Ribosome Profiling Reveals Pervasive Translation Outside of Annotated Protein-Coding Genes

•Assembled 80S ribosomes occupy many 5′ UTRs and some lncRNAs•Fragment length metric (FLOSS) identifies true ribosome footprints bioinformatically•Large ribosome subunit pull-down confirms ribosome footprints experimentally•Noncanonical translation produces peptides, including novel antigens

SummaryRibosome 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.

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