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. 2013 Sep;41(17):8308-18.
doi: 10.1093/nar/gkt587. Epub 2013 Jul 1.

Dyskerin depletion increases VEGF mRNA internal ribosome entry site-mediated translation

Laura Rocchi  1 , Annalisa Pacilli, Rajni Sethi, Marianna Penzo, Robert J Schneider, Davide Treré, Maurizio Brigotti, Lorenzo Montanaro
Affiliations

Dyskerin depletion increases VEGF mRNA internal ribosome entry site-mediated translation

Laura Rocchi et al. Nucleic Acids Res. 2013 Sep.

Abstract

Dyskerin is a nucleolar protein encoded by the DKC1 gene that (i) stabilizes the RNA component of the telomerase complex, and (ii) drives the site-specific pseudouridilation of rRNA. It is known that the partial lack of dyskerin function causes a defect in the translation of a subgroup of mRNAs containing internal ribosome entry site (IRES) elements such as those encoding for the tumor suppressors p27 and p53. In this study, we aimed to analyze what is the effect of the lack of dyskerin on the IRES-mediated translation of mRNAs encoding for vascular endothelial growth factor (VEGF). We transiently reduced dyskerin expression and measured the levels of the IRES-mediated translation of the mRNA encoding for VEGF in vitro in transformed and primary cells. We demonstrated a significant increase in the VEGF IRES-mediated translation after dyskerin knock-down. This translational modulation induces an increase in VEGF production in the absence of a significant upregulation in VEGF mRNA levels. The analysis of a list of viral and cellular IRESs indicated that dyskerin depletion can differentially affect IRES-mediated translation. These results indicate for the first time that dyskerin inhibition can upregulate the IRES translation initiation of specific mRNAs.

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Figures

Figure 1.
Figure 1.
Dyskerin KD stimulates VEGF-IRES–mediated translation. Transient transfection of DKC1-specific siRNA strongly reduced DKC1 mRNA and protein level in MCF7 and MDA-MB231 cells (A, left and B, left, respectively). [3H]-leucine incorporation indicates that the total protein synthesis is not compromised (A and B, center) after DKC1 KD. IRES-mediated translation was assessed by measuring the FLuc and RLuc activity in MCF-7 and MDA-MB231 (A, right, and B, right, respectively) cells 8 h after the transfection with the bicistronic mRNA transcribed from pRL-VEGF-IRES. siRNA transfection was performed 96 h before cell harvesting. Histograms represent means and SDs from at least three independent experiments. P < 0.05 are considered significant. NS = not significant.
Figure 2.
Figure 2.
Dyskerin KD drives VEGF mRNA translation in breast cancer cells. Total (left) and polysome-associated (right) VEGF mRNA levels assessed by real-time PCR after DKC1 KD in MCF7 (A) and MDA-MB231 cells (C). Representative polysomal profiles are shown. VEGF protein levels in supernatant and in whole cell extracts are also reported for MCF-7 (B) and MDA-MB231 cells (D). siRNA transfection was performed 96 h before cell harvesting. Histograms represent means and SDs from three independent experiments. P < 0.05 is considered significant.
Figure 3.
Figure 3.
The increased VEGF secretion in DKC1 KD cells is due to the up-regulation of VEGF mRNA IRES-mediated translation. (A) Histograms show the level of VEGF protein detected using an ELISA array. Experiments were performed in MCF-7 control (left) and DKC1 KD (right) cells treated with 15 nM PTC299 for 72 h. VEGF secretion of PTC299 treated cells is normalized on the correspondent untreated cells; for the effect of DKC1 KD on VEGF secretion on MCF7 cells see Figure 2A (B) VEGF-IRES–mediated translation measured in MCF7 control and DKC1 KD cells after 72 h of 100 nM PTC299 treatment. siRNA transfection was performed 96 h before cell harvesting. (C) Representative image (left) and a summarizing graph (right) from clonogenic assays performed with control (empty vector) and shDKC1 MCF7 cells. Histograms represent means and SDs from three independent experiments. P < 0.05 is considered significant. NS = not significant.
Figure 4.
Figure 4.
DKC1 KD increases VEGF-IRES recruitment to 48S preinitiation complex. (A) Left: representative profile at 260 nM O.D. obtained from MCF7 cytoplasmic extracts: fractions 18–21 were considered to correspond to the small ribosomal subunits. Right: Representative profile of a sucrose density gradient reporting the radioactive intensity per fraction in from DKC1 KD (circles) and control (SCR—triangles) cells extracts, respectively. Peaks of radioactivity coinciding with the identified fractions containing the 48S complexes was generated when MCF7 cytoplasmic extracts were incubated with a [32P]VEGF IRES mRNA probe. (B) Histogram represent mean and SD of the radioactivity measured in the identified peaks for DKC1 KD and control (SCR) cells extracts. siRNA transfection was performed 96 h before cell harvesting. P < 0.05 is considered significant.
Figure 5.
Figure 5.
Reduction of dyskerin levels differentially affects IRES-mediated translation of viral and cellular IRESs. (A) IRES-mediated translation assessed by measuring the FLuc and RLuc activity in MCF-7 DKC1 KD cells, 8 h after transfection with a bicistronic mRNA transcribed from viral pR-CrPV-IRES-F (left), pR-HCV-IRES-F (center) and pF-EMCV-IRES-R (right). (B) IRES-mediated translation assessed by measuring the FLuc and RLuc activity in MCF-7 DKC1 KD cells 8 h after transfection with a bicistronic mRNA transcribed from cellular pR-HSP70-IRES-F (left), pR-c-MYC-IRES-F (center) and pR-p53IRES-F (right). siRNA transfection was performed 96 h before cell harvesting. Histograms represent means and SDs from three independent experiments. P < 0.05 is considered significant. NS = not significant.
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