Ribosome sequencing (Ribo-seq), also known as ribosome profiling, is a method that sequences ribosome-protected mRNA fragments to monitor the process of translation.
Ribo-seq is also known as active mRNA translation sequencing (ART-seq) or global translation initiation sequencing (GTI-seq). It is used to identify all active ribosomes in a cell at a given moment which helps to understand which proteins are being produced. This provides detailed information about how ribosomes translate mRNAs into proteins and helps us understand gene expression better than traditional RNA sequencing.
Traditional RNA sequencing cannot reveal which mRNAs are being translated into proteins. It only measures how much mRNA is present in a cell and just because an mRNA is there does not mean it is being used to make a protein. Unlike traditional methods, Ribo-seq focuses only on the ribosome-protected mRNAs that are being actively translated.
It is important to understand the process of translation to understand gene expression and cellular functions. The process of translation converts mRNAs into proteins and ribosomes are involved in this process. They read the mRNA in sets of three nucleotides (codons) and link the corresponding amino acids to form a protein. So, Ribo-seq is an important method for studying protein synthesis and gene regulation in living cells with greater accuracy than traditional methods. This is useful in many areas of biological research.
Historical Developments of Ribosome Sequencing
- Ribo-seq was first introduced in 2009 by Nicholas Ingolia and Jonathan Weissman. Their study showed that sequencing ribosome-protected mRNA fragments could provide a detailed understanding of translation in living cells.
- Traditional methods for studying translation that were used before Ribo-seq lacked the precision needed to fully understand ribosome positions, translation rates, and protein synthesis.
- Ribo-seq overcomes this limitation by providing more accurate and high-resolution information about active translation.
- Improvements in library preparation and data analysis have further enhanced the accuracy of Ribo-seq.
- Ribo-seq has also been combined with other data including transcriptomics and proteomics to better understand gene regulation and protein synthesis.
- Traditional ribosome profiling cannot detect differences among individual cells. This is important to consider because ribosomes are not identical across different cell types and this can affect which mRNAs get translated.
- So, researchers have developed single-cell ribosome profiling using a method called dual-ligation to study translation in individual cells.
- To improve ribosome sequencing in small cell samples, researchers have also developed a low-input ribosome profiling method using the template-switch method which allows profiling with just a few hundred cells.
- New methods like RIBOMap further improve single-cell Ribo-Seq by increasing mRNA capture. This method was developed to address the problem of cell stress and translation changes when isolating cells. RIBOMap allows translation studies without cell isolation.
Principle of Ribosome Sequencing
Ribo-seq is based on the principle that actively translating ribosomes protect specific regions of mRNA from ribonuclease digestion. These ribosome-protected mRNA fragments are called ribosome footprints and the number of ribosome footprints indicates the level of active translation. This can be used to identify actively translating ribosomes which is useful for studying protein synthesis. When these fragments are sequenced, ribosome positions on mRNAs can be precisely mapped and translation activity can be studied in detail.
Ribo-seq initially involves freezing the ribosomes and treating cells with ribonuclease enzymes to digest unprotected mRNAs and isolate ribosome footprints. These ribosome-protected mRNA fragments are converted into compatible cDNA sequencing libraries and analyzed using next-generation sequencing methods.
Process of Ribosome Sequencing
1. Cell Lysis
Initially, cells are lysed to release mRNA molecules bound to ribosomes. Methods like detergents, mechanical disruption, and enzymatic digestion can be used for cell lysis. It is also necessary to freeze translation to preserve ribosome positions. Chemicals like cycloheximide can be used to stop translation and make the complexes immobile. Cells can also be frozen using liquid nitrogen. After cell lysis, the extract contains a mixture of ribosomes, mRNA, proteins, and other cellular components.
- Ribo-seq provides a better understanding of gene regulation as it identifies mRNAs that are actively being translated.
- It can identify previously unknown proteins, small peptides, and alternative isoforms of known proteins.
- It is more accurate for studying protein synthesis. It can reveal the exact location of ribosomes on mRNA and gives an accurate view of active translation. This is useful for identifying low-level translation activity.
- It does not require prior knowledge of RNA sequences which is useful for discovering novel coding regions.
- Ribo-seq provides more detailed information than traditional RNA-seq as it focuses specifically on mRNA that are being actively translated.
- Ribo-seq generates large amounts of data so data analysis can be challenging. It requires advanced bioinformatics tools to handle and interpret the data accurately.
- It requires high-quality RNA and well-prepared cell extracts. It also requires large RNA amounts which makes it unsuitable for low-input samples.
- It is sensitive but can face challenges when detecting low-abundance mRNAs that have fewer ribosomes bound to them.
- RNase used in the process favors certain sequences and may affect translation analysis.
- It involves labor-intensive steps like RNA extraction, digestion, and library preparation. This requires advanced equipment and is expensive compared to other methods.
- Ribo-seq is used to identify mRNAs that are actively being translated by ribosomes. This provides a detailed view of gene expression at the translational level.
- This method can be used to observe how newly synthesized proteins fold.
- It can be used to discover novel proteins and isoforms.
- Ribo-seq also provides information about translation initiation sites where ribosomes start protein synthesis on mRNA.
- It also detects locations on mRNA where ribosomes temporarily pause during translation which can affect protein folding and gene regulation.
- It is used to track protein production and predict protein abundance in cells.
- Ribo-seq also has applications in identifying translation-related changes in diseases by comparing translation process in healthy and diseased tissues.
- Analysis of translatome by Ribo-Seq (Ribosome Profiling). (2024, April 23). 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 Footprint by Ribo-SEq: Principle, workflow, and applications to understand the mechanism of human diseases. Cells, 11(19), 2966. https://doi.org/10.3390/cells11192966
- Brar, G. A., & Weissman, J. S. (2015). Ribosome profiling reveals the what, when, where and how 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 Views of Translation. Cold Spring Harbor Perspectives in Biology, 11(5), a032698. https://doi.org/10.1101/cshperspect.a032698
- Power, L. (2022). Beginners guide to ribosome profiling. The Biochemist, 44(2), 30–34. https://doi.org/10.1042/bio_2021_196
- Ribosome Profiling (Ribo-Seq) – CD Genomics. (n.d.). Retrieved from https://www.cd-genomics.com/ribosome-profiling.html
- Ribosome Profiling | Ribo-Seq/ART-Seq for ribosome-protected mRNA. (n.d.). Retrieved from https://www.illumina.com/techniques/sequencing/rna-sequencing/ribosome-profiling.html
- Wang, Q., & Mao, Y. (2023). Principles, challenges, and advances in ribosome profiling: from bulk to low-input and single-cell analysis. Advanced Biotechnology, 1(4). https://doi.org/10.1007/s44307-023-00006-4
- What is Ribo-Seq (Ribosome footprinting)? | CD Genomics blog. (2022, February 23). Retrieved from https://www.cd-genomics.com/blog/what-is-ribo-seq-ribosome-footprinting/
2. Ribonuclease Digestion
Then, the sample is treated with ribonuclease (RNase) enzymes to digest unprotected RNA, leaving only the fragments bound by ribosomes. This step isolates ribosome-protected mRNA fragments. The most commonly used enzymes are RNase I or micrococcal nuclease (MNase) which selectively degrades RNA molecules not protected by ribosomes. This ensures only actively translated mRNA fragments are analyzed.
3. RNA Isolation
After digestion, the mRNA-ribosome complex is separated from other cellular components by using methods like centrifugation or chromatography. Then, mRNA fragments are size-selected and purified. Ribosome footprints are around 28-30 nucleotides in length. Polyacrylamide gel electrophoresis is used for size selection to isolate RNA fragments within this range.
4. rRNA Depletion
The purified ribosome footprints still contain various RNA molecules including the abundant ribosomal RNA (rRNA) which need to be removed to focus only on the mRNA footprints. rRNAs can be removed by using biotinylated oligos and streptavidin-coated magnetic beads.
5. Library Preparation
The extracted RNA is converted into a library of complementary DNA (cDNA) using reverse transcription. Then, the cDNA is amplified by PCR to generate enough material for sequencing.
6. Sequencing
The cDNA library is sequenced using next-generation sequencing methods to generate reads containing ribosome footprints.
7. Data Analysis
The sequencing data is analyzed to determine which mRNAs are being actively translated and to obtain detailed information about gene regulation and the translation process. The general steps involved in data analysis include quality control, trimming adapter sequences and rRNA contaminants, sequence alignment, and visualizing ribosome footprints for further analysis.
Advantages of Ribosome Sequencing
Limitations of Ribosome Sequencing
Applications of Ribosome Sequencing
References
