Chemical-Induced Read-Through at Premature Termination Codons Determined by a Rapid Dual-Fluorescence System Based on S. cerevisiae

Abstract

Nonsense mutations generate in-frame stop codons in mRNA leading to a premature arrest of translation. Functional consequences of premature termination codons (PTCs) include the synthesis of truncated proteins with loss of protein function causing severe inherited or acquired diseases. A therapeutic approach has been recently developed that is based on the use of chemical agents with the ability to suppress PTCs (read-through) restoring the synthesis of a functional full-length protein. Research interest for compounds able to induce read-through requires an efficient high throughput large scale screening system. We present a rapid, sensitive and quantitative method based on a dual-fluorescence reporter expressed in the yeast Saccharomyces cerevisiae to monitor and quantitate read-through at PTCs. We have shown that our novel system works equally well in detecting read-through at all three PTCs UGA, UAG and UAA.

Fig 1. Read-through reporter systems.

(A) Plasmids of the YEpGR series harboring the yEGFP and yEmRFP coding sequences separated by either an in frame sense or nonsense codon; (B) Constructs bearing the same yEmRFP and yEGFP ORFs cloned in the inverted order; (C) Fluorescence microscope images of yeast cells transformed with plasmids expressing a yEGFP-sense-yEmRFP construct (CGA) or yEmRFP-nonsense-yEGFP (UGA) configuration; (D) plasmids expressing a yEmRFP-sense-yEGFP (CGA) or yEmRFP-nonsense-yEGFP (UGA) read-through cassette.

Fig 2. Read-through mediated by G418 determined by using a dual-laser fluorescence scanner.

(A) Yeast strain CW04 was transformed with plasmids (YEpGR series) harboring the indicated read-through cassette UGA or the corresponding sense control CGA. Independent clones were inoculated in quadruplicates in 96 wells microplates to perform two read-through assays (RT1 and RT2). Geneticin (G418) was added at the concentrations indicated. Microplates OD was measured at 595 nm and fluorescence was acquired by using the dual-laser scanner Typhoon 8600 after 24h incubation at 30°C. (B) Read-through levels as a function of the presence of increasing concentrations of G418 were quantitate as described in the Materials and Methods. Quantitative data were obtained from two independent experiments and are expressed as mean values and indicated with standard deviation.

Fig 3. Expression of YEpRG reporters in yeast.

Yeast cells were transformed with plasmids of the YEpRG series based on the constructs depicted (Fig 1B) which are expressing the read-through cassettes indicated. Yeast cells transformed with a YEpRG expressing the yEmRFP and yEGFP ORFs with an in frame sense codon (CGA, CAG or CAA) display both the red and green fluorescence, whereas only red fluorescence could be expressed from those plasmids into which a nonsense codon (UGA, UAG or UAA) was placed between the yEmRFP and yEGFP ORFs. Yeast transformants were selectively grown on solid synthetic minimal medium in the absence of uracil. Imaging was performed by using the dual laser scanner and acquired after overnight incubation of the plate at 30°C.

Fig 4. Read-through efficiency at UGA, UAG and UAA premature stop codons mediated by aminoglycoside G418 determined by YEpRG dual fluorescent reporters.

Yeast transformants harboring the YEpRG series (Figs 1B and 3), bearing each UGA, UAG or UAA premature stop codon, or the corresponding sense codon controls, inserted between the yEmRFP and yEGFP ORFs were grown in liquid selective medium and inoculated in quadruplicate in 96 wells microplates in the absence or presence of G418 (8–16 μg/ml). Dual fluorescence was acquired as in Fig 2 (see also text). A) Shown is a representative image of yEGFP acquired by a Typhoon 9600 FLA after 19h incubation at 30°C related to a G418 mediated read-through assay at the UGA stop codon. G418 was added at 8 μg/ml (lanes 2, 5 and 8) or 16 μg/ml (lanes 3, 6 and 9) (primary data and experimental scheme are available in the Supporting Information (S2 Fig). B) Read-through percentage is calculated as described in the Materials and Methods. Data are expressed as mean values and indicated with standard deviation.

Fig 5. Read-trough efficiency at UGA, UAG and UAA premature stop codons mediated by Gentamicin.

Yeast transformants with a plasmid harboring each of the nonsense or the correspondent sense codon, were prepared and cultivated in quadruplicates as described (Fig 4), in the absence or presence of aminoglycoside gentamicin, added at 200 μg/ml (lanes 2, 5 and 8) or 400 μg/ml (lanes 3, 6 and 9. A) Shown is a representative image of yEGFP acquired by a Typhoon 9600 FLA after 19h incubation at 30°C related to a gentamicin mediated read-through assay at the UGA stop codon (see also S2 Fig). B) Read-through percentage is calculated as described in the Materials and Methods. Data are expressed as mean values and indicated with standard deviation.

Fig 6. Read-through assay as a function of abolished NMD.

WT and Δupf1, Δupf2 and Δupf3 strains were transformed with the YepRG-UGA or YepRG-CGA reporters. A) Yeast transformants were inoculated incubated at 30°C for 24 h in microplate and analysed by the dual fluorescence scanner (Typhoon FLA9600) as described in the previous experiments. B) Read-through percentage was calculated as described in the Materials and Methods. Data are related to identical clones replicates and expressed as mean values and indicated with standard deviation. Wild type and upfs deleted strains are described in Materials and Methods.

Fig 7. Expression products of the YepRG-UGA and YepRG-CGA reporters in the wild type and Δupf1, Δupf2 and Δupf3 strains.

(A) Quantification (RT-qPCR) of full length yEmRFP-yEGFP mRNAs in WT and Δupf1, Δupf2 and Δupf3 mutants transformed with YepRG-UGA or YepRG-CGA. The relative values (RFP-GFP mRNA/β-actin) are indicated with black boxes (UGA clones) and white boxes (CGA clones). (B) Western blotting with cellular extracts obtained from WT and upfs mutants transformants. Western blotting was performed using an antibody against RFP protein, detecting the full-length yEmRFP-yEGFP (blue arrowed) as well as truncated yEmRFP-stop (red arrowed) proteins. Yeast anti-Actin antibody was used for densitometry normalization (data not shown). UGA, clones containing the read-through UGA cassette; CGA, clones containing the sense control CGA cassette; V, clones containing the empty vector; M, markers.

Fig 8. Dual fluorescence reporter response to NMD is recapitulated in flow cytometry analysis.

A) WT and Δupf1, Δupf2 and Δupf3 strains harboring the YepRG-UGA or YepRG-CGA reporters were analyzed by a flow cytometer. yEmRFP fluorescence was measured by observing 10.000 yeast cells as described in Materials and Methods. B) UGA/CGA fluorescence ratio derived from data in A; C) UGA/CGA ratio calculated from RT-qPCR analysis (Fig 7A); D) UGA/CGA ratio calculated from microplate assay (Fig 6A). Data are expressed as mean values and indicated with standard deviation.