Article series: Applications of next-generation sequencing

Ribosome profiling: new views of translation, from single codons to genome scale

Journal name:
Nature Reviews Genetics
Volume:
15,
Pages:
205–213
Year published:
DOI:
doi:10.1038/nrg3645
Published online

Abstract

Genome-wide analyses of gene expression have so far focused on the abundance of mRNA species as measured either by microarray or, more recently, by RNA sequencing. However, neither approach provides information on protein synthesis, which is the true end point of gene expression. Ribosome profiling is an emerging technique that uses deep sequencing to monitor in vivo translation. Studies using ribosome profiling have already provided new insights into the identity and the amount of proteins that are produced by cells, as well as detailed views into the mechanism of protein synthesis itself.

At a glance

Figures

  1. Ribosome footprint profiling.
    Figure 1: Ribosome footprint profiling.

    RNase digestion of polysomes that are carrying out translation in vivo yields ribosome-protected mRNA fragments, which are known as ribosome footprints. These footprints are recovered and converted into a DNA library through ligation of a linker followed by reverse transcription and circularization PCR. cDNA libraries are then analysed by deep sequencing. Figure is reproduced, with permission, from Ref. 65 © (2012) Macmillan Publishers Ltd. All rights reserved.

  2. Analysis of ribosome occupancy data.
    Figure 2: Analysis of ribosome occupancy data.

    a | The positions of ribosomes on many copies of an mRNA in a cell population are converted into nuclease-protected footprints that correspond to ribosome positions in vivo. Ribosome profiling data indicate the density of ribosomes at each position on the transcript; the thick line represents the protein-coding sequence and the thin lines represent the untranslated regions. b | The amount of protein synthesized corresponds to ribosome density across the protein-coding sequence of a transcript. Ribosome footprints indicate the total number of ribosomes that are engaged in synthesizing a protein, which is determined by the number of mRNAs in the cell and by the number of ribosomes that translate each transcript. Changes in either mRNA abundance (for example, having fewer mRNAs) or ribosome loading (for example, having fewer ribosomes per mRNA) will affect the number of ribosome footprints that are recovered from the gene.

  3. Alternative reading frames.
    Figure 3: Alternative reading frames.

    a | Alternative sites of translation initiation may be found in the 5′ transcript leader and may initiate translation of a short upstream open reading frame (uORF, shown in orange). These uORFs can overlap the start codon of the protein-coding gene, which leads to out-of-frame translation that extends into the coding sequence. Alternative reading frame translation can also begin within the protein-coding gene. An in-frame start codon within a gene can lead to a truncated protein, whereas an in-frame start codon upstream of a gene without an intervening stop codon can produce an extended protein product. The shaded regions represent the alternative mRNA sequences that are being translated. b | Alternative translation start sites may result either from non-canonical initiation, such as 'leaky scanning' past the first start codon, or from canonical initiation on alternative mRNA variants.

  4. Ribosomal pausing and co-translational processes.
    Figure 4: Ribosomal pausing and co-translational processes.

    a | Ribosomal stalling causes an accumulation of ribosome footprints at a specific position within a gene and thus a peak of ribosome footprint density. b | Footprints of ribosomes can be purified by pulldown assays through their association with co-translational protein biogenesis factors, such as folding chaperones, which indicate the time during translation and the position in the protein at which these factors act.

  5. Deconvolving transcription and translation.
    Figure 5: Deconvolving transcription and translation.

    Matched ribosome profiling and mRNA sequencing (RNA-seq) data distinguish between alternative modes of gene expression regulation. Transcriptional induction results in matched increases in mRNA abundance, as measured by RNA-seq, and protein synthesis, as measured by ribosome profiling. Translational induction manifests as an increase in ribosome profiling measurements without a corresponding change in mRNA abundance.

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Affiliations

  1. Department of Embryology, Carnegie Institution, 3520 San Martin Drive, Baltimore, Maryland 21218, USA. Present address: Department of Molecular and Cell Biology, University of California, Berkeley, Barker Hall 422, Berkeley, California 94720, USA.

    • Nicholas T. Ingolia

Competing interests statement

N.T.I. is Inventor on patent application covering ribosome profiling.

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Author details

  • Nicholas T. Ingolia

    Nicholas T. Ingolia recently joined the Molecular and Cell Biology department at the University of California, Berkeley, USA, as Assistant Professor. Previously, he was a staff member at the Department of Embryology, Carnegie Institution for Science, Baltimore, Maryland, USA. He received his Ph.D. from Harvard University, Cambridge, Massachusetts, USA, and went on to do postdoctoral work at the University of California, San Francisco, USA, where he developed ribosome profiling. His laboratory seeks to better understand the molecular basis and cellular roles of translational regulation. Nicholas T. Ingolia's homepage.

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