Length of the ORF, position of the first AUG and the Kozak motif are important factors in potential dual-coding transcripts

Abstract

A single mammalian transcript normally encodes one protein, but the transcript of GNAS (G-protein α-subunit) contains two reading frames and produces two structurally unrelated proteins, XLαs and ALEX. No other confirmed GNAS-like dual-coding transcripts have been reported to date, even though many such candidate genes have been predicted by bioinformatics analysis. In this study, we constructed a series of vectors to test how two protein products were translated from a single transcript in vitro. The length of the ORF (open reading frame), position of the first AUG and the Kozak motif were found to be important factors. These factors, as well as 55-bp NMD (nonsense-mediated mRNA decay) rule, were used in a bioinformatics search for candidate dual-coding transcripts. A total of 1307, 750 and 474 two-ORF-containing transcripts were found in human, mouse and rat, respectively, of which 170, 89 and 70, respectively, were found to be potential dual-coding transcripts. Most transcripts showed low conservation among species. Interestingly, dual-coding transcripts were significantly enriched for transcripts from the zinc-finger protein family, which are usually DNA-binding proteins involved in regulation of the transcription process.

Decoding the dual-coding region: key factors influencing the translational potential of a two-ORF-containing transcript

“…For each configuration, the combinations of strong and/or weak Kozak motifs [6] were respectively introduced to flank the two AUG codons. Thus, Xu et al [5] were able to systematically examine the impact of three key factors on the translational activity of a two-ORF-containing transcript: the position of the start codon, the ORF length and the strength of the Kozak motif. Using fluorescence imaging techniques and western blots, they detected dual protein products in four out of the eight transcript subtypes under survey.…”

“…Using the rules inferred from their experimental studies, Xu et al [5] further performed a bioinformatic analysis to screen the dual-coding transcripts in the human and mouse genomes. They found that about 170 human transcripts are potentially dual coding and only 18 are conserved in the mouse genome.…”

“…A fundamental question is what factors determine the on/off function of the two peptide products potentially encoded in a two-ORF-containing transcript. Xu and colleagues address this question in a recently published article [5], in which they describe an elegant in vitro dual-coding expression system they developed. In their study, two fluorescent proteins (green fluorescent protein [GFP] and red [RFP]) were encoded in an artificial transcript.…”

“…The study of Xu et al [5] provides crucial insights into characterizing dual-coding transcripts in mammalian genomes, and raises some interesting questions. First, to what extent can the observation based on the artificial dual-coding transcript be generalized to other transcripts?…”

“…The translation of cellular messenger RNA in vivo is a more complex process and often involves many factors that are specific to a given tissue or cell type. Nevertheless, Xu et al [5] obtain a set of experimentally-determined and biologically-sensible rules to identify dual-coding transcripts, which provides a valuable starting point for further investigation.…”