Selective Constraint on the Upstream Open Reading Frames That Overlap with Coding Sequences in Animals

Abstract

Upstream open reading frames (uORFs) are translational regulatory elements located in 5′ untranslated regions. They can significantly repress the translation of the downstream coding sequences (CDS), and participate in the spatio-temporal regulations of protein translation. Notwithstanding this biological significance, the selective constraint on uORFs remains underexplored. Particularly, the uORFs that partially overlap with CDS with a different reading frame (overlapping uORFs, or “VuORFs”) may lead to strong translational inhibition or N-terminal truncation of the peptides encoded by the affected CDS. By analyzing VuORF-containing transcripts (designated as “VuORF transcripts”) in human, mouse, and fruit fly, we demonstrate that VuORFs are in general slightly deleterious - the proportion of genes that encode at least one VuORF transcript is significantly smaller than expected in all of the three examined species. In addition, this proportion is significantly smaller in fruit fly than in mammals, indicating a higher efficiency of removing VuORFs in the former species because of its larger effective population size. Furthermore, the deleterious effect of a VuORF depends on the sequence context of its start codon (VuAUG). VuORFs with an optimal VuAUG context are more strongly disfavored than those with a suboptimal context in all of the three examined species. And the propensity to remove optimal-context VuAUGs is stronger in fruit fly than in mammals. Intriguingly, however, the currently observable optimal-context VuAUGs (but not suboptimal-context VuAUGs) are more conserved than expected. These observations suggest that the regulatory functions of VuORFs may have been gained fortuitously in organisms with a small effective population size because the slightly deleterious effect of these elements can be better tolerated in these organisms, thus allowing opportunities for the development of novel biological functions. Nevertheless, once the functions of VuORFs were established, they became subject to negative selection.

Ribosome profiling reveals translational regulation of mammalian cells in response to hypoxic stress

“…uORFs are emerging as important mediators of transcript-specific translational control of their downstream coding sequences [ 47 ]. Typically, uORFs lead to a significant reduction of the translation efficiency of the main downstream ORF under unstressed conditions [ 48 ], however, when eIF2 was phosphorylated and global translation was consequently inhibited under stress conditions, the presence of uORF(s) in a transcript promotes an increase in the corresponding protein levels [ 40 ]. Using ribosome-profiling technology to investigate translational regulation under normoxia and sublethal hypoxic stress conditions in seedlings of Arabidopsis thaliana , Juntawong et al reported that uORFs are translated and that these uORFs act as a barrier to mORF (main ORF) translation under conditions of normoxia [ 13 ].…”

Genome-Wide Transcriptional Excavation of Dipsacus asperoides Unmasked both Cryptic Asperosaponin Biosynthetic Genes and SSR Markers

“…To further predict and rank possible functions, we attentively analyzed the result of the above comparison. If the comparison was successful, the ORF information box containing the CDS (Hsu and Chen, 2012 ) of this transcript could be extracted from the results, then this conserved domain could be translated into an amino acid sequence (from 5′to 3′) in accordance with the standard codon table; if the unigene was not mapped to the above protein databases, the CDS could be predicted by Estscan (Iseli et al, 1999 ; 3.0.3 version) software, thereby obtaining the nucleic acid or amino acid sequences of this conserved domain.…”

Multilevel regulation of the glass locus during Drosophila eye development

“…However, since ribosomes cannot backup, the overlapping open reading frame should profoundly inhibit Glass translation [29, 30]. It was shown that overlapping upstream open reading frames; and particularly those that have an optimal AUG context, are efficiently removed from the Drosophila genome [31], suggesting that those that can be found and that are even conserved outside of the most closely related species, have been selected due to a specific function. In the case of Glass, we found evidence that translation from the upstream start codon strongly reduces GFP translation and also interferes with Glass translation when overexpressed.…”