Ribo-Seq or ARTSeq
Ribosome Profiling Sequencing
Ribosome profiling (Ribo-Seq), also called active mRNA translation sequencing (ARTseq), isolates RNA that is being processed by the ribosome in order to monitor the translation process (Ingolia et al., 2009). In this method, ribosome-bound RNA first undergoes digestion. The RNA is extracted, and the rRNA is depleted. The extracted RNA is reverse-transcribed to cDNA. Deep sequencing of the cDNA provides the sequences of RNAs bound by ribosomes during translation. This method has been refined to improve both the qualitative and quantitative nature of the results. Careful attention should be paid to: 1) generation of cell extracts in which ribosomes have been faithfully halted along the mRNA they are translating in vivo; 2) nuclease digestion of RNAs that are not protected by the ribosome, followed by recovery of the ribosome-protected mRNA fragments; and 3) quantitative conversion of the protected RNA fragments into a DNA library that can be analyzed by deep sequencing. The addition of harringtonine (an alkaloid that inhibits protein biosynthesis) causes ribosomes to accumulate precisely at initiation codons and assists in their detection.
Advantages:
- Reveals a snapshot with the precise location of ribosomes on the RNA
- More closely reflects the rate of protein synthesis than mRNA levels
- No prior knowledge of the RNA or open-reading frames (ORFs) is required
- Surveys the whole genome
- Can identify protein-coding regions
Disadvantages:
- Initiation from multiple sites within a single transcript makes it challenging to define all ORFs
- Does not provide the kinetics of translational elongation
Reagents:
Illumina Library prep and Array Kit Selector
Reviews:
References:
Zur H., Aviner R. and Tuller T. Complementary Post Transcriptional Regulatory Information is Detected by PUNCH-P and Ribosome Profiling. Sci Rep. 2016;6:21635
Arribere J. A., Cenik E. S., Jain N., et al. Translation readthrough mitigation. Nature. 2016;534:719-723
Jeong Y., Kim J. N., Kim M. W., et al. The dynamic transcriptional and translational landscape of the model antibiotic producer Streptomyces coelicolor A3(2). Nat Commun. 2016;7:11605
Nissley D. A., Sharma A. K., Ahmed N., et al. Accurate prediction of cellular co-translational folding indicates proteins can switch from post- to co-translational folding. Nat Commun. 2016;7:10341
Bennett C. G., Riemondy K., Chapnick D. A., Bunker E., Liu X., et al. Genome-wide analysis of Musashi-2 targets reveals novel functions in governing epithelial cell migration. Nucleic Acids Res. 2016;44:3788-3800
Dar D., Shamir M., Mellin J. R., et al. Term-seq reveals abundant ribo-regulation of antibiotics resistance in bacteria. Science. 2016;352:aad9822
Gawron D., Ndah E., Gevaert K. and Van Damme P. Positional proteomics reveals differences in N-terminal proteoform stability. Mol Syst Biol. 2016;12:858
Lin L., Jiang P., Park J. W., et al. The contribution of Alu exons to the human proteome. Genome Biol. 2016;17:15
Olexiouk V., Crappe J., Verbruggen S., Verhegen K., Martens L. and Menschaert G. sORFs.org: a repository of small ORFs identified by ribosome profiling. Nucleic Acids Res. 2016;44:D324-329
Sin C., Chiarugi D. and Valleriani A. Quantitative assessment of ribosome drop-off in E. coli. Nucleic Acids Res. 2016;44:2528-2537
Singh A. R., Sivadas A., Sabharwal A., et al. Chamber Specific Gene Expression Landscape of the Zebrafish Heart. PLoS One. 2016;11:e0147823
Tichon A., Gil N., Lubelsky Y., et al. A conserved abundant cytoplasmic long noncoding RNA modulates repression by Pumilio proteins in human cells. Nat Commun. 2016;7:12209
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History: Ribo-Seq or ARTSeq
Revision by sbrumpton on 2017-06-21 07:50:25 - Show/Hide
Ribosome Profiling Sequencing
Ribosome profiling (Ribo-Seq), also called active mRNA translation sequencing (ARTseq), isolates RNA that is being processed by the ribosome in order to monitor the translation process (Ingolia et al., 2009). In this method, ribosome-bound RNA first undergoes digestion. The RNA is extracted, and the rRNA is depleted. The extracted RNA is reverse-transcribed to cDNA. Deep sequencing of the cDNA provides the sequences of RNAs bound by ribosomes during translation. This method has been refined to improve both the qualitative and quantitative nature of the results. Careful attention should be paid to: 1) generation of cell extracts in which ribosomes have been faithfully halted along the mRNA they are translating in vivo; 2) nuclease digestion of RNAs that are not protected by the ribosome, followed by recovery of the ribosome-protected mRNA fragments; and 3) quantitative conversion of the protected RNA fragments into a DNA library that can be analyzed by deep sequencing. The addition of harringtonine (an alkaloid that inhibits protein biosynthesis) causes ribosomes to accumulate precisely at initiation codons and assists in their detection.
Advantages:- Reveals a snapshot with the precise location of ribosomes on the RNA
- More closely reflects the rate of protein synthesis than mRNA levels
- No prior knowledge of the RNA or open-reading frames (ORFs) is required
- Surveys the whole genome
- Can identify protein-coding regions
Disadvantages:- Initiation from multiple sites within a single transcript makes it challenging to define all ORFs
- Does not provide the kinetics of translational elongation
Reagents:Illumina Library prep and Array Kit SelectorReviews:References:Zur H., Aviner R. and Tuller T. Complementary Post Transcriptional Regulatory Information is Detected by PUNCH-P and Ribosome Profiling. Sci Rep. 2016;6:21635Arribere J. A., Cenik E. S., Jain N., et al. Translation readthrough mitigation. Nature. 2016;534:719-723Jeong Y., Kim J. N., Kim M. W., et al. The dynamic transcriptional and translational landscape of the model antibiotic producer Streptomyces coelicolor A3(2). Nat Commun. 2016;7:11605Nissley D. A., Sharma A. K., Ahmed N., et al. Accurate prediction of cellular co-translational folding indicates proteins can switch from post- to co-translational folding. Nat Commun. 2016;7:10341Bennett C. G., Riemondy K., Chapnick D. A., Bunker E., Liu X., et al. Genome-wide analysis of Musashi-2 targets reveals novel functions in governing epithelial cell migration. Nucleic Acids Res. 2016;44:3788-3800Dar D., Shamir M., Mellin J. R., et al. Term-seq reveals abundant ribo-regulation of antibiotics resistance in bacteria. Science. 2016;352:aad9822Gawron D., Ndah E., Gevaert K. and Van Damme P. Positional proteomics reveals differences in N-terminal proteoform stability. Mol Syst Biol. 2016;12:858Lin L., Jiang P., Park J. W., et al. The contribution of Alu exons to the human proteome. Genome Biol. 2016;17:15Olexiouk V., Crappe J., Verbruggen S., Verhegen K., Martens L. and Menschaert G. sORFs.org: a repository of small ORFs identified by ribosome profiling. Nucleic Acids Res. 2016;44:D324-329Sin C., Chiarugi D. and Valleriani A. Quantitative assessment of ribosome drop-off in E. coli. Nucleic Acids Res. 2016;44:2528-2537Singh A. R., Sivadas A., Sabharwal A., et al. Chamber Specific Gene Expression Landscape of the Zebrafish Heart. PLoS One. 2016;11:e0147823Tichon A., Gil N., Lubelsky Y., et al. A conserved abundant cytoplasmic long noncoding RNA modulates repression by Pumilio proteins in human cells. Nat Commun. 2016;7:12209