title: Evidence of efficient stop codon readthrough in four mammalian genes.
abstract: 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.
authors.affiliation: School of Biochemistry and Cell Biology, University College Cork, Cork, Ireland g.loughran@ucc.ie.
authors.forename: Gary
authors.initials: G
authors.lastname: Loughran
authors.affiliation: School of Biochemistry and Cell Biology, University College Cork, Cork, Ireland.
authors.forename: Ming-Yuan
authors.initials: MY
authors.lastname: Chou
authors.affiliation: School of Biochemistry and Cell Biology, University College Cork, Cork, Ireland.
authors.forename: Ivaylo P
authors.initials: IP
authors.lastname: Ivanov
authors.affiliation: CSAIL, Massachusetts Institute of Technology, Cambridge, MA 02139-4307, USA.
authors.forename: Irwin
authors.initials: I
authors.lastname: Jungreis
authors.affiliation: CSAIL, Massachusetts Institute of Technology, Cambridge, MA 02139-4307, USA.
authors.forename: Manolis
authors.initials: M
authors.lastname: Kellis
authors.affiliation: School of Biochemistry and Cell Biology, University College Cork, Cork, Ireland.
authors.forename: Anmol M
authors.initials: AM
authors.lastname: Kiran
authors.affiliation: School of Biochemistry and Cell Biology, University College Cork, Cork, Ireland.
authors.forename: Pavel V
authors.initials: PV
authors.lastname: Baranov
authors.affiliation: School of Biochemistry and Cell Biology, University College Cork, Cork, Ireland Department of Human Genetics, University of Utah, Salt Lake City, UT 84112-5330, USA g.loughran@ucc.ie.
authors.forename: John F
authors.initials: JF
authors.lastname: Atkins
mesh_terms: D000617:Aminoglycosides / Q000494:pharmacology; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D051401:Aquaporin 4 / Q000235:genetics; D018388:Codon, Terminator; D017124:Conserved Sequence; D057809:HEK293 Cells; D006801:Humans; D048057:Mitogen-Activated Protein Kinase 10 / Q000235:genetics; D059372:Nucleotide Motifs; D010802:Phylogeny; D014176:Protein Biosynthesis / Q000187:drug effects; D011957:Receptors, Opioid / Q000235:genetics; D017473:Receptors, Opioid, kappa / Q000235:genetics; D000094942:Nociceptin Receptor
chemical_list: C496030:AQP4 protein, human; D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D051401:Aquaporin 4; D018388:Codon, Terminator; C515653:OPRK1 protein, human; D011957:Receptors, Opioid; D017473:Receptors, Opioid, kappa; D048057:Mitogen-Activated Protein Kinase 10; D000094942:Nociceptin Receptor; C000722283:OPRL1 protein, human