2.2 | Ribosome Profiling
Deep sequencing of the protected mRNA footprints of actively translating ribosomes (ribosome profiling or Ribo-seq) has been extensively reviewed and provides a powerful technology for detection of translated sORFs. Ribo-seq is carried out by isolating translating ribosomes associated with mRNA transcripts, using either elongation inhibitors like cycloheximide or rapid freezing. Because translating 80S ribosomes protect bound mRNA fragments from digestion by RNase, sequencing the ribosome-protected fragments (RPFs) reports on translated regions of mRNA. Furthermore, the codon-by-codon elongation of 80S ribosome gives RPFs a characteristic 3-nucleotide periodic distribution, which can be used to infer the reading frame and confidently differentiate translated ORFs from noise. Furthermore, translation efficiency can be assessed by comparing the frequency of ribosome footprint reads to mRNA transcript levels. Rigorous data analysis, high-resolution datasets, and analysis of replicates are essential for calling sORF translation using Ribo-seq, because their short lengths and translation by monosomes lead to lower signal-to-noise in sORF-mapped reads relative to longer canonical protein coding sequences.
While Ribo-seq is powerful in profiling the footprints of elongating ribosomes and identifying novel coding regions, elongation inhibitors like cycloheximide are not well-suited to deconvolute some translation initiation sites, especially for ORFs with multiple start sites or overlapping reading frames. As a result, a specialized method called translation initiation sequencing (TI-seq) has been developed for inhibition and profiling of the footprints of initiating ribosomes that leverage molecules like puromycin, harringtonine and lactimidomycin in eukaryotes, and retapamulin, tetracycline and Onc112 in prokaryotes. The enrichment of ribosome footprints at canonical and non-canonical start codons in TI-seq datasets generates peaks at the beginning of putative sORFs as well as canonical protein coding sequences. This allows deconvolution of sORF translation initiation from larger main ORFs in multicistronic mRNAs, and is especially important for detection of nested and out of frame sORFs. TI-Seq can also be combined with Ribo-Seq to call translated ORFs with higher confidence.