RIBO-seq (Ribosome Profiling) is the sequencing of mRNA fragments covered by ribosomes. These ribosome-protected mRNA fragments or ‘footprints’ allow the positions of ribosomes to be mapped across the entire transcriptome, providing a global snapshot of active translation in vivo (1).
Ribosome footprint densities across the coding regions of transcripts is the best available proxy for active protein synthesis. These measurements bridge the gap that exists between traditional gene expression metrics of mRNA and protein abundances to provide a more comprehensive understanding of gene expression (2).
RIBO-seq uniquely provides accurate positional information on ribosome mRNA locations allowing for exact monitoring of the reading frame being translated as well as the codon identity residing within the A-site of each individual ribosome to be determined (3). This nucleotide level of precision enables exploration of a range of mechanistic features of protein synthesis and translational control.
Overview: Cells are lysed and translating ribosomes are pharmacologically ‘frozen’ in place on transcripts. Nucleases digest any unprotected RNA, while the RNA within the ribosome (approximately 28 nt) is protected from digestion. Ribosome protected fragments are purified and size selected on Urea PAGE gels. Fragments are converted into a cDNA library which undergoes deep sequencing.
Complete Gene Expression | Global Translation Rates | Codon Decoding Rates
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uORF Regulatory Activity | Ribosome Pausing | Stop Codon Readthrough
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Open Reading Frame & Translation Start Site Delineation | Alternative Proteoform & Micropeptide Prediction
Explore translatomics annotations to help pinpoint novel therapeutic targets and biomarkers hidden within the proteome.
Advantages over RNA-seq
Reveals translated regions: Ribo-seq captures only the ribosome-protected fragments (RPFs) of mRNA, which represent the translated regions of the transcriptome. In contrast, RNA-seq measures the abundance of all transcripts, including non-coding RNA, regardless of whether they are translated and contributing to the proteome..
Quantifies protein synthesis: By capturing only the RPFs, Ribo-seq allows for more accurate quantification of protein synthesis than RNA-seq, which measures the abundance of mRNA transcripts. This is particularly useful in studying gene expression in specific cell types or under specific physiological conditions.
Resolves ribosome footprints: Ribo-seq provides higher resolution than RNA-seq, as it resolves the ribosome footprints of the mRNA transcriptome. This allows for precise mapping of translation initiation and elongation, and identification of potential translation pauses or ribosome stalling.
1. Ingolia, N.T., Ghaemmaghami, S., Newman, J.R. and Weissman, J.S. (2009). Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science. 324(5924):218-223.
2. Brar, G.A. and Weissman, J.S. (2015). Ribosome profiling reveals the what, when, where, and how of protein synthesis. Nature reviews. Molecular Cell Biology. 16(11):651.
3. Michel, A.M. and Baranov, P.V. (2013). Ribosome profiling: A Hi‐Def monitor for protein synthesis at the genome‐wide scale. Wiley Interdisciplinary Reviews: RNA. 4(5):473-490.