Evidence of abundant stop codon readthrough in Drosophila and other metazoa

Abstract

While translational stop codon readthrough is often used by viral genomes, it has been observed for only a handful of eukaryotic genes. We previously used comparative genomics evidence to recognize protein-coding regions in 12 species of Drosophila and showed that for 149 genes, the open reading frame following the stop codon has a protein-coding conservation signature, hinting that stop codon readthrough might be common in Drosophila. We return to this observation armed with deep RNA sequence data from the modENCODE project, an improved higher-resolution comparative genomics metric for detecting protein-coding regions, comparative sequence information from additional species, and directed experimental evidence. We report an expanded set of 283 readthrough candidates, including 16 double-readthrough candidates; these were manually curated to rule out alternatives such as A-to-I editing, alternative splicing, dicistronic translation, and selenocysteine incorporation. We report experimental evidence of translation using GFP tagging and mass spectrometry for several readthrough regions. We find that the set of readthrough candidates differs from other genes in length, composition, conservation, stop codon context, and in some cases, conserved stem-loops, providing clues about readthrough regulation and potential mechanisms. Lastly, we expand our studies beyond Drosophila and find evidence of abundant readthrough in several other insect species and one crustacean, and several readthrough candidates in nematode and human, suggesting that functionally important translational stop codon readthrough is significantly more prevalent in Metazoa than previously recognized.

Evolution at two levels of gene expression in yeast

“…Two functional C-terminal extensions were previously identified in S. cerevisiae: Extension of the PDE2 gene decreases its stability, resulting in accumulation of cyclic AMP (Namy et al 2002); and readthrough of IMP3, involved in ribosomal biogenesis, destabilizes its interaction with the U3 snoRNA (Cosnier et al 2011). A recent systematic study of conserved protein-coding potential in candidate C-terminal extensions in eukaryotes failed to identify any candidates in yeasts (Jungreis et al 2011); however, the investigators required strong sequence conservation of the extension across five sensu stricto species. Multispecies riboprofiling data provide an excellent opportunity to search for direct evidence of translation in putative Cterminal extensions at the transcriptomewide level.…”

Cyclin-dependent kinase 4 may be expressed as multiple proteins and have functions that are independent of binding to CCND and RB and occur at the S and G2/M phases of the cell cycle

“…On the other hand, if the translation reads through the 1137-1139th TGA canonical stop codon, it should stop at the 1275-1277th TAA, appending to the C terminus 45 AA, or 46 AA if the TGA is not nonsense but instead encodes an AA, either arginine, cysteine, serine, tryptophan, pyrrolysine, or selenocysteine. 13,14 Similarly, mouse CDK4 (mCDK4) mRNA has an upstream CTG at its 33-35th nt, which initiates an in-frame ORF that ends at the 144-146th TAG (Fig. 1).…”

Avoidance of reporter assay distortions from fused dual reporters

“…Therefore, Firefly activities should faithfully reflect Firefly abundance which in turn can be reliably normalized to Renilla activity to provide a more accurate estimate of recoding efficiency. To assess this prediction, we tested in pSGDluc, several known human stop codon readthrough signals that had previously been identified by comparative genomics analysis (Jungreis et al 2011;Lindblad-Toh et al 2011;Loughran et al 2014). These were compared against the traditional vector system, pDluc (Fixsen and Howard 2010) (a derivative of p2luc [Grentzmann et al 1998]).…”