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. 2009 Apr 10;324(5924):218-23.
doi: 10.1126/science.1168978. Epub 2009 Feb 12.

Genome-wide Analysis in Vivo of Translation With Nucleotide Resolution Using Ribosome Profiling

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Free PMC article

Genome-wide Analysis in Vivo of Translation With Nucleotide Resolution Using Ribosome Profiling

Nicholas T Ingolia et al. Science. .
Free PMC article

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.

Figures

Fig. 1
Fig. 1
Quantifying mRNA abundance and ribosome footprints by means of deep sequencing. (A) Schematic of the protocol for converting ribosome footprints or randomly fragmented mRNA into a deep-sequencing library. (B) Internal reproducibility of mRNA-abundance measurements. CDSs were conceptually divided as shown, and the mRNA counts on the two regions are plotted. The error estimate is based on the χ2 statistic.
Fig. 2
Fig. 2
Ribosome footprints provide a codon-specific measurement of translation. (A) Total number of ribosome footprints falling near the beginning or end of CDSs. (B) The offset between the start of the footprint and the P- and A-site codons at translation initiation and termination (34). (C) Positionof28-nt ribosome footprints relative to the reading frame. (D) Ribosome footprint densities in two complete biological replicates. Density in terms of reads per kilobase per million (rpkM) is corrected for total reads and CDS length (21). (Inset) Histogram of log2 ratios between replicates for genes with low counting statistics error (fig. S7) along with the normal error curve (mean = 0.084, SD = 0.291 in log2 units; σ is SD expressed as a fold change). (E) Histogram of translational efficiency, the ratio of ribosome footprint density to mRNA density. The error shows actual ratios between biological replicates (SD = 0.367 in log2 units). (F) Read density as a function of position. Well-expressed genes were each individually normalized and then averaged with equal weight (14).
Fig. 3
Fig. 3
Ribosome occupancy of upstream open-reading frames and other sequences. (A) Density of mRNA fragments and ribosome footprints on non—protein-coding sequences relative to the associated CDS. (B) Histogram of translational efficiencies for different classes of sequences. (C) Ribosome and mRNA density showing the uORF in the ICY1 5′UTR. (D) Ribosome and mRNA density showing non-AUG uORFs in the PRE2 5′ UTR. The proposed AAAUUG translational initiation site is shown along with the subsequent open reading frame and stop codon (indicated by a vertical line).
Fig. 4
Fig. 4
Translational response to starvation. (A) Changes in mRNA abundance and translational efficiency in response to starvation. (B) Distribution of translational efficiency changes in response to starvation. Measurement error was estimated from the actual distribution of ratios between biological replicates. A false discovery rate threshold of 10% corresponds to a twofold change in translational efficiency.
Fig. 5
Fig. 5
Changes in 5′UTR translation during starvation. (A) Ribosome and mRNA densities in the GCN4 5′UTR in repressive and inducing conditions. The four known uORFs are indicated along with the proposed initiation sites for upstream translation. (B) Non-AUGuORF upstream of GCN4. Shown is an enlargement of the gray boxed area in (A). (C) Ribosome occupancy of noncoding sequences. The number of ribosome footprints mapping to different classes of regions is shown relative to the number of CDS reads. (D) Aggregate translational efficiency of uORFs (14).

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