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.