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Mastering RIBO-seq: going beyond transcription by measuring translation

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Most of our current knowledge about gene regulation is based on studying mRNA expression, despite both the greater functional importance of protein abundance and evidence that post-transcriptional regulation is pervasive. Ribosome profiling is a high throughput technique that allows to measure translation of mRNA. In this way, measuring protein synthesis closes the gap to measuring protein abundance, routinely done with MS-based proteomics methods. Ribosome profiling allows to reveal translated regions, quantify protein synthesis and resolves ribosome footprints towards translation initiation, elongation and/or stalling.

What is RIBO-seq (Ribosome Profiling)?

RIBO-seq, also known as ribosome profiling or ribosome sequencing, is a specialized sequencing technique used to study the translation of mRNA into proteins. It provides a snapshot of the actively translated regions of the transcriptome by capturing the positions of ribosomes on mRNA molecules. RIBO-seq allows researchers to gain insights into the dynamics of translation, ribosome occupancy, and translational regulation at a genome-wide scale.



The RIBO-seq workflow involves the following steps:

1. Sample Preparation
Cells or tissues are treated with a translation inhibitor, such as cycloheximide, or immediately snap frozen, to freeze the ribosomes in their positions on the mRNA molecules. This ensures the capture of ribosome-protected fragments (RPFs) that represent the regions being actively translated.



2. Ribosome Capture and RNA Extraction
The cells or tissues are lysed, and the lysate is treated with RNase enzymes to digest unprotected RNA fragments. Ribosomes and their associated mRNA fragments (RPFs) are isolated using techniques like sucrose gradient centrifugation or ribosome immunoprecipitation.



3. Library Preparation
The isolated RPFs are converted into a sequencing library by adding adapters to their ends. This involves steps such as reverse transcription, adapter ligation, and PCR amplification.



4. Sequencing
The prepared RIBO-seq library is sequenced using next-generation sequencing (NGS) platforms. The sequencing generates short reads representing the RPFs derived from the ribosome-bound mRNA fragments.



5. Data Analysis
The RIBO-seq data analysis involves aligning the sequenced reads to a reference genome or transcriptome to determine their origin and mapping positions. The read distribution and density along mRNA molecules indicate the regions undergoing active translation. Additional bioinformatics analyses, such as quantification of translation efficiency, identification of translation initiation sites, and analysis of ribosome dynamics, can be performed to extract biological insights.



Significance of RIBO-seq:

1. Translational Regulation
RIBO-seq provides information about ribosome occupancy on mRNA molecules, allowing the identification of translationally active regions and differentially translated genes. It helps uncover translational regulatory mechanisms influencing protein synthesis and post-transcriptional gene regulation.



2. Translation Initiation Sites
RIBO-seq enables the identification of translation initiation sites, providing insights into the precise locations where ribosomes begin protein synthesis. This information aids in the characterization of alternative translation initiation events, upstream open reading frames (uORFs), and the impact of sequence features on translation initiation efficiency.



3. Translational Dynamics and Efficiency
By comparing RIBO-seq data with corresponding mRNA-seq data, researchers can analyze translation efficiency, ribosome pausing, and ribosome release rates. This allows the study of translational kinetics, co-translational protein folding, and the impact of regulatory elements on translation dynamics.



4. Translational Regulation in Disease
RIBO-seq has applications in understanding translational dysregulation in various diseases, including cancer, neurodegenerative disorders, and viral infections. It helps uncover alterations in translation patterns, identify disease-specific translational signatures, and explore therapeutic targets related to translation control.

RIBO-seq provides a valuable tool for investigating the translation process and deciphering the complexities of gene regulation at the protein synthesis level. It offers insights into the spatial and temporal aspects of translation, expanding our understanding of gene expression and its regulation in health and disease.

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What are the advantages of ribosome profiling?

Study Translational Control

Ribosome profiling allows researchers to investigate how gene expression is regulated at the translational level. It provides information about the positions of ribosomes along mRNA molecules, revealing regions undergoing active translation. By comparing ribosome occupancy between different conditions or cell types, researchers can identify genes and pathways subject to translational control.

Uncover Translational Regulation Mechanisms

Ribosome profiling helps uncover the mechanisms underlying translational regulation. It provides information about translation initiation sites, ribosome pausing, and ribosome release rates. These data can shed light on cis-regulatory elements, trans-acting factors, and RNA structures that influence translation efficiency and dynamics.

Assess Translational Efficiency

Ribosome profiling enables the quantification of translation efficiency by comparing ribosome footprint densities with mRNA expression levels. It provides insights into the relationship between mRNA abundance and the number of ribosomes engaged in translation. This information is essential for understanding the efficiency of protein synthesis and identifying factors that modulate translation rates.

Identify Translational Start Sites and Alternative Reading Frames

Ribosome profiling data can be used to accurately determine translation start sites, which are crucial for accurate annotation of protein-coding genes. Additionally, it helps identify alternative reading frames and upstream open reading frames (uORFs) that play a role in translational regulation, protein isoform diversity, and cellular stress responses.

Study Codon Usage and Translation Efficiency

Ribosome profiling data allows researchers to investigate codon usage biases and their impact on translation efficiency. By analyzing ribosome occupancy patterns at different codons, researchers can gain insights into codon preferences, ribosome stalling, and the effects of codon usage on protein production and folding.

Investigate Translational Regulation in Disease

Ribosome profiling has significant applications in understanding translational dysregulation in various diseases. It helps identify altered translation patterns, uncover disease-specific translational signatures, and explore the impact of mutations or genetic variations on translation efficiency. Such insights can contribute to understanding disease mechanisms and discovering potential therapeutic targets.

Integration with Other Omics Data

Ribosome profiling data can be integrated with other omics data, such as RNA-seq and proteomics, to gain a comprehensive understanding of gene expression regulation. This integration enables researchers to examine the relationships between transcriptional activity, translational efficiency, and protein abundance, providing a more holistic view of cellular processes.

Ribosome profiling provides a unique and powerful approach to investigate translational control and unravel the intricacies of gene expression regulation. It offers insights into the dynamics of protein synthesis, translational regulatory mechanisms, and their relevance in normal cellular processes and disease conditions.

Ribosome profiling workflow

The ribosome profiling workflow

Ribosome profiling relies on deep sequencing of ribosome footprints—these short (typically, ∼30 nucleotides) fragments of mRNA that are physically enclosed by the ribosome and shielded from nuclease digestion.

These footprints are converted into a library of DNA fragments and analyzed by one of our Illumina sequencers. The technical quality of the sequencing run is monitored in real-time.
Each sequenced footprint reports on the position of one ribosome, revealing what transcript that ribosome was translating and where along the coding sequence it was captured during cell lysis, often with single-nucleotide resolution.

Current deep-sequencing technologies analyze hundreds of millions of individual short reads in one experiment. When applied to libraries of ribosome footprints, this sequencing yields a comprehensive view of the translational landscape that can address many fundamental questions about translations.
Several specific antibiotic treatments during sample preparation result in genome-wide maps of elongating and initiating ribosomes to help delineate the ORF by also capturing the translation initiation events.

We request between 25 – 50 million cells per sample and provide a flash-frozen cell pellet. We perform cell lysis ourselves.

Raw sequencing data can be transferred to you via the server (sFTP download).

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What is the difference between RNA-seq and RIBO-seq?

RNA-seq and RIBO-seq are two distinct sequencing techniques used in molecular biology to study different aspects of RNA molecules. Here’s a breakdown of the differences between the two:

RNA-seq (RNA sequencing):

Purpose: RNA-seq is primarily used to measure the abundance and identify the different types of RNA molecules present in a sample.

Focus: It provides a comprehensive snapshot of the transcriptome, including all types of RNA such as messenger RNA (mRNA), non-coding RNA, and small RNA molecules.

Methodology: RNA-seq involves isolating and sequencing the entire population of RNA molecules present in a sample, followed by computational analysis to determine the sequence and quantity of RNA molecules.

Key applications: Gene expression analysis, identification of novel transcripts, alternative splicing analysis, transcriptome profiling, differential gene expression analysis, etc.

RIBO-seq (ribosome profiling sequencing):

Purpose: RIBO-seq is specifically designed to study the activity of ribosomes on mRNA molecules, providing insights into translation dynamics and protein synthesis.

Focus: It captures the ribosome-protected fragments (RPFs) of mRNA molecules, which represent the positions where ribosomes are actively translating.

Methodology: RIBO-seq involves treating cells or tissues with ribosome-stalling drugs, followed by nuclease digestion to isolate ribosome-protected mRNA fragments. These fragments are then subjected to sequencing and subsequent analysis to determine ribosome occupancy and translation efficiency.

Key applications: Identification of translated ORFs (open reading frames), characterization of translated regions, quantification of translation efficiency, investigation of translational regulation, ribosome profiling of specific cell types or conditions, etc.

In summary, RNA-seq provides a global overview of RNA molecules present in a sample, whereas RIBO-seq specifically focuses on the ribosome-associated mRNA fragments, enabling the study of translation and protein synthesis.

How can RIBO-seq help to explain drug mode-of-action (MOA)?

RIBO-seq, or ribosome profiling sequencing, can provide valuable insights into drug mechanisms of action (MOA) by examining how drugs affect translation dynamics and protein synthesis.
Here are a few ways in which RIBO-seq can contribute to understanding drug MOA:

Identifying direct targets

RIBO-seq can help identify the specific mRNA targets that are affected by a drug. By comparing RIBO-seq profiles of treated and untreated cells, researchers can determine which mRNA molecules show altered ribosome occupancy or translation efficiency upon drug treatment. These differentially regulated transcripts may represent direct targets of the drug.

Uncovering off-target effects

RIBO-seq can reveal unintended off-target effects of drugs. By analyzing RIBO-seq data from cells treated with different drugs, researchers can identify transcripts that exhibit altered translation profiles common to multiple drugs. This analysis can help identify potential off-target effects or shared targets among different drugs.

Characterizing translational regulation

RIBO-seq provides information about translation dynamics, such as translation initiation and elongation rates. By comparing RIBO-seq profiles between drug-treated and control samples, researchers can gain insights into how a drug affects translation regulation. Changes in ribosome occupancy patterns or translation efficiency can indicate alterations in specific steps of translation, providing clues about the drug’s MOA.

Assessing drug-induced stress responses

Some drugs can induce cellular stress responses, affecting translation and protein synthesis. RIBO-seq can help characterize these stress responses by analyzing changes in ribosome occupancy and translation efficiency in response to drug treatment. This information can shed light on how drugs impact cellular processes and signaling pathways.

Predicting drug targets and pathways

Integrating RIBO-seq data with other omics data, such as transcriptomics and proteomics, can provide a comprehensive view of drug effects. By correlating changes in ribosome occupancy with changes in gene expression or protein abundance, researchers can identify drug targets, affected pathways, and potential downstream effects.

Overall, RIBO-seq can offer a mechanistic understanding of drug effects at the translational level, aiding in the identification of drug targets, off-target effects, stress responses, and alterations in cellular processes. This knowledge can contribute to a better understanding of drug MOA and facilitate the development of more effective therapeutic interventions.

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How can Ribosome Profiling help to discover disease targets?

RIBO-seq, or ribosome profiling sequencing, can contribute to the discovery of disease targets by providing insights into dysregulated translation and identifying disease-associated changes in protein synthesis.
Here are some ways RIBO-seq can help in the discovery of disease targets:

Identification of disease-associated transcripts

RIBO-seq can identify specific mRNA transcripts that show altered ribosome occupancy or translation efficiency in disease conditions compared to healthy controls. By comparing RIBO-seq profiles between diseased and healthy samples, researchers can identify transcripts that are differentially translated in the disease state. These dysregulated transcripts may represent potential disease targets.

Characterization of disease-specific translation signatures

RIBO-seq can reveal disease-specific translation signatures by analyzing changes in ribosome occupancy patterns and translation dynamics. By comparing RIBO-seq data from different disease states, researchers can identify commonalities and differences in translation profiles. These disease-specific translation signatures can help uncover key regulatory pathways and potential therapeutic targets.

Identification of disease-associated alternative translation events

RIBO-seq can detect alternative translation events, such as upstream open reading frames (uORFs) and alternative translation initiation sites. Dysregulation of such events has been implicated in various diseases. RIBO-seq can provide information on the translation patterns of these alternative events, aiding in the identification of disease-associated translation alterations and potential therapeutic targets.

Mapping disease-associated mutations affecting translation

RIBO-seq can be used to map the effects of disease-associated mutations on translation. By integrating RIBO-seq data with genomic information, researchers can identify mutations that disrupt translation initiation, elongation, or termination. Understanding the impact of these mutations on protein synthesis can help identify disease targets and pathways affected by the mutations.

Integrating RIBO-seq with other omics data

Integrating RIBO-seq data with other omics data, such as transcriptomics, proteomics, and genomics, can provide a comprehensive view of disease-related molecular changes. By correlating changes in ribosome occupancy with changes in gene expression, protein abundance, and genomic alterations, researchers can uncover disease targets, affected pathways, and potential therapeutic interventions.

Overall, RIBO-seq enables the identification of dysregulated translation events, disease-specific translation signatures, and disease-associated targets, providing valuable insights into the molecular mechanisms underlying diseases. This information can guide the development of targeted therapies and interventions for various diseases.

How can RIBO-seq help in the field of immune therapy?

RIBO-seq can contribute to the field of immune therapy by providing insights into immune cell activation, immune response regulation, and the identification of potential therapeutic targets.
Here are several ways RIBO-seq can be beneficial in the context of immune therapy:

Characterizing immune cell activation

RIBO-seq can help characterize the translational changes occurring during immune cell activation. By profiling ribosome occupancy in immune cells before and after activation, researchers can identify genes and pathways that are translationally upregulated or downregulated. This information can provide a deeper understanding of the molecular events driving immune cell activation and guide the development of targeted therapies.

Identifying immune response regulators

RIBO-seq can aid in the discovery of key regulators of immune responses. By comparing RIBO-seq profiles between different immune cell types or states, researchers can identify differentially translated genes and pathways associated with immune regulation. These findings can uncover potential targets for modulating immune responses, leading to the development of novel immunotherapies.

Investigating immune cell heterogeneity

RIBO-seq can be used to study the translational profiles of specific immune cell subsets or rare cell populations. By isolating and profiling ribosome-protected mRNA fragments from specific cell types, researchers can gain insights into the translational landscape of distinct immune cell populations. This information can help understand the functional differences between immune cell subsets and guide the development of cell-specific therapies.

Identifying immune-related drug targets

RIBO-seq can aid in the identification of potential therapeutic targets for immune-related diseases or conditions. By analyzing ribosome occupancy and translation efficiency in immune cells under different treatment conditions, researchers can identify genes and pathways that are translationally modulated by immune-related drugs or therapies. These targets can be further explored for therapeutic interventions.

Personalizing immune therapy

RIBO-seq can contribute to precision medicine approaches in immune therapy. By profiling ribosome occupancy in patient samples, researchers can identify patient-specific translational profiles and biomarkers associated with therapeutic response or resistance. This information can guide the selection of appropriate immune therapies and aid in treatment decision-making.

Overall, RIBO-seq can provide valuable insights into immune cell activation, immune response regulation, and the identification of therapeutic targets in the field of immune therapy. These findings can contribute to the development of novel immunotherapies and personalized treatment strategies.

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Ribosome Profiling services with

At, we take pride in offering state-of-the-art Ribosome Profiling (RIBO-seq) services.

With over 50 RIBO-seq-related projects and more than 20 publications spanning various fields and applications, our protocols and pipelines have been optimally designed to cater to your specific needs, ensuring precise results and comprehensive insights.

Feel free to reach out to our team with any inquiries or to discuss potential collaborations. Together, we can unlock the full potential of Nanopore sequencing and drive scientific advancements that benefit us all.

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