Decision letter: Extensive translation of small Open Reading Frames revealed by Poly-Ribo-Seq

Abstract

To produce a protein, a stretch of DNA must first be transcribed to produce a molecule of messenger RNA (mRNA). The genetic information copied from the DNA is then read three letters at a time, in groups called codons. Each codon either encodes a particular amino acid to be added into a protein or provides further instructions: ‘start codons’ mark the beginning of a protein; ‘stop codons’ mark its end. The DNA between these two points is called an open reading frame (or ORF)—however, not all ORFs produce proteins. Most proteins are made of several hundred amino acids, but the genomes of animals contain thousands of ORFs that would generate much smaller proteins made of fewer than 100 amino acids, if they were translated. It is, however, unclear how many of these small ORFs are converted into mRNA molecules and functional proteins. Ribosomes are large molecular machines that translate the code in mRNA molecules and join together the appropriate amino acids in the right order to make a protein. Ribosome profiling is a technique that identifies which mRNA molecules are translated into proteins by determining the sequences of all the mRNA molecules bound to ribosomes at a particular moment. The mRNA sequences can then be compared with the sequence of the whole genome to work out which ORFs they correspond to. Ribosome profiling has been used to detect translated small ORFs, but the method yields a relatively high false positive rate as some mRNAs can bind to ribosomes without being translated. To better detect small protein-producing ORFs, Aspden et al. developed a technique based on ribosome profiling called Poly-Ribo-Seq. The method takes advantage of the fact that during active translation, clusters of multiple ribosomes, called polysomes, bind mRNAs. Poly-Ribo-Seq isolates these polysomes and determines the sequence bound by each of the ribosomes, thereby reducing the number of false positives. Applying Poly-Ribo-Seq to cells from the fruit fly Drosophila allowed Aspden et al. to identify two types of short ORF. The first type codes for proteins that are around 80 amino acids long and are translated with the same efficiency as larger ORFs. The sequences of these ORFs are found in other species, match at least in part sequences of known functional ORFs, and the proteins produced are found in specific locations inside cells. These small proteins may contribute to membrane integrity or function. Together, these properties suggest that these mRNAs create functional small proteins. The second pool consists of very small ORFs (‘dwarf smORFs’) that code for around 20 amino acids, which are not translated so often and do not show many similarities with other species. As the findings of Aspden et al. suggest that a large fraction of Drosophila small ORFs are translated into proteins, the next challenge will be to determine the roles of these small proteins in cells.