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

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.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.Translation termination is a crucial step in the process of information decoding. Its accuracy reaches about 10-4 and ensures that only very few abnormal products are synthesised under normal conditions [1]. Conversely, translation termination is widely used by both animal and plant viruses as a mean of controling expression, through recoding events [2, 3]. Readthrough is the process by which a stop codon is misread as sense by the translational apparatus, allowing the synthesis of an extended polypeptide which carries novel activities [2]. Up to now, no specific gene products have been implicated in the control of translational readthrough, strongly suggesting that only normal interac