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

• Published in: Nature reviews. Molecular cell biology Vol. 16; no. 11; pp. 651 - 664
• Main Authors: Brar, Gloria A., Weissman, Jonathan S.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.11.2015; Nature Publishing Group
• Subjects: 38/39; 45/91; 49/39; 631/1647/2067; 631/337/475; 631/337/574/1789; 82/47; Base Sequence; Biochemistry; Cancer Research; Cell Biology; Developmental Biology; High-Throughput Nucleotide Sequencing; Humans; Life Sciences; Physiological aspects; Protein biosynthesis; Protein Biosynthesis - genetics; Protein synthesis; Proteins - metabolism; review-article; Ribosomes; Ribosomes - genetics; RNA, Messenger - genetics; Sequence Analysis, RNA; Stem Cells; United States
• ISSN: 1471-0072; 1471-0080
• DOI: 10.1038/nrm4069

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. Ribosome profiling has the power to interrogate — in vivo and on a global scale — what is being translated, how this translation is regulated and where in the cell the translation of specific sets of proteins occurs. 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.