Standardized annotation of translated open reading frames

Techniques for systematically monitoring protein translation have lagged far behind methods for measuring messenger RNA (mRNA) levels. Here, we present a ribosome-profiling strategy that is based on the deep sequencing of ribosome-protected mRNA fragments and enables genome-wide investigation of translation with subcodon resolution. We used this technique to monitor translation in budding yeast under both rich and starvation conditions. These studies defined the protein sequences being translated and found extensive translational control in both determining absolute protein abundance and responding to environmental stress. We also observed distinct phases during translation that involve a large decrease in ribosome density going from early to late peptide elongation as well as widespread regulated initiation at non-adenine-uracil-guanine (AUG) codons. Ribosome profiling is readily adaptable to other organisms, making high-precision investigation of protein translation experimentally accessible.

Motivation: As high-throughput transcriptome sequencing provides evidence for novel transcripts in many species, there is a renewed need for accurate methods to classify small genomic regions as protein coding or non-coding. We present PhyloCSF, a novel comparative genomics method that analyzes a multispecies nucleotide sequence alignment to determine whether it is likely to represent a conserved protein-coding region, based on a formal statistical comparison of phylogenetic codon models. Results: We show that PhyloCSF's classification performance in 12-species Drosophila genome alignments exceeds all other methods we compared in a previous study. We anticipate that this method will be widely applicable as the transcriptomes of many additional species, tissues and subcellular compartments are sequenced, particularly in the context of ENCODE and modENCODE, and as interest grows in long non-coding RNAs, often initially recognized by their lack of protein coding potential rather than conserved RNA secondary structures. Availability and Implementation: The Objective Caml source code and executables for GNU/Linux and Mac OS X are freely available at http://compbio.mit.edu/PhyloCSF Contact: mlin@mit.edu; manoli@mit.edu

The ability to sequence genomes has far outstripped approaches for deciphering the information they encode. Here we present a suite of techniques, based on ribosome profiling (the deep sequencing of ribosome-protected mRNA fragments), to provide genome-wide maps of protein synthesis as well as a pulse-chase strategy for determining rates of translation elongation. We exploit the propensity of harringtonine to cause ribosomes to accumulate at sites of translation initiation together with a machine learning algorithm to define protein products systematically. Analysis of translation in mouse embryonic stem cells reveals thousands of strong pause sites and unannotated translation products. These include amino-terminal extensions and truncations and upstream open reading frames with regulatory potential, initiated at both AUG and non-AUG codons, whose translation changes after differentiation. We also define a class of short, polycistronic ribosome-associated coding RNAs (sprcRNAs) that encode small proteins. Our studies reveal an unanticipated complexity to mammalian proteomes. Copyright © 2011 Elsevier Inc. All rights reserved.