DEAD-box protein DDX3 associates with eIF4F to promote translation of selected mRNAs

Abstract

Here, we have characterized a step in translation initiation of viral and cellular mRNAs that contain RNA secondary structures immediately at the vicinity of their m(7)GTP cap. This is mediated by the DEAD-box helicase DDX3 which can directly bind to the 5' of the target mRNA where it clamps the entry of eIF4F through an eIF4G and Poly A-binding protein cytoplasmic 1 (PABP) double interaction. This could induce limited local strand separation of the secondary structure to allow 43S pre-initiation complex attachment to the 5' free extremity of the mRNA. We further demonstrate that the requirement for DDX3 is highly specific to some selected transcripts, cannot be replaced or substituted by eIF4A and is only needed in the very early steps of ribosome binding and prior to 43S ribosomal scanning. Altogether, these data define an unprecedented role for a DEAD-box RNA helicase in translation initiation.

Figure 1

DDX3 promotes translation of complex mRNAs. (A) Western blotting showing knockdown of DDX3 from HeLa cells transfected with a control siRNA (siCtrl) or an anti-DDX3 siRNA (siDDX3). eIF4E was used as a loading control. (B) Schematic representation of DDX3 helicase core indicating the sites that were mutated for functional analysis (upper cartoon). Control (siCtrl) and DDX3-depleted (siDDX3) HeLa cells were transfected with 0.3 μg of HIV-1-Renilla proviral DNA together 1 μg of a control vector coding for EGFP (in siCtrl and siDDX3). For rescue of siDDX3 cells, 1 μg of vectors encoding wild-type DDX3R or the indicated mutants were included. Translation of the HIV-1 genomic RNA (gRNA) was determined at 24 h.p.t. by reading the Renilla luciferase activity normalized to cytoplasmic gRNA levels. Results were normalized to the siCtrl/EGFP transfection (indicated as siCtrl in the figure, arbitrary set to 100%) and are presented as mean±s.d. of three duplicated experiments. (C) Control (siCtrl) and DDX3-depleted (siDDX3) HeLa cells were transfected with 0.3 μg of HIV-1-Renilla proviral DNA together with 1 μg of vectors expressing EGFP (siCtrl and siDDX3) or DDX3R, yeast Ded1 or human eIF4A (in siDDX3). HIV-1 gRNA translation was determined at 24 h.p.t. by reading the Renilla luciferase activity normalized to cytoplasmic gRNA levels. Results were normalized to the siCtrl/EGFP DNA transfection (siCtrl in the figure, arbitrary set to 100%) and are presented as mean±s.d. of three duplicated experiments. (D) Control (siCtrl) and DDX3-depleted (siDDX3) HeLa cells were transfected with 0.125 pmol of Renilla luciferase reporter mRNAs carrying the indicated 5′-UTR (see cartoon on the left). Renilla luciferase activity was analysed at 3 h.p.t. Results were normalized to siCtrl (arbitrary set to 100%) and are presented as mean±s.d. of three duplicated experiments.

Figure 2

DDX3 acts on structures located close to the m⁷GTP cap. (A) Control (siCtrl) and DDX3-depleted (siDDX3) HeLa cells were transfected with 0.125 pmol of Renilla luciferase reporter mRNAs carrying the indicated 5′-UTR. Renilla luciferase activity was analysed at 3 h.p.t. Results were normalized to siCtrl (arbitrary set to 100%) and are presented as mean±s.d. of three duplicated experiments. (B) Schematic representation of the HIV-1 and HIV-2 TAR structures. In HIV-1, the m⁷GTP cap is base paired with the C residue at position 57 while in HIV-2 it is base paired with the C residue at position 123. (C–E) Control (siCtrl) and DDX3-depleted (siDDX3) HeLa cells were transfected with 0.125 pmol of Renilla luciferase reporter mRNAs carrying the indicated 5′-UTR. Renilla luciferase activity was analysed at 3 h.p.t. Results were normalized to siCtrl (arbitrary set to 100%) and are presented as mean±s.d. of three duplicated experiments. (F) Control (siCtrl) and DDX3-depleted (siDDX3) HeLa cells were transfected with 0.125 pmol of the indicated Renilla luciferase reporter mRNA. Renilla luciferase activity was analysed at 3 h.p.t. Results were normalized to siCtrl (arbitrary set to 100%) and are presented as mean±s.d. of three duplicated experiments.

Figure 3

DDX3 binds RNA in a non-specific manner. (A) 1.5% agarose gel showing results from an EMSA. In vitro transcribed, [³²P]-labelled TAR RNA was incubated in buffer (lanes 1–3), recombinant DDX3 (lanes 4–6) or eIF4A (lanes 7–9) in the absence (lanes 1, 4 and 7) or presence of ATP (lanes 2, 5 and 8) or AMP-PNP (lanes 3, 6 and 9). TAR and TAR/DDX3 complexes are indicated at the left of the image. Binding reactions were carried out at a 1:12 RNA:protein ratio. Result shown is representative of at least three independent experiments. (B) 1.5% agarose gel showing results from an EMSA. In vitro transcribed, [³²P]-labelled HIV-1 5′-UTR or ΔTAR 5′-UTR was incubated in buffer (lanes 1 and 6) or with increasing amounts of rDDX3 (lanes 2–5 and 7–10). The free RNA is indicated on the left and the RNA/DDX3 complexes are indicated at the right. Binding reactions were carried out at 1:0.2 (lanes 2 and 7); 1:0.6 (lanes 2 and 8); 1:3 (lanes 4 and 9) and 1:6 (lanes 5 and 10) RNA:protein ratio. Results shown are representative of at least three independent experiments. (C) Toeprints reactions preformed at 30 or 37°C were resolved as indicated in Materials and methods. Positions of two binding sites are indicated at the right. Their positions within the overall 5′-UTR structure are shown in Supplementary Figure 3. The image shown is representative of results obtained in three independent experiments. (D) 1.5% agarose gel showing results from an EMSA. In vitro transcribed, [³²P]-labelled 5′-UTR was incubated in buffer (lanes 1, 4, 7, 10 and 13), with recombinant DDX3 (lanes 2, 5, 8, 11 and 14) or with recombinant eIF4A (lanes 3, 6, 9, 12 and 15). Only DDX3 is able to form complexes with RNA. Binding reactions were carried at a 1:1.5 RNA:protein ratio. Results shown are representative of at least three independent experiments. Binding with the globin RNA was carried out in the same experiment but run in a different gel. Figure source data can be found with the Supplementary data.

Figure 4

DDX3 cooperates with eIF4A during TAR unwinding. (A) Indirect TAR unwinding assay ex vivo. Control (siCtrl) or DDX3-depleted (siDDX3) HeLa cells were transfected with 0.125 pmol of the indicated Renilla luciferase reporter mRNA and Renilla luciferase activity was analysed at 3 h.p.t. Efficiency of TAR unwinding (as determined by the Renilla luciferase activity) in siCtrl cells is showed on the left graph. The effect of DDX3 knockdown in TAR unwinding is showed on the right graph. Results were normalized to TAR wt (left, arbitrary set to 1; where average RLU for TAR wt, TAR-AUG and CAA-TAR-AUG are 7098, 126 461 and 111 792, respectively) or the siCtrl transfection (right, arbitrary set to 100%) and are presented as mean±s.d. of three duplicated experiments. (B) HeLa cells previously treated with DMSO or 200 nM hippuristanol for 2 h were transfected with 0.125 pmol of the indicated Renilla luciferase reporter mRNA and Renilla luciferase activity was analysed at 3 h.p.t. Results were normalized to DMSO (arbitrary set to 100%) and are presented as mean±s.d. of three duplicated experiments. (C) Control (siCtrl) or DDX3-depleted (siDDX3) HeLa cells were treated with DMSO or hippuristanol as described above and then transfected with 0.125 pmol of the indicated Renilla luciferase reporter mRNA and Renilla luciferase activity was analysed at 3 h.p.t. Results were normalized to siCtrl/DMSO condition (arbitrary set to 100%) and are presented as mean±s.d. of three duplicated experiments. Normalized values obtained for HIV-1 under siCtrl/DMSO, siCtrl/Hipp, siDDX3/DMSO and siDDX3/Hipp are 100, 4, 18 and 0.4%, respectively.

Figure 5

DDX3 is associated with eIF4F and plays a role before ribosomal scanning. (A) HeLa cells transfected with vectors coding for GFP-DDX3 and the indicated eIF4F member were treated with 0.5 mM sodium arsenite for 45 min and then fixed and subjected to indirect immunofluorescence and confocal microscopy analyses. White arrows indicate colocalization between GFP-DDX3, HA-tagged eIF4F components and endogenous PABP in cytoplasmic stress granules. Scale bars 25 μm. (B) HeLa cells expressing HA-tagged DDX3 were subjected to IP using mouse IgG (lane 2) or a mouse anti-HA antibody (lane 3) and associated proteins were analysed by western blotting. Input (lane 1) corresponds to a 1/10 of the total cell extract. The asterisk (*) and the double asterisk (**) in the eIF4E and eIF4A panels show cross-reactivity of the anti-rabbit secondary antibody with the mouse IgG light chain and heavy chain, respectively. Bands observed upper and lower eIF4B in lanes 2 and 3 most probably correspond to unspecific recognition of our anti-goat secondary antibody. (C) S7 nuclease-treated HeLa cell extracts (100 μg) were incubated with an m⁷GTP cap affinity matrix. Proteins retained after washing were analysed by western blotting (lane 2 in left panels). In the right panel, cell extracts were incubated in the absence or presence of m⁷GpppG cap analogue (lanes 2 and 3, respectively) and proteins retained after washing were analysed by western blotting. Input (lane 1 in each panel) corresponds to 100 μg of cell extract. Bands shown in each panel correspond to the same western blot and were cropped for simplicity. (D, E) Control (siCtrl) and DDX3-depleted (siDDX3) or DMSO and Hippuristanol-treated HeLa cells were transfected with 0.125 pmol of the indicated Renilla luciferase reporter mRNA. Renilla luciferase activity was analysed at 3 h.p.t. Results were normalized to siCtrl (set to 100%) and are presented as mean±s.d. of three independent experiments. (F) HeLa cells previously treated with DMSO or 200 nM hippuristanol for 2 h were transfected with 0.125 pmol of the indicated Renilla luciferase reporter mRNA and Renilla luciferase activity was analysed at 3 h.p.t. Results were normalized to DMSO (arbitrary set to 100%) and are presented as mean±s.d. of three duplicated experiments. Figure source data can be found with the Supplementary data.