Human DDX3 functions in translation and interacts with the translation initiation factor eIF3

Abstract

The conserved RNA helicase DDX3 is of major medical importance due to its involvement in numerous cancers, human hepatitis C virus (HCV) and HIV. Although DDX3 has been reported to have a wide variety of cellular functions, its precise role remains obscure. Here, we raised a new antibody to DDX3 and used it to show that DDX3 is evenly distributed throughout the cytoplasm at steady state. Consistent with this observation, HA-tagged DDX3 also localizes to the cytoplasm. RNAi of DDX3 in both human and Drosophila cells shows that DDX3 is required for cell viability. Moreover, using RNAi, we show that DDX3 is required for expression of protein from reporter constructs. In contrast, we did not detect a role for DDX3 in nuclear steps in gene expression. Further insight into the function of DDX3 came from the observation that its major interaction partner is the multi-component translation initiation factor eIF3. We conclude that a primary function for DDX3 is in protein translation, via an interaction with eIF3.

Figure 1.

Characterization of human DDX3 antipeptide antibody. (A) The N-terminal amino acid sequence of DDX3 used for raising an antipeptide rabbit polyclonal antibody. (B) Western blot of HeLa whole cell lysate using the DDX3 antibody. (C) Immunoprecipitation from whole cell lysate using α-DDX3 or α-cntl (SAP 130) antibody followed by western analysis with α-DDX3.

Figure 2.

DDX3 is even distributed throughout the cytoplasm using IF. (A) DDX3 was detected in the cytoplasm by IF using our α-DDX3 antibody. (B) DDX3 localization is not cell cycle dependent. IF of HeLa cells using α-DDX3 antibody at lower (×100) magnification is shown. (C) Western analysis of whole cell lysate using α-DDX3 (lane 1). Immunoprecipitation using α-HA antibody from HeLa whole cell lysate (lane 2) or HeLa whole cell lysates expressing HA-DDX3 (lane 3) followed by western analysis with α-DDX3 antibody. (D) HA-DDX3 localized in the cytoplasm. IF of HeLa cells expressing HA-DDX3 was carried out using an HA antibody followed by an Alexa 647-conjugated mouse secondary antibody.

Figure 3.

DDX3 RNA interference inhibits cell growth and expression from a β-globin reporter in HeLa cells. (A) Schematic of HA-tagged β-globin reporter. Boxes indicate exons and lines indicate introns. The CMV promoter and BGH polyA sites are shown. (B, C) Efficiency of DDX3 knockdown (using DDX3-a siRNA) in HeLa cells was examined by western analysis (B) and IF (C). Knockdown control (cntl) was lipofectamine alone. Loading control in panel b is UAP56. (D–G) Knockdown of DDX3 inhibits expression of β-globin reporter. Femtoliter aliquots of plasmid DNA (50 μg/ml) containing HA-tagged β-globin was microinjected together with a nuclear injection marker (FITC-conjugated 70 KD-dextran) into the nucleus of siRNA-treated HeLa cells. After incubation at 37°C for 2.5 h, β-globin protein expression was detected by IF using an HA antibody. (H) Knockdown of DDX3 inhibits cell growth. Two sets of siRNAs against DDX3 (DDX3-a, DDX3-b) or lipofectamine alone (cntl) were transfected into HeLa cells. Cell viability was determined using a hemocytometer after staining cells with trypan blue.

Figure 4.

DDX3 RNA interference has no apparent effects on nuclear steps in gene expression of β-globin reporter. (A) Schematic of β-globin reporter construct. The FISH probe is indicated (see Methods section). (B, C) The RNAi efficiencies after knockdown of DDX3 (B) or UAP56 (C) in HeLa cells were analyzed by western blotting. Loading controls were eIF4A3 (B) and CBP80 (C). Knockdown control (cntl) in panels a and b was lipofectamine alone. (D−F) The β-globin reporter construct was microinjected into the nucleus of the knockdown cells together with FITC-conjugated 70 KD-dextran as an injection marker. Thirty minutes after microinjection, α-amanitin was added to inhibit further transcription. Cells were fixed after 2 h incubation at 37°C and the distribution of β-globin mRNA was visualized by FISH using an Alexa Fluor 546 labeled probe. (G) Knockdown of DDX3 has no apparent effect on pre-mRNA splicing of β-globin reporter. After knockdown using siRNAs targeting DDX3 (lane 1), cntl1 (eIF4A3, lane 2), cntl2 (Skar, lane 3) or using lipofectamine alone (lane 4) or untreated cells (lane 5), RT–PCR was carried out on total RNA using PCR primers that flank intron 1 of the β-globin reporter. A band of the expected size for spliced mRNA (248 bp) is indicated. Marker sizes (base pair) are shown to the right of the gel. (H) Quantification of the total levels of FISH signal for β-globin mRNA was carried out using NIH ImageJ and is shown in the graph. Error bars represent standard deviations (n = 11).

Figure 5.

Evidence that Drosophila DDX3 (Belle) functions in translation in S2 cells. (A, B) RT–PCR (A) and western analysis (B) of knockdown of LacZ, DDX3 and Elp1 in a stable S2 cell line containing a copper-inducible lacZ gene. 18S rRNA was used as a loading control, and RT cntl (no reverse transcription was carried out) was used as a control for RT–PCR. A loading control protein (Skar) was used for the western blot analysis. (C) Knockdown of DDX3 inhibits cell growth. Cells were transfected with the indicated dsRNAs and then cell numbers were counted each day using a hemocytometer after staining with trypan blue. (D) Protein expression levels were determined by β-galactosidase activity. Error bars represent standard deviations (n = 3). (E) FISH of mRNA export in the indicated knockdown cells using an oligo dT probe for polyA tail mRNA and FITC-conjugated wheat germ agglutinin to stain the nuclear envelope. (F) Quantification of the total levels of FISH signal for polyA tail mRNA was carried out using NIH ImageJ and is shown in the graph. Error bars represent standard deviations (n = 18).

Figure 6.

DDX3 associates with the translation initiation factor, eIF3, via protein–protein interactions. (A) Immunoprecipitations were carried out using antibodies against DDX3, eIF3b or a negative control antibody (SAP 130) from HeLa cytoplasmic extract pretreated with RNase A. (B) Western blots of the indicated immunoprecipitations were first probed with α-DDX3 (left panel) followed by α-eIF3b, and finally α-eIF3e. The right panel shows the blot after probing with both α-eIF3b and α-eIF3e. The band designated by the asterisk in the right panel is remaining DDX3 signal.