Ribosome profiling
Ribosome profiling, or Ribo-Seq, is a technique developed by Nick Ingolia and Jonathan Weissman that uses specialized messenger RNA (mRNA) sequencing to determine which mRNAs are being actively translated.[1] It produces a “global snapshot” of all the ribosomes active in a cell at a particular moment, known as a translatome. Consequently, this enables researchers to identify the location of translation start sites, the complement of translated ORFs in a cell or tissue, the distribution of ribosomes on a messenger RNA, and the speed of translating ribosomes.[2] Ribosome profiling involves similar sequencing library preparation and data analysis to RNA-Seq, but unlike RNA-Seq, which sequences all of the mRNA of a given sequence present in a sample, ribosome profiling targets only mRNA sequences protected by the ribosome during the process of decoding by translation.[1]
History
Ribosome profiling is based on the discovery that the mRNA within a ribosome can be isolated through the use of nucleases that degrade unprotected mRNA regions. This technique analyzes the regions of mRNAs being converted to protein, as well as the levels of translation of each region to provide insight into global gene expression. Prior to its development, efforts to measure translation in vivo included microarray analysis on the RNA isolated from polysomes, as well as translational profiling through the affinity purification of epitope tagged ribosomes. These are useful and complementary methods, but neither allows the sensitivity and positional information provided by ribosome profiling.[2]
Procedure
- Lyse the cells or tissue and isolate the mRNA molecules bound to ribosomes.
- Immobilize complexes. This is commonly performed with cycloheximide but other chemicals can be employed. It is also possible to forgo translation inhibitors with translation-incompetent lysis conditions.
- Using ribonucleases, digest the RNA not protected by ribosomes.
- Isolate the mRNA-ribosome complexes using sucrose gradient density centrifugation or specialized chromatography columns.
- Phenol/chloroform purification of mixture to remove proteins.
- Size-select for previously-protected mRNA fragments.
- Ligate 3' adapter to fragments.
- Subtract known rRNA contaminants (optional).
- Reverse transcribe RNA to cDNA using reverse transcriptase.
- Amplify in strand-specific manner.
- Sequence reads.
- Align sequence results to genomic sequence to determine translational profile.[3]
Materials
- RNA-ribosome complexes
- Cycloheximide
- Nucleases
- Phenol/Chloroform
- Reverse transcriptase
- dNTPs
- Sequencing method-cDNA library.[3]
References
- 1 2 Ingolia, Nicholas T. (28 January 2014). "Ribosome profiling: new views of translation, from single codons to genome scale". Nature Reviews Genetics 15 (3): 205–213. doi:10.1038/nrg3645. PMID 24468696.
- 1 2 Weiss, R. B.; J. F. Atkins (2011). "Translation Goes Global". Science 334 (6062): 1509–1510. doi:10.1126/science.1216974. ISSN 0036-8075.
- 1 2 Ingolia, N. T.; S. Ghaemmaghami; J. R. S. Newman; J. S. Weissman (2009). "Genome-Wide Analysis in Vivo of Translation with Nucleotide Resolution Using Ribosome Profiling". Science 324 (5924): 218–223. doi:10.1126/science.1168978. ISSN 0036-8075. PMC 2746483. PMID 19213877.