Ribosome profiling reveals pervasive and regulated stop codon readthrough in Drosophila melanogaster

Abstract

Ribosomes can read through stop codons in a regulated manner, elongating rather than terminating the nascent peptide. Stop codon readthrough is essential to diverse viruses, and phylogenetically predicted to occur in a few hundred genes in Drosophila melanogaster, but the importance of regulated readthrough in eukaryotes remains largely unexplored. Here, we present a ribosome profiling assay (deep sequencing of ribosome-protected mRNA fragments) for Drosophila melanogaster, and provide the first genome-wide experimental analysis of readthrough. Readthrough is far more pervasive than expected: the vast majority of readthrough events evolved within D. melanogaster and were not predicted phylogenetically. The resulting C-terminal protein extensions show evidence of selection, contain functional subcellular localization signals, and their readthrough is regulated, arguing for their importance. We further demonstrate that readthrough occurs in yeast and humans. Readthrough thus provides general mechanisms both to regulate gene expression and function, and to add plasticity to the proteome during evolution.

DOI: http://dx.doi.org/10.7554/eLife.01179.001

eLife digest

For a gene to give rise to a protein, its DNA is first used as a template to produce a messenger RNA molecule. Each group of three nucleotides within the messenger RNA encodes an amino acid, and structures called ribosomes assemble the protein by joining together amino acids in the correct order. The nucleotide triplets are called codons, and some are known as stop codons because they typically instruct the ribosome to stop adding amino acids.

Sometimes ribosomes interpret stop codons as amino acid insertion signals, giving rise to an extended protein with a modified structure or function. This phenomenon is known as stop codon readthrough, and is required for many viruses to complete their reproductive cycles. However, much less is known about stop codon readthrough in other organisms.

Now, Dunn et al. have used a technique called ribosome profiling to analyze stop codon readthrough across the entire genome of the fruit fly Drosophila melanogaster. An enzyme was used to fragment messenger RNA, and those fragments that were specifically engaged by ribosomes—and thus likely to encode protein—were sequenced. Stop codon readthrough occurred much more often than had been expected based on previous studies. Indeed, computational analysis strongly suggests that evolution has favored this process for certain fruit fly genes. Moreover, stop codon readthrough was also observed in yeast and human cells, suggesting that it is important in many organisms, not just the fruit fly.

Stop codon readthrough thus provides a novel way for organisms to tune the expression levels and functions of their genes, both throughout the lifetime of an individual, and the evolution of a species.

DOI: http://dx.doi.org/10.7554/eLife.01179.002