Ribosome sequencing (Ribo-seq), also known as ribosome profiling, is a technique that sequences ribosome-protected mRNA fragments to monitor the process of translation.
Ribo-seq is also referred to as active mRNA translation sequencing (ART-seq) or global translation initiation sequencing (GTI-seq).
It identifies all active ribosomes in a cell at a specific moment, providing insights into which proteins are being produced.
This method offers detailed information about how ribosomes translate mRNAs into proteins, giving a clearer understanding of gene expression than traditional RNA sequencing.
Traditional RNA sequencing only measures the quantity of mRNA present in a cell and does not reveal whether those mRNAs are being translated into proteins.
The presence of mRNA in a cell does not necessarily indicate active protein synthesis.
Ribo-seq specifically targets ribosome-protected mRNAs that are actively undergoing translation.
Understanding the process of translation is crucial for comprehending gene expression and cellular functions.
During translation, ribosomes read mRNA sequences in codons (sets of three nucleotides) and assemble the corresponding amino acids into a protein chain.
Ribo-seq is a powerful method for studying protein synthesis and gene regulation with greater precision compared to traditional approaches.
This technique is valuable in many areas of biological research.
Historical Developments of Ribosome Sequencing
Ribosome sequencing (Ribo-seq) was first introduced in 2009 by Nicholas Ingolia and Jonathan Weissman.
Their study demonstrated that sequencing ribosome-protected mRNA fragments could provide a detailed understanding of translation in living cells.
Traditional methods used before Ribo-seq lacked the precision needed to accurately determine ribosome positions, translation rates, and protein synthesis activity.
Ribo-seq addressed these limitations by offering more accurate and high-resolution insights into active translation processes.
Advancements in library preparation techniques and data analysis methods have further improved the accuracy and effectiveness of Ribo-seq.
Ribo-seq has been integrated with other datasets, such as transcriptomics and proteomics, to provide a more comprehensive view of gene regulation and protein synthesis.
Traditional ribosome profiling methods cannot detect differences between individual cells, which is a limitation because ribosomes vary between cell types and can influence which mRNAs are translated.
To study translation in individual cells, researchers developed single-cell ribosome profiling using a technique known as dual-ligation.
For profiling translation in small cell samples, a low-input ribosome profiling method has been developed using the template-switch technique, enabling analysis with as few as a few hundred cells.
Innovative methods like RIBOMap have been introduced to further enhance single-cell Ribo-seq by increasing mRNA capture efficiency.
RIBOMap was specifically designed to overcome the challenge of translation changes and cell stress caused by cell isolation, allowing researchers to study translation without needing to isolate individual cells.
Principle of Ribosome Sequencing
Ribo-seq works on the principle that actively translating ribosomes protect specific regions of mRNA from degradation by ribonuclease enzymes.
These protected mRNA regions are known as ribosome footprints, and their presence indicates areas of active translation.
The number of ribosome footprints directly reflects the level of active protein synthesis occurring in the cell.
By identifying ribosome footprints, Ribo-seq enables the detection of actively translating ribosomes, which is essential for studying protein synthesis.
Sequencing these ribosome-protected fragments allows precise mapping of ribosome positions on mRNAs and provides detailed insights into translation activity.
The process begins by freezing ribosomes in place and treating cells with ribonucleases to digest unprotected mRNA regions, isolating only the ribosome-protected fragments.
These isolated mRNA fragments are then converted into cDNA libraries that are compatible with next-generation sequencing platforms.
The resulting sequencing data is analyzed to study translation dynamics at high resolution.
Process of Ribosome Sequencing
1. Cell Lysis
Cells are first lysed to release mRNA molecules bound to ribosomes. This can be achieved using detergents, mechanical disruption, or enzymatic digestion.
To preserve ribosome positions during lysis, translation is halted using chemicals like cycloheximide or by rapidly freezing the cells with liquid nitrogen.
The resulting extract contains ribosomes, mRNA, proteins, and other cellular components.
2. Ribonuclease Digestion
The lysate is treated with ribonuclease (RNase) enzymes to digest all RNA not protected by ribosomes. This leaves behind only the ribosome-protected mRNA fragments.
Enzymes like RNase I or micrococcal nuclease (MNase) are commonly used to selectively degrade unprotected RNA, ensuring that only actively translated mRNA fragments remain for analysis.
3. RNA Isolation
After digestion, the mRNA-ribosome complexes are separated from the rest of the cellular material using centrifugation or chromatography.
The protected mRNA fragments, known as ribosome footprints, are then size-selected—typically around 28–30 nucleotides in length—using polyacrylamide gel electrophoresis for purification.
4. rRNA Depletion
Even after purification, the sample still contains abundant ribosomal RNA (rRNA), which must be removed to focus on the ribosome-protected mRNA fragments.
This is done using biotinylated oligonucleotides that bind rRNA, followed by removal with streptavidin-coated magnetic beads.
5. Library Preparation
The isolated RNA fragments are converted into complementary DNA (cDNA) through reverse transcription.
The resulting cDNA is then amplified by PCR to generate sufficient material for sequencing.
6. Sequencing
The prepared cDNA library is sequenced using next-generation sequencing technologies to produce short reads corresponding to ribosome footprints.
7. Data Analysis
The sequencing data is processed to identify which mRNAs are actively being translated.
This includes steps such as quality control, removal of adapter sequences and rRNA contaminants, sequence alignment, and visualization of ribosome footprints to analyze gene regulation and translation dynamics.
Advantages of Ribosome Sequencing
Ribo-seq provides a better understanding of gene regulation by identifying mRNAs that are actively undergoing translation.
It can detect previously unknown proteins, small peptides, and alternative isoforms of known proteins.
The method offers higher accuracy for studying protein synthesis by revealing the exact locations of ribosomes on mRNA, enabling precise analysis of active translation.
It is especially useful for identifying low-level translation activity that might be missed by other methods.
Ribo-seq does not require prior knowledge of RNA sequences, making it effective for discovering novel coding regions.
Compared to traditional RNA-seq, Ribo-seq offers more detailed and focused information by targeting only mRNAs that are actively being translated.
Limitations of Ribosome Sequencing
Ribo-seq generates large volumes of data, making data analysis complex and requiring advanced bioinformatics tools for accurate interpretation.
It demands high-quality RNA and carefully prepared cell extracts to ensure reliable results.
The method typically requires large amounts of RNA, making it less suitable for low-input or limited sample sizes.
Although sensitive, Ribo-seq may struggle to detect low-abundance mRNAs with few ribosomes bound, potentially missing subtle translation events.
The RNase enzymes used can have sequence preferences, which may introduce bias and affect the accuracy of translation analysis.
The procedure involves multiple labor-intensive steps such as RNA extraction, enzymatic digestion, and library preparation.
Ribo-seq requires specialized equipment and is more expensive than many other gene expression analysis methods.
Applications of Ribosome Sequencing
Ribo-seq is used to identify mRNAs that are actively being translated by ribosomes, offering a detailed view of gene expression at the translational level.
It allows observation of how newly synthesized proteins fold during translation.
The method can be used to discover novel proteins and alternative isoforms of known proteins.
Ribo-seq provides information about translation initiation sites, showing where ribosomes begin synthesizing proteins on mRNA.
It detects locations on mRNA where ribosomes temporarily pause during translation, which can influence protein folding and gene regulation.
It is used to monitor protein production and predict protein abundance in cells.
Ribo-seq has applications in identifying translation-related changes in diseases by comparing translation activity between healthy and diseased tissues.
References
University of Geneva. (2024, April 23). Analysis of the translatome by Ribo-Seq (ribosome profiling). Retrieved from https://www.unige.ch/medecine/ppr2p-platforms/biocode-rna-proteins/services/analysis-translatome-ribosome-profiling-ribo-seq
Bagheri, A., Astafev, A., Al-Hashimy, T., & Jiang, P. (2022). Tracing translational footprints with Ribo-Seq: Principles, workflow, and applications for understanding human diseases. Cells, 11(19), 2966. https://doi.org/10.3390/cells11192966
Brar, G. A., & Weissman, J. S. (2015). Ribosome profiling reveals the timing, location, and mechanisms of protein synthesis. Nature Reviews Molecular Cell Biology, 16(11), 651–664. https://doi.org/10.1038/nrm4069
Ingolia, N. T., Hussmann, J. A., & Weissman, J. S. (2018). Ribosome profiling: Global perspectives on translation. Cold Spring Harbor Perspectives in Biology, 11(5), a032698. https://doi.org/10.1101/cshperspect.a032698
Power, L. (2022). A beginner’s guide to ribosome profiling. The Biochemist, 44(2), 30–34. https://doi.org/10.1042/bio_2021_196
CD Genomics. (n.d.). Ribosome profiling (Ribo-Seq). Retrieved from https://www.cd-genomics.com/ribosome-profiling.html
Illumina. (n.d.). Ribosome profiling | Ribo-Seq/ART-Seq for ribosome-protected mRNA. Retrieved from https://www.illumina.com/techniques/sequencing/rna-sequencing/ribosome-profiling.html
Wang, Q., & Mao, Y. (2023). Ribosome profiling: Principles, challenges, and recent advances from bulk to single-cell analysis. Advanced Biotechnology, 1(4). https://doi.org/10.1007/s44307-023-00006-4
CD Genomics. (2022, February 23). What is Ribo-Seq (ribosome footprinting)? Retrieved from https://www.cd-genomics.com/blog/what-is-ribo-seq-ribosome-footprinting/
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