Article Figures & Data
Figures
- FIG 1
DHX33 knockdown markedly reduces the ratio of polysomes/monosomes. Polysome profiling was performed on HCC1806 cells, BT549 cells, BJ primary fibroblasts, and MDAMB361 cells after lentiviral infection by DHX33-specific shRNA. shScrambled was used as a control. (Left) Polysome profiles; (right) protein knockdown efficiency for each cell line analyzed by Western blotting. Control and knockdown samples were aligned on the basis of the peak position of the 40S ribosome and labeled with distinct colors. (A) Black line, shScrambled (shSCR); red line, #2-shDHX33; blue line, #4-shDHX33. (B) Blue line, shScrambled; red line, #4-shDHX33. (C and D) Black line, shScrambled; red line, #2-shDHX33; blue line, #4-shDHX33. (E) Polysome profiles were determined with 1 M NaCl after HCC1806 cells were infected with lentivirus encoding #2-shDHX33. Blue line, shScrambled; red line, #4-shDHX33.
- FIG 2
DHX33 is required for new protein synthesis. (A) Five different shRNAs (#1-shDHX33 to #5-shDHX33) were delivered into HCC1806, BT549, and MDAMB361 cells by use of a lentiviral vector. DHX33 protein levels were analyzed by Western blotting to determine the knockdown efficiency. scr, shScrambled. The numbers between the gels are fold changes. (B) A 35S incorporation assay was performed for the above-mentioned cells infected by use of a lentiviral vector; the radioactivity from each sample was analyzed by scintillation counting after normalization for equal total cellular protein levels. Quantitation data are shown. Bars represent the standard deviations from three separate experiments. P was <0.005 for all of the DHX33 knockdown samples compared to the results for the shScrambled control.
- FIG 3
The nucleoside triphosphatase activity of DHX33 promotes mRNA translation. (A) HCC1806 cells were infected with lentiviruses encoding shDHX33or shScrambled (shSCR) as a control. Equal numbers of cells were then transiently transduced with pGL3-5′-UTR-F-luc-3′-UTR-GAPDH. F-luc activity was analyzed by IVIS imaging. Western blot analysis was performed to determine the levels of knockdown of the DHX33 protein. (B) Quantitation data for F-luc activity after normalization of the luciferase transcript levels in each sample. The data shown are the results from three independent experiments, and bars represent standard deviations. P was <0.001 for DHX33 knockdown samples compared to the results for the shScrambled control. (C) HCC1806 cells were infected with lentiviruses encoding wild-type (WT) DHX33, the DHX33 K94R mutant, or the empty vector (EV). Cells were then infected with lentiviruses encoding shDHX33 to knock down endogenous DHX33. Western blot analysis was performed to determine the levels of knockdown of the DHX33 protein as well as the level of overexpression of DHX33 (wild-type and K94R mutant DHX33). (D) Equal numbers of cells were then transiently transduced with pGL3-5′-UTR-F-luc-3′-UTR-GAPDH. F-luc activity was analyzed by IVIS imaging. The F-luc activity of each cell sample, after normalization of the F-luc transcript levels, is shown. Data represent the results from three independent experiments, and bars represent standard deviations. (E) HCC1806 cells were infected with lentiviruses encoding wild-type DHX33, the DHX33 Δ1–80 mutant (the D1 mutant), or the empty vector. Cells were then infected with lentiviruses encoding DHX33-specific shRNA to knock down endogenous DHX33. Western blot analysis was performed to determine the levels of knockdown of the DHX33 protein as well as the overexpression of DHX33 (wild-type and mutant D1 DHX33). (F) Equal numbers of cells were then transiently transduced with pGL3-5′-UTR-F-luc-3′-UTR-GAPDH. F-luc activity was analyzed by IVIS imaging. The F-luc activity of each cell sample, after normalization of the F-luc transcript levels, is shown. Data represent the results from three independent experiments, and bars represent standard deviations. (G) Cells were then harvested; total RNA was isolated and analyzed by quantitative RT-PCR for 47S pre-rRNA levels. The bar graph shows 47S rRNA levels after normalization to the amount of total RNA for each sample, and bars represent standard deviations from three independent experiments.
- FIG 4
DHX33 participates in nucleolar/cytoplasmic shuttling. (A) T47D, HCC1806, SKBR3, and HeLa cells were fractionated into cytosolic and nuclear fractions, and the DHX33 protein levels in each fraction were analyzed by immunoblotting. SOD1 was used as a cytosolic marker, and lamin A/C was used as a nuclear marker. (B) Human primary fibroblast BJ cells were fractionated into cytosolic and nuclear fractions, and approximately 100 μg each of the cytosolic and nuclear extracts was loaded onto an SDS-polyacrylamide gel for Western blot analysis using antibodies to the indicated proteins. (C) HeLa cells were transiently transfected by pLVX carrying FLAG-tagged wild type-DHX33. At 24 h posttransfection, NIH 3T3 cells were seeded onto these HeLa cells, and the cells were cocultured in the presence of cycloheximide. The cells were then treated with polyethylene glycol to fuse neighboring cells. Indirect immunofluorescence was performed with antibodies recognizing DHX33 to visualize DHX33 protein expression in mouse donor cells (m) and human recipient cells (h). DHX33 staining is red, and the blue DAPI staining demarcates human and mouse nuclei (dominated by heterochromatin). The heterokaryon formed between two mouse cells and a single human cell is outlined in white. DIC, differential interference contrast.
- FIG 5
DHX33 cosediments with ribosome subunits and monosomes. Approximately 3 × 106 cells each of HCC1806, SKBR3, and HeLa cells were centrifuged over a sucrose gradient for polysome profiling using a continuous 254-nm monitoring system to detect RNA across the gradient. Fractions were collected and TCA precipitated. Proteins that precipitated from the fractions were analyzed by Western blotting for antibody to each of the indicated proteins.
- FIG 6
DHX33 associates mRNA translation initiation factors and ribosomes. (A) Experimental flowchart for tandem affinity purification of DHX33 from cytosolic extracts doubly tagged with 3× FLAG at the N terminus and streptavidin (Strep) at the C terminus. (B) Proteins identified by MS analysis. Ribosomal proteins and mRNA translation initiation factors were detected in the DHX33 immunoprecipitates. None of these proteins were detected in precipitates from the sample transfected with lentiviruses encoding the empty vector. (C) HCC1806 cells were transduced with pCMV-3×FLAG-DHX33, and cells transfected with lentiviruses encoding the empty vector were used as a negative control. Cell lysates were then immunoprecipitated with anti-eIF3G or anti-FLAG antibodies or with IgG as a control and then immunoblotted with the indicated antibody to detect an association between DHX33 and eIF3G. (D and E) rpL27, rpL7, and rpL26 were found to be coimmunoprecipitated with FLAG-DHX33 in HCC1806 cells. HCC1806 cells were transfected by pCMV-3×FLAG-DHX33; cells transfected with lentiviruses encoding the empty vector were used as a negative control. Cell lysates were then immunoprecipitated with anti-FLAG antibody or with IgG and immunoblotted with antibodies to the indicated proteins. IB, immunoblotting; IP, immunoprecipitation.
- FIG 7
DHX33 helicase activity and RNA interactions are dispensable for DHX33 complex formation. (A) HCC1806 cells were transduced with pCMV-3×FLAG-DHX33 (carrying wild-type or K94R helicase-dead mutant DHX33) and the empty vector as a control. Cell lysates were then immunoprecipitated using an anti-FLAG antibody and immunoblotted with antibodies to the indicated proteins. (B) HCC1806 cells were transduced with pCMV-3×FLAG-DHX33 (wild-type or K94R mutant DHX33) and the empty vector as a control. Cell lysates were then immunoprecipitated by anti-FLAG antibody with 10 μg/ml RNase and without 10 μg/ml RNase and immunoblotted with antibodies to the indicated proteins.
- FIG 8
DHX33 binds to eIF3G and ribosomal proteins through shared protein domains. (A) A series of deletion mutants of DHX33 was generated. Diagrams of the sequences of a panel of deletion mutants compared to the sequence of wild-type DHX33 are shown. Helic C, helicase C; OB-NTP-bind, oligonucleotide/oligosaccharide-nucleoside triphosphate binding. (B) HCC1806 cells were transduced with pCMV-3×FLAG-DHX33 carrying the DHX33 wild type and deletion mutants, and cells transduced with the empty vector were used as a control. Cells were then fixed and incubated with anti-FLAG antibody for immunofluorescence detection of mutant DHX33. Anti-NPM was used to mark nucleoli, and DAPI was used to mark nuclei. (C and D) Coimmunoprecipitations were performed using the deletion mutants after transient transduction of each mutant into HCC1806 cells. (C and D) Initiation factor eIF3G required DHX33 residues 480 to 580 (C), while rpL26 required DHX33 residues 340 to 580 (D).
- FIG 9
DHX33 interacts with numerous mRNAs and promotes translation initiation after 80S ribosome assembly. (A) Experimental flowchart describing the immunoprecipitation of DHX33 and analysis of its interacting mRNAs. (B) The immunoprecipitated complex was immunoblotted with anti-DHX33 to confirm that DHX33 is pulled down from cell lysates. (C) FLAG-DHX33 was immunoprecipitated with anti-FLAG antibody, followed by RNA extraction from the immunoprecipitated complex. Quantitative RT-PCR was performed with a primer set defined for each indicated mRNA with the empty vector control (Vc) or DHX33 sample. The data listed are the mean results from three separate experiments, and standard errors are shown. nt, number of nucleotides. (D) HCC1806 cells were infected with lentiviruses encoding shScrambled or DHX33-specific shRNA. The efficiency of knockdown of the DHX33 protein was analyzed by Western blotting with GAPDH as an internal control. (E) Equal cell numbers from the above-mentioned samples were then treated and used for polysome profiling. Fractions from the polysome profiles were collected, and total RNA was extracted from each fraction. These RNA samples were then converted into cDNA and used as the templates for analysis of the GAPDH mRNA distribution. The experiment was repeated three times, and a representative distribution pattern for GAPDH mRNA is shown. (F) Quantitation data from three independent experiments show that DHX33 knockdown redistributes GAPDH mRNA from heavy polysomes to light polysomes (P < 0.001; n = 3). (G) Quantitation data from three independent experiments show that DHX33 knockdown causes the accumulation of GAPDH mRNA in 80S monosomes (*, P < 0.005; n = 3). (H to J) Experiments similar to those described above were performed to analyze the polysome distribution of DDX5 mRNA after DHX33 knockdown. The numbers on the x axis in panel H are fraction numbers.
- FIG 10
Purified DHX33 does not enhance luciferase mRNA translation in vitro. (A) The open reading frame of mouse DHX33 was cloned into the BamHI/HindIII sites in the pET32M-3C vector. The following primers were used to amplify DHX33 PCR products: forward primer 5′-ATTATAGGATCCATGCCGGAGGAGGCGAGCCT-3′ and reverse primer 5′-ATAAATAAGCTTGTTTCTGGCCGTTCTCAGCTT-3′. Overexpressed wild-type recombinant DHX33 with a thioredoxin (Trx) tag at its N terminus and a 6× His tag at its C terminus was affinity purified from E. coli through an Ni-nitrilotriacetic acid column (Qiagen). (Left) Coomassie blue-stained proteins showing the purity of crude DHX33, which was further verified by Western blotting. Approximately 0.5 μg of this DHX33 recombinant protein was added into a rabbit reticulocyte lysate system (catalog no. L4960; Promega) to monitor in vitro protein translation activity with firefly luciferase mRNA as a control. (Right) After DHX33 was added, protein translational activity was decreased slightly. (B) FLAG-tagged DHX33 overexpressed in HeLa cells was immunoprecipitated by anti-FLAG beads and was then added into a rabbit reticulocyte system (catalog no. L4960; Promega) to assay in vitro protein translation activity. (Left) The DHX33 protein was immunoprecipitated from the cell lysates by Western blotting; (right) the addition of wild-type DHX33 inhibits luciferase mRNA translation in this experimental setting.
