Genome-Wide Analysis in Vivo of Translation with Nucleotide Resolution Using Ribosome Profiling

Abstract

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.The ability to monitor the identity and quantity of proteins that a cell produces would inform nearly all aspects of biology. Microarray-based measurements of mRNA abundance have revolutionized the study of gene expression (1). However, for several reasons there is a critical need for techniques that directly monitor protein synthesis. First, mRNA levels are an imperfect proxy for protein production because mRNA translation is subject to extensive regulation (2-4). Second predicting the exact protein product from the transcript sequence is not possible because of effects such as internal ribosome entry sites, initiation at non-AUG codons, and nonsense read-through (5,6). Finally, programmed ribosomal pausing during protein synthesis is thought to aid the cotranslational folding and secretion of some proteins (7-9).Polysome profiling, in which mRNAs are recovered from translating ribosomes for subsequent microarray analysis, can provide a useful estimate of protein synthesis (10). However, this approach suffers from limited resolution and accuracy. Additionally, upstream open reading frames (uORFs)-short translated sequences found in the 5′ untranslated region (5′UTR) of many genes-result in ribosomes that are bound to an mRNA and yet are not translating the encoded gene (11). Advances in quantitative proteomics circumvent some of these problems (2,3), but there currently are substantial limits on their ability to independently determine protein sequences and measure low-abundance proteins.The position of a translating ribosome can be precisely determined by using the fact that a ribosome protects a discrete footprint [∼30 nucleotides (nt)] on its mRNA template from nuclease digestion (12). We reasoned that advances in deep-sequencing technology, which NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript make it possible to read tens of millions of short (∼35 base pairs) DNA sequences in parallel (13), would allow the full analysis of ribosome footprints from cells. Here, we present a ribosome-...

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“…Secondly, a computational prediction of the ribosome density along mRNAs, based on this hypothesis, was subsequently confirmed experimentally by ribosome profiling (Ingolia et al 2009). …”

Global translational reprogramming is a fundamental layer of immune regulation in plants

“…Translatome fold changes (RFfc) for protein-coding genes were measured using reads assigned to CDS by gene. TE was calculated by combining reads for all genes that passed RPKM ≥ 1 in CDS threshold in two biological replicates and normalizing Ribo-seq RPKM to RNA-seq RPKM as reported 12 . The criteria used for uORF prediction are shown in Extended Data Fig.…”

“…1a). We then calculated TE values according to a previously reported formula 12 (Extended Data Fig. 4c, e, Supplementary Table 2), using the endogenous TBF1 as a positive control by counting reads to exon2 to distinguish reads from the 35S:uORFs TBF1 -LUC reporter (Extended Data Fig.…”

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“…Castello et al, 2012). A modification of this approach to specifically pull-down nascent ribosomes or RNA polymerase molecules provides a snapshot of translating mRNAs (Ingolia et al, 2009) or nascent transcripts (Churchman and Weissman, 2011), respectively, at a given time point in the cell. Finally, even the secondary structure of cellular transcripts can be investigated with the help of deepsequencing, namely by analyzing RNA samples that were treated with different structurespecific enzymes (Kertesz et al, 2010).…”