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.
Highlights
Identifying the genomic regions that are transcribed and translated is a fundamental step in annotating a genome and understanding its expression
Protein-coding sequences were discovered by search for long (>100 codon) open reading frames, which are unlikely to occur in the absence of selection against stop codons
In ribosome-profiling data, the overall size distribution of fragments derived from protein-coding sequences, which should predominantly reflect true ribosome footprints, differs from the lengths of the abundant rRNA contamination found in profiling samples (Ingolia et al, 2009, 2011)
Summary
Identifying the genomic regions that are transcribed and translated is a fundamental step in annotating a genome and understanding its expression. Protein-coding sequences were discovered by search for long (>100 codon) open reading frames, which are unlikely to occur in the absence of selection against stop codons Widespread use of this approach has been based on the assumption that short peptides are unlikely to fold into stable structures and perform robust biological functions. More-sophisticated conservation-based metrics, such as PhyloCSF, were developed for the computational identification of sequences that appear to encode proteins over a broad size range (Lin et al, 2008, 2011). These approaches focus on identifying regions of the genome experiencing selective pressure to maintain a reading frame encoding a functional protein. The questions of which parts of the genome are translated and whether or not the protein product has an adaptive function in the cell are related but distinct; the former can be answered by experimentally finding the locations of ribosomes on mRNAs
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