UAG readthrough in mammalian cells: effect of upstream and downstream stop codon contexts reveal different signals

Abstract

Background: Translation termination is mediated through an interaction between the release factors eRF1 and eRF3 and the stop codon within its nucleotide context. Although it is well known that the nucleotide contexts both upstream and downstream of the stop codon, can modulate readthrough, little is known about the mechanisms involved.

Results: We have performed an in vivo analysis of translational readthrough in mouse cells in culture using a reporter system that allows the measurement of readthrough levels as low as 10(-4). We first quantified readthrough frequencies obtained with constructs carrying different codons (two Gln, two His and four Gly) immediately upstream of the stop codon. There was no effect of amino acid identity or codon frequency. However, an adenine in the -1 position was always associated with the highest readthrough levels while an uracil was always associated with the lowest readthrough levels. This could be due to an effect mediated either by the nucleotide itself or by the P-site tRNA. We then examined the importance of the downstream context using eight other constructs. No direct correlation between the +6 nucleotide and readthrough efficiency was observed.

Conclusions: We conclude that, in mouse cells, the upstream and downstream stop codon contexts affect readthrough via different mechanisms, suggesting that complex interactions take place between the mRNA and the various components of the translation termination machinery. Comparison of our results with those previously obtained in plant cells and in yeast, strongly suggests that the mechanisms involved in stop codon recognition are conserved among eukaryotes.

Figure 1

Description of the pRSVL74 vector and of the cloning strategy. The sequence of the 5' end of the luc gene coding sequence is shown with the common sequence of the inserted oligonucleotide containing the stop with its surrounding context. This sequence is inserted in place of the boxed "T" in the luc gene sequence. For each test construct, a control with the same sequence where a CAG replaced the TAG stop codon was used to evaluate the luciferase specific molecular activity.

Figure 2

Readthrough efficiencies of upstream mutants. The different stop codon context mutations were inserted at the beginning of the luc gene, as illustrated in Figure 1. Each construct was used to transfect NIH3T3 mouse cells and luciferase activity was determined. Each value is the mean of at least 3 independent experiments. Readthrough is expressed as percentage of the corresponding control where the stop codon has been replaced by a sense CAG glutamine codon. A: CAN UAG CAA mutants B: GGN UAG CAA mutants C: Nucleotide usage at the -1 position relative to the stop.

Figure 3

Readthrough efficiencies of downstream mutants Same legend as Figure 2. A: CAA UAG CAN mutants B: CAA UAG GGN mutants C: Nucleotide usage at the +6 position relative to the stop.

Figure 4

Readthrough efficiencies in the presence of a suppressor tRNA. Readthrough efficiencies were determined as described in Figure 1. To compare the values that differed by several orders of magnitude between the two sets of experiments, results are given as percentage of the highest readthrough obtained in each condition.