Ribosome profiling reveals the what, when, where and how of protein synthesis

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Ribosome profiling, which involves the deep sequencing of ribosome-protected mRNA fragments, is a powerful tool for globally monitoring translation in vivo. The method has facilitated discovery of the regulation of gene expression underlying diverse and complex biological processes, of important aspects of the mechanism of protein synthesis, and even of new proteins, by providing a systematic approach for experimental annotation of coding regions. Here, we introduce the methodology of ribosome profiling and discuss examples in which this approach has been a key factor in guiding biological discovery, including its prominent role in identifying thousands of novel translated short open reading frames and alternative translation products.

Key Points

  • Ribosome profiling is a deep-sequencing-based tool that allows the detailed measurement of translation globally and in vivo.

  • The method provides quantification of levels of new protein synthesis, as well as information about ribosome positions that can be used to infer details about translation mechanism or to identify translated open reading frames (ORFs).

  • Ribosome profiling enables instantaneous rather than steady-state measurement and is thus a particularly valuable tool for the study of gene expression over dynamic processes.

  • Proximity-specific ribosome profiling is based on localized labelling of ribosome populations within cells and enables in vivo measurement of translation at specific organelles or subcellular structures.

  • Ribosome profiling is the first tool available for the experimental annotation of translated ORFs and has enabled the discovery of a wide range of new translation products. These include novel short peptides and alternative isoforms of characterized proteins, the vast majority of which are currently of unknown function.

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Figure 1: An overview of ribosome profiling.
Figure 2: Qualitative and quantitative data provided by ribosome profiling.
Figure 3: Ribosome profiling facilitates quantitative proteomic discovery in diverse systems.
Figure 4: Dom34 facilitates the release of 80S ribosomes from a subset of 3′ untranslated regions (UTRs).
Figure 5: Proximity-specific ribosome profiling at the endoplasmic reticulum (ER).
Figure 6: Proposed cellular roles for the peptide products of translated short open reading frames (sORFs) identified by ribosome profiling.


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The authors wish to thank C. Jan and E. Ünal for helpful comments on this manuscript and N. Ingolia for development of the original ribosome profiling protocol and helpful discussions. This work was partially supported by the Winkler Family Biological Sciences Award to G.A.B. and Howard Hughes Medical Institute and Center for RNA Systems Biology funding to J.S.W.

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Correspondence to Gloria A. Brar or Jonathan S. Weissman.

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Competing interests

J.S.W. is an inventor on a patent application for ribosome profiling.

Supplementary information

Supplementary information S1 (Figure)

FLOSS analysis enables post-hoc curation of ribosome profiling data. (PDF 197 kb)

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Ribosome footprints

mRNA fragments of 30 nucleotides that result from nuclease treatment of translating ribosomes. These are mRNA regions that are protected by the ribosome as the mRNA is decoded to a protein sequence.

Upstream open reading frames

(uORFs). ORFs in the 5′ leader region of a characterized mRNA transcript. Translation of uORFs may regulate translation of a downstream ORF. Ribosome profiling allows for the empirical identification of all translated uORFs in vivo under a condition of interest. Although uORFs are short, here we do not include them in the class of 'short ORFs', which are on an mRNA that was not previously thought to encode a protein.

Polysome gradients

A method for fractionating ribosomes that are bound to mRNAs by velocity centrifugation of cell extract on sucrose gradients, allowing for the separation of mRNAs that are associated with one ribosome (monosome) from those being translated by multiple ribosomes (polysome). Sucrose gradient fractionation facilitates qualitative analysis of the translation status of cells.

Ribosome P site

The site within an actively translating ribosome that is usually associated with the tRNA attached to the growing peptide chain.

Codon periodicity

The three-nucleotide pattern of ribosome occupancy, reflecting mRNA translocation in the ribosome by codon as translation occurs.

Fragment length organization similarity score (FLOSS) analysis

A metric for determining the probability that ribosome footprints over a given region (or set of regions) result from translation. This analysis involves comparing size distributions of footprints over a query region and over validated coding regions and is based on the concept that the biophysical properties of translating ribosomes result in characteristic signatures in ribosome footprint sizes.


The proteinaceous tunnel through which nascent proteins cross the endoplasmic reticulum membrane.

Translating ribosome affinity capture

(TRAP). A method that allows identification of translated mRNAs on the basis of their in vivo association with a tagged ribosomal subunit that is expressed in a cell type-specific manner. This method is a valuable tool for assaying tissue-specific translation in animal and plant systems.

Nonsense-mediated decay

mRNA degradation, which has traditionally been thought to result from stop codons that terminate translation more 5′ than is usual on an mRNA.

Short ORFs

(sORFs). Open reading frames of fewer than 100 codons on mRNAs that are not known to encode a canonical (long) protein. sORFs are a class of ORF that have not traditionally been thought to be frequently translated, although ribosome profiling and other approaches have recently validated the translation of thousands of sORFs in a range of organisms.

ORFs encoding alternative isoforms of known proteins

Open reading frames (ORFs) that differ from another ORF at the same locus in either the start codon or the stop codon position but share the same reading frame. Translation of these ORFs may result in, for example, different subcellular targeting for a similar protein.

Signatures of protein-coding conservation

Purifying evolutionary selection results in higher levels of synonymous than nonsynonymous substitutions, specifically among homologous coding sequences. The pattern of nonsynonymous to synonymous differences among homologous regions compared in a phylogenetic group can be used to predict the likelihood that a genomic locus encodes a translated open reading frame (ORF).

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Brar, G., Weissman, J. Ribosome profiling reveals the what, when, where and how of protein synthesis. Nat Rev Mol Cell Biol 16, 651–664 (2015) doi:10.1038/nrm4069

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