Suppression of nonsense mutations in cell culture and mice by multimerized suppressor tRNA genes

Abstract

We demonstrate here the first experimental suppression of a premature termination codon in vivo by using an ochre suppressor tRNA acting in an intact mouse. Multicopy tRNA expression plasmids were directly injected into skeletal muscle and into the hearts of transgenic mice carrying a reporter gene with an ochre mutation. A strategy for modulation of suppressor efficiency, applicable to diverse systems and based on tandem multimerization of the tRNA gene, is developed. The product of suppression (chloramphenicol acetyltransferase) accumulates linearly with increases in suppressor tRNA concentration to the point where the ochre-suppressing tRNA(Ser) is in four- to fivefold excess over the endogenous tRNA(Ser). The subsequent suppressor activity plateau seems to be attributable to accumulation of unmodified tRNAs. These results define many salient variables for suppression in vivo, for example, for tRNA suppression employed as gene therapy for nonsense defects.

FIG. 1

tRNA^sersu⁺ ochre gene multimerization strategy. ptRNA 510 and 105 were obtained by recloning the tRNAsu⁺ gene into plasmid pUC18 as described in Materials and Methods. They carry different 5′ regions, respectively 468 and 63 nt (vertically striped boxes) and the same 36-nt 3′ region (open boxes). Plasmids containing two tRNAsu⁺ copies (ptRNA2mer/510 and ptRNA2mer/105) were generated after endonuclase digestion and ligation as shown schematically. Since the BamHI/BglII junction [B/B2] becomes resistant to the cleavage of either enzyme, the BamHI-BglII sites can be reused for a new round of multimerization. Unique restriction sites are indicated by the following abbreviations: E, EcoRI; H, HindIII; B, BamHI; B2, BglII. Plasmids containing 8 or 16 tRNAsu⁺ copies were generated from constructs containing two tRNAsu⁺ copies after two or three rounds of the described multimerization steps. The final constructs were designated ptRNA8mer/105, ptRNA16mer/105, ptRNA8mer/510, and ptRNA16mer/510 (numbers before the slash represent the copies of tRNAsu⁺ gene present in each plasmid; numbers after the slash indicate the nucleotide length of the spacer separating each tRNAsu⁺ gene).

FIG. 2

CAT activity in COS 7 cells cotransfected with the CAT ochre reporter gene and different tRNAsu⁺ constructs. (A) COS 7 cells were cotransfected with 4 μg of ViCAT(oc27) and 6 μg of each tRNAsu⁺ construct. Data represent mean values of CAT activity from four independent transfections. In order to maintain the assay in the linear range, extracts were diluted in 1% bovine serum albumin. Relative CAT activity corresponds to the percentage of acetylated chloramphenicol relative to 10 μg of total protein multiplied by the dilution factor of the cell lysate. tRNAsu⁺ multimers are designated as in Fig. 1; the monomer corresponds to the plasmid ptRNA 510. By analysis of variance, P < 0.05. By Fisher's protected least-square difference post hoc analysis, for 8mer/510 and 16mer/510 P = 0.76 and for 8mer/510 and 8mer/105 P = 0.034. (B) COS 7 cells were cotransfected with 4 μg of ViCAT(oc27) and 1, 3, 6, and 12 μg of each tRNAsu⁺ construct. Data points represent mean values of CAT activity from three independent transfections.

FIG. 3

Northern blot analysis of RNA purified from COS 7 cells transfected with different tRNAsu⁺ constructs by the DEAE-dextran procedure. COS 7 cells were transfected according to the DEAE-dextran procedure with 6 μg of different tRNAsu⁺ constructs. After 36 h RNA was isolated, separated on an 8% polyacrylamide–8 M urea gel, and electroblotted as described in Materials and Methods. tRNAsu⁺ constructs are designated as in Fig. 1. +control, in vitro-transcribed tRNAsu⁺ (2 ng), which contains three additional nucleotides (Materials and Methods); −control, RNA purified from untransfected cells. Hybridization signals were normalized to the level of 5.8S rRNA and quantified as described in Materials and Methods. (A) The membrane at the left was probed with the 5′-end-labeled oligonucleotide α (α*) complementary to the anticodon loop of tRNAsu⁺ in the presence of an excess of unlabeled oligonucleotide T complementary to the TΨC arm and variable arm. Oligonucleotide α* (black line) and oligonucleotide T (gray line) are depicted at the left. RNA purified from COS 7 cells transfected with the 16mer/105 construct was blotted on the membrane on the right. The membrane was hybridized with oligonucleotide J (black line) complementary to the last 8 nt of the 3′ end of the mature tRNA and the subsequent 8 nt located in the tRNA gene. (B) The membrane from panel A was rehybridized using 5′-end-labeled oligonucleotide T* (left) as described in Materials and Methods. A short exposure of the portion of the gel containing the mature tRNAs is shown at the bottom.

FIG. 4

Limiting steps affecting tRNAsu⁺ overexpression. (A) Northern blot analysis of RNA purified from COS 7 cells transfected with different tRNAsu⁺ constructs by the FuGENE procedure. RNA was isolated and analyzed as for Fig. 3. tRNAsu⁺ constructs are designated as in Fig. 1. +control, in vitro-transcribed tRNAsu⁺ (2 ng); −control, RNA purified from untransfected cells. Oligonucleotides α* and T are at the left. (B) The membrane from panel A was rehybridized using the 5′-end-labeled oligonucleotide T* (left) as described in Materials and Methods. (C) Extent of tRNAsu⁺ in vivo aminoacylation. COS 7 cells were transfected with 12 μg of different tRNAsu⁺ constructs using the FuGENE method. RNA isolated in acidic conditions (pH 5) was separated on an acid-urea gel, blotted, and analyzed by Northern hybridization as for Fig. 3A. tRNAsu⁺ constructs are designated as in Fig. 1. +control, nonaminoacylated in vitro-transcribed tRNAsu⁺; −, samples loaded without any treatment; +, samples treated with 0.2 M Tris-HCl (pH 9) at 37°C for 30 min before gel electrophoresis.

FIG. 5

In vivo suppression of CAT ochre reporter gene coinjected in skeletal muscles with different tRNAsu⁺ constructs. CAT activity in 10% of muscle extract was determined 1 week after plasmid injection. The highest and lowest percentages of chloramphenicol conversion obtained for each construct are shown at the bottom of the panels (n = 5). Since the CAT activity of ViCAT was out of the linear range, the extracts were diluted in 1% bovine serum albumin. tRNAsu⁺ constructs are designated as in Fig. 1. (A) Two-week-old mouse TA muscles were injected with 12.5 μg of ViCAT(oc27) and 40 μg of different tRNAsu⁺ constructs in 50 μl of sterile normal saline. (B) Four-week-old mouse TA muscles undergoing regeneration were injected with 12.5 μg of ViCAT(oc27) and 40 μg of different tRNAsu⁺ constructs in 100 μl of sterile normal saline. Muscle regeneration was induced 5 days before DNA injections by treatment with 0.75% bupivacaine solution. (C) Four-week-old mouse tongues were injected with 12.5 μg of ViCAT(oc27) and 40 μg of different tRNAsu⁺ constructs. Tongues were reinjected a second time 30 min after the first injection. Each injection was performed in 60 μl of sterile normal saline.

FIG. 6

In vivo suppression in the α-CAT ochre gene-expressing transgenic mouse. (A) Northern blot analysis of α-CAT ochre gene mRNA. Total RNA (10 μg) extracted from the heart of an α-CAT ochre gene-expressing transgenic mouse was separated on a 1.5% agarose–6% formaldehyde gel and blotted on a nylon membrane. The filter was sequentially hybridized with α-CAT and actin_αc mRNA-specific oligonucleotide probes, labeled at the same specific activity. After each hybridization, the filter was exposed for 6 h and signals were quantified by a Storm 860 image analyzer. Similar results have been obtained by analyzing several transgenic mice. (B) Levels of nonsense suppression in the α-CAT ochre gene-expressing transgenic mouse. Four-month-old transgenic mouse hearts were injected with 50 μg of different tRNAsu⁺ constructs in 20 μl of normal saline. tRNAsu⁺ constructs are designated as in Fig. 1. Control, α-CAT ochre gene-expressing transgenic mice injected with normal saline alone (n = 10 for the monomer and 3 for each of the other constructs). CAT activity in 10% of heart extract was measured 1 week after plasmid injection. The highest percentage of chloramphenicol conversion is shown below each construct. tRNAsu⁺ constructs are designated as in Fig. 1.