF4G HEAT domain Ts ؊ mutation by eIF4A overexpression. In addition, excess eIF1 inhibits growth of a second eIF4G mutant defective in eIF4E binding, which was also reversed by cooverexpression of eIF4A. Interestingly, excess eIF1 carrying the sui1-1 mutation, known to relax the accuracy of start site selection, did not inhibit the growth of the eIF4G mutant, and sui1-1 reduced the interaction between eIF4G and eIF1 in vitro. Moreover, a HEAT domain mutation altering eIF4G moderately enhances translation from a non-AUG codon. These results strongly suggest that the binding of the eIF4G HEAT domain to eIF1 and eIF5 is important for maintaining the integrity of the scanning ribosomal preinitiation complex.
Hepatocellular carcinoma (HCC) is a common malignant tumor that severely threatens human health. The poor prognosis of HCC is mainly attributed to intrahepatic and extrahepatic metastases. HOXD9 proteins belong to a superfamily that regulates the development and control of many cellular processes, including proliferation, apoptosis, cell shape, and cell migration. HOXD9 can also function as an oncogene in several cancer cells. However, its biological function in human HCC requires further investigation. In this study, HOXD9 exhibited high expression in invasive HCC cells. HOXD9 overexpression can significantly enhance HCC cell migration, invasion, and metastasis, whereas silencing HOXD9 inhibits these processes. HOXD9 also promotes the epithelial–mesenchymal transition (EMT) of HCC cells. Microarray analysis suggests that ZEB1 can function as a downstream factor of HOXD9. HOXD9 can interact with the promoter region of ZEB1 and promotes ZEB1 expression. ZEB1 knockdown inhibits HOXD9-induced migration and invasion, as well as EMT in HCC cells. This study helps elucidates the oncogenic functions of HOXD9 in HCC.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-015-0245-3) contains supplementary material, which is available to authorized users.
BackgroundATAD2 is associated with many cellular processes, such as cell growth, migration and invasion. However, no studies have been conducted on the molecular biological function of the ATAD2 gene in hepatocellular carcinoma (HCC).MethodsThe protein and mRNA level expression of ATAD2 was examined in tissues and cell lines. Prognostic significance was analyzed by the Kaplan-Meier survival method and Cox regression. ATAD2 knockdown was used to analyze cell proliferation and invasion. The upstream and downstream of ATAD2 was analyzed by RT2 Profiler™ PCR array and luciferasex fluorescence system.ResultsATAD2 was highly expressed in liver cancer samples and correlated with poor survival. High ATAD2 expression was positively correlated with metastasis (P = 0.005) and was an independent prognostic factor in HCC (P = 0.001). ATAD2 depletion by RNA interference reduced their capacity for invasion and proliferation and led to a G1 phase arrest in vitro. Further study revealed that miR-372 was an upstream target of ATAD2 as miR-372 was bound directly to its 3′ untranslated region (3′ UTR). In addition, ATAD2 knockdown was found to extremely up-regulate APC expression and down-regulate CTNNA1 at the mRNA level.ConclusionsThe findings demonstrated that miR-372 suppressed the expression of ATAD2, which was highly expressed in HCC and exerted a proto-oncogene effect in hepatic carcinogenesis. In conclusion, ATAD2 may promote HCC progression.
Current extraction methods often extract DNA and RNA separately, and few methods are capable of co-extracting DNA and RNA from sputum. We established a nucleic acid co-extraction method from sputum based on magnetic beads and optimized the method by evaluating influencing factors, such as the guanidinium thiocyanate (GTC) and dithiothreitol (DTT) concentrations, magnetic bead amount, incubation temperature, lysis buffer pH and RNA carrier type. The feasibility of the simultaneous nucleic acid co-extraction method was evaluated by amplifying DNA and RNA viruses from a single clinical specimen with a multiplex RT-qPCR method. Both DNA and RNA were most efficiently extracted when the GTC and DTT concentrations were 2.0 M and 80 mM, respectively, 20 μl magnetic beads were added, the incubation temperature was 80 °C, the pH was 8 or 9, and RNA carrier A was used. Therefore, we established a simple method to extract nucleic acids from two important respiratory viruses compared with other commercial kits. This magnetic beads-based co-extraction method for sputum followed by a multiplex RT-qPCR can rapidly and precisely detect DNA and RNA viruses from a single clinical specimen and has many advantages, such as decreased time, low cost, and a lack of harmful chemicals.
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