title: Ribosome profiling reveals pervasive translation outside of annotated protein-coding genes.
abstract: Ribosome profiling suggests that ribosomes occupy many regions of the transcriptome thought to be noncoding, including 5' UTRs and long noncoding RNAs (lncRNAs). Apparent ribosome footprints outside of protein-coding regions raise the possibility of artifacts unrelated to translation, particularly when they occupy multiple, overlapping open reading frames (ORFs). Here, we show hallmarks of translation in these footprints: copurification with the large ribosomal subunit, response to drugs targeting elongation, trinucleotide periodicity, and initiation at early AUGs. We develop a metric for distinguishing between 80S footprints and nonribosomal sources using footprint size distributions, which validates the vast majority of footprints outside of coding regions. We present evidence for polypeptide production beyond annotated genes, including the induction of immune responses following human cytomegalovirus (HCMV) infection. Translation is pervasive on cytosolic transcripts outside of conserved reading frames, and direct detection of this expanded universe of translated products enables efforts at understanding how cells manage and exploit its consequences.
authors.affiliation: Department of Embryology, Carnegie Institution for Science, Baltimore, MD 21218, USA. Electronic address: ingolia@berkeley.edu.
authors.forename: Nicholas T
authors.initials: NT
authors.lastname: Ingolia
authors.affiliation: Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, Center for RNA Systems Biology, California Institute for Quantitative Biosciences, University of California, San Francisco, San Francisco, CA 94158, USA.
authors.forename: Gloria A
authors.initials: GA
authors.lastname: Brar
authors.affiliation: Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, Center for RNA Systems Biology, California Institute for Quantitative Biosciences, University of California, San Francisco, San Francisco, CA 94158, USA.
authors.forename: Noam
authors.initials: N
authors.lastname: Stern-Ginossar
authors.affiliation: Department of Embryology, Carnegie Institution for Science, Baltimore, MD 21218, USA; Department of Biology, The Johns Hopkins University, Baltimore, MD 21218, USA.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Harris
authors.affiliation: Department of Embryology, Carnegie Institution for Science, Baltimore, MD 21218, USA; Department of Biology, The Johns Hopkins University, Baltimore, MD 21218, USA.
authors.forename: Gaëlle J S
authors.initials: GJ
authors.lastname: Talhouarne
authors.affiliation: Department of Medicine, University of Cambridge, Cambridge CB2 0QQ, UK.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Jackson
authors.affiliation: Department of Medicine, University of Cambridge, Cambridge CB2 0QQ, UK.
authors.forename: Mark R
authors.initials: MR
authors.lastname: Wills
authors.affiliation: Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, Center for RNA Systems Biology, California Institute for Quantitative Biosciences, University of California, San Francisco, San Francisco, CA 94158, USA.
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Weissman
mesh_terms: D020121:5' Untranslated Regions; D000465:Algorithms; D000818:Animals; D018387:Codon, Initiator; D017124:Conserved Sequence; D004474:Ecthyma, Contagious / Q000235:genetics* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D058977:Molecular Sequence Annotation; D011485:Protein Binding; D014176:Protein Biosynthesis; D023302:Protein Footprinting / Q000379:methods*; D062085:RNA, Long Noncoding / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D059467:Transcriptome
chemical_list: D020121:5' Untranslated Regions; D018387:Codon, Initiator; D062085:RNA, Long Noncoding