Four mammalian genes displayed efficient stop codon readthrough, with OPRL1 showing ~31% readthrough in HEK-293T cells, driven by a highly conserved UGA-CUAG motif.
The study identifies efficient stop codon readthrough in mammalian genes, highlighting the importance of a conserved UGA-CUAG motif.
Stop codon readthrough is used extensively by viruses to expand their gene expression. Until recent discoveries in Drosophila, only a very limited number of readthrough cases in chromosomal genes had been reported. Analysis of conserved protein coding signatures that extend beyond annotated stop codons identified potential stop codon readthrough of four mammalian genes. Here we use a modified targeted bioinformatic approach to identify a further three mammalian readthrough candidates. All seven genes were tested experimentally using reporter constructs transfected into HEK-293T cells. Four displayed efficient stop codon readthrough, and these have UGA immediately followed by CUAG. Comparative genomic analysis revealed that in the four readthrough candidates containing UGA-CUAG, this motif is conserved not only in mammals but throughout vertebrates with the first six of the seven nucleotides being universally conserved. The importance of the CUAG motif was confirmed using a systematic mutagenesis approach. One gene, OPRL1, encoding an opiate receptor, displayed extremely efficient levels of readthrough (∼31%) in HEK-293T cells. Signals both 5' and 3' of the OPRL1 stop codon contribute to this high level of readthrough. The sequence UGA-CUA alone can support 1.5% readthrough, underlying its importance.
Loughran et al. (Thu,) conducted a other in Stop codon readthrough. Reporter constructs with potential readthrough signals was evaluated on Stop codon readthrough efficiency. Four mammalian genes displayed efficient stop codon readthrough, with OPRL1 showing ~31% readthrough in HEK-293T cells, driven by a highly conserved UGA-CUAG motif.