Fig 1.
Schematic representation of constructs.
(A) Coding sequence of firefly luciferase, used in this study. The initiation codon is underlined. The codon used to generate a nonsense mutation is framed. The intron sequence is in small letters and highlighted in gray. (B) Schematic representation of the firefly luciferase (Fluc) constructs: Fluc-WT is the original cDNA encoding the firefly luciferase; Fluc-int is the same cDNA with an added intron (horizontal thick black line), giving rise to a splicing-prone RNA with a wild-type open reading frame. Fluc-int-UGA, Fluc-int-UAG, and Fluc-int-UAA were derived from Fluc-int by introducing the DNA sequences corresponding to the premature termination codons UGA, UAG, and UAA, respectively.
Fig 2.
Validation of the reporter genes used in the screen.
(A) The Fluc-int-WT reporter RNA is spliced. PCR amplification was performed on the Fluc-WT or Fluc-int-WT construct (lanes 1 and 3, respectively), or on the reverse-transcription reaction (RT) performed with extracted RNAs from HeLa cells transfected with the Fluc-int-WT construct (lane 2) in the presence of radioactive dCTP(α33P). The amplified fragments were electrophoresed through an acrylamide gel to demonstrate that the intron introduced in the firefly luciferase cDNA is efficiently spliced out. The position of each species is indicated on the right side of the gel and a quantification of the relative proportion of pre-mRNA (black box) and mRNA (spotted box) is shown on the right panel. (B) Luciferase expression associated with the constructs used in this study. Firefly luciferase activity normalized by renilla luciferase measured in wells of a 96-well plate containing untransfected HeLa cells, or HeLa cells transfected with pRluc and pFluc-WT, pFluc-int-WT, pFluc-int-UGA, pFluc-int-UAG, or pFluc-int-UAA constructs. (C) Measure of the firefly luciferase (Fluc) and renilla luciferase (Rluc) mRNAs by RT-PCR from HeLa cells transfected with pRluc and pFluc-WT, Fluc-int-WT, Fluc-int-UGA, Fluc-int-UAG, or Fluc-int-UAA constructs. (D) The extent of NMD is shown on a bar plot depicting levels of Fluc-int-PTC RNAs measured by quantitative RT-PCR in the absence and in the presence of cycloheximide (+CHX). The values shown are from two independent experiments. Error bar = S.D., Student t-test: **P<0.01; ***P<0.001.
Fig 3.
Identification of the H7 extract by screening.
Fungal and marine invertebrate extracts at 10 ng/μl (A) or at 100 ng/μl (B) were tested for their capacity to induce luciferase activity from constructs Fluc-int-UGA (upper panels), Fluc-int-UAA (middle panels), and Fluc-int-UAG (lower panels). (A) The extract in well H7 promoted high luciferase activity reflecting good correction of the UGA or UAA nonsense mutation. G418 at 1000 ng/μl in wells B1, C1, and D1 was used as positive control and served to distinguish strong nonsense mutation correctors (luciferase activity higher than that induced by G418) from weaker ones (luciferase activity equal to or lower than that induced by G418). (B) H7 at 100 ng/μl was introduced in well G1 and G418 at 1000 ng/μl in wells C1 and D1. (C) Western-blot analysis to detect the presence of the firefly luciferase (F-Luc) protein after transfection of HeLa cells with pFluc-int-UGA (lanes 1–4), pFluc-int-UAA (lanes 5–8), pFluc-int-UAG (lanes 9–12), pFluc-int-WT (lane 13) or pFluc-WT (lane 14). CBP80 was used as a loading control.
Fig 4.
H7 restores expression of the TP53 gene harboring a UGA or UAA nonsense mutation but not of the TP53 gene harboring a UAG mutation.
(A) Calu-6 (UGA nonsense mutation at codon 196 of the TP53 gene), (B) Caov-3 (UAA nonsense mutation at codon 136 of the TP53 gene), or (C) Caco-2 (UAG nonsense mutation at the codon 204 of TP53 gene) cells were incubated for 24 h with DMSO as control or with H7 in increasing amounts (from 0.2 to 125 ng/μl) or G418 (from 25 to 1000 ng/μl for (A) and (B) or from 25 to 2000 ng/μl for (C)) before protein extraction and analysis. Western blotting with anti-p53 antibody raised against the N-terminal part of the protein or with anti-CBP80 antibody as a loading control. Truncated (p53 TR) and full-length p53 (p53 FL) are indicated on the right side of each gel and the molecular weight (MW) is shown on the left side of each gel. The three leftmost lanes show twofold serial dilutions of untreated HeLa cell extract. The results presented in Figure are representative of three independent experiments.
Fig 5.
Calu-6 (A), Caov-3 (B) cells and HeLa cells transfected with pFluc-int-PTC and pIE-MUP (C) were incubated with DMSO, H7 extract at 25 ng/μl, or G418 at 1000 ng/μl for 24 h before RNA extraction and RT-qPCR. Quantification based on two independent experiments is shown. Error bar = S.D., Student t-test: *P<0.05.
Fig 6.
Cell toxicity in the presence of DMSO, H7, or G418.
Cell proliferation was measured in the presence of DMSO, H7 extract, or G418. Calu-6 cells were counted and treated with DMSO (dashed line), H7 extract (H7) at 25 ng/μl (black line) or G418 at 1000 ng/μl (dotted line) every two days from day 0 (D0) to day 10 (D10). Error bars represent standard deviations. The results presented in the figure are based on two independent experiments.
Fig 7.
H7 extract promotes the synthesis of functional p53 protein in Calu-6 cells.
(A) Calu-6 cells were transfected or not (untransfected) with an expression vector carrying a luciferase gene under the transcriptional control of a p53 response element. The cells were then incubated with DMSO, H7, or G418. Luciferase activity in the cells was measured 24 h after the start of treatment. Activities are expressed in arbitrary units. (B) The level of p21 mRNA was measured in Calu-6 cells exposed to DMSO, H7, or G418. The results presented in the figure are representative of three independent experiments. Error bar = S.D., Student t-test: *P<0.05; ***P<0.001.
Fig 8.
H7 extract promotes the synthesis of functional CFTR in cystic fibrosis patient cells.
Patient cells harboring a nonsense mutation on each allele of the CFTR gene were treated with DMSO (blue line), H7 (green line), or G418 (red line) before measuring CFTR-mediated transmembrane ion transport (iodide efflux). The fluorescence intensity, due to the halide-sensitive dye SPQ, depends on the quantity of functional CFTR protein at the cell membrane. The arrow indicates the moment when a cAMP-stimulating cocktail was added to the cells. The upper panel corresponds to R1158X (UGA)/R1162X (UGA) patient cells (PF1 cells). The middle panel corresponds to W1282X (UGA) /W1282X (UGA) patient cells (LY1 cells). The lower panel corresponds to Y122X (UAA)/Y122X (UAA) patient cells (FONJU1 cells).