Ribosome Complex Profiling Service
Are you currently facing challenges in accurately quantifying protein synthesis, deciphering complex translational control mechanisms, or identifying novel therapeutic targets hidden within the translatome? Our ribosome complex profiling service at Creative Biolabs helps you gain unprecedented insights into active translation events by precisely mapping ribosome positions on mRNA, utilizing advanced nuclease footprinting techniques coupled with high-throughput sequencing.
Introduction of Ribosome Complex Profiling
Ribosome complex profiling, often termed Ribo-seq or translation complex profiling, is a powerful technique that provides a global snapshot of active protein synthesis within a cell at a specific moment. By sequencing mRNA fragments protected by translating ribosomes, it reveals not just which genes are expressed, but how actively they are being translated into proteins. This method allows for precise mapping of translation start and stop sites, identification of novel translated regions, and quantification of ribosome density, offering deeper insights than transcriptomics alone into the dynamics of gene expression, including initiation, elongation, and termination phases.
Fig.1 Biological processes and functional pathway senriched in the differentially translated gene sidentified by different methods.1
Simplified Process
Our workflow is designed for clarity and robust results, providing you with actionable data.
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Sample Preparation & Lysis
Cells or tissues are gently lysed under conditions that preserve ribosome-mRNA complexes. Specific inhibitors are used to halt ribosome movement, capturing a snapshot of active translation. -
Nuclease Footprinting
Optimized concentrations of RNase are used to digest mRNA regions not protected by ribosomes. This step precisely trims away exposed RNA, leaving behind ribosome-protected fragments (RPFs).
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Ribosome Isolation & RPF Purification
Ribosome complexes (monosomes) containing the RPFs are isolated, typically via sucrose gradient ultracentrifugation or size-exclusion chromatography. The RPFs are then carefully extracted from the ribosomes.
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Library Preparation & Deep Sequencing
The RPFs are transformed into a cDNA library that is compatible with next-generation sequencing (NGS). This process entails adapter ligation, reverse transcription, and PCR amplification. The library is then sequenced to generate millions of reads.
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Data Analysis & InterpretationSequencing reads are quality-controlled, adapters trimmed and aligned to the relevant reference genome or transcriptome. Advanced bioinformatics analyses are performed to map footprint density, identify translated open reading frames (ORFs), quantify ribosome occupancy, and, if RNA-seq data is available, calculate translational efficiency.
Final Deliverables
- Raw and Processed Sequencing Data: Including FASTQ files and alignment files.
- Quantitative Ribosome Footprint Maps: Genome-wide visualization of ribosome occupancy and identification of translated ORFs.
- Differential Translation Analysis Report: Statistical analysis identifying genes with significant changes in translation efficiency between experimental conditions, often correlated with matched transcriptomic data.
Applications
Ribosome complex profiling offers a wide array of applications across various biological disciplines.
- Drug Discovery and Development
Ribosome complex profiling is pivotal in drug discovery. It helps identify molecular targets of drugs affecting translation, offering insights into drug-cell interactions. It also assesses how drug candidates impact global and specific protein synthesis, aiding in efficacy and safety evaluations. Moreover, it uncovers translational reprogramming-related-resistance mechanisms, crucial for improving long-term drug effectiveness.
- Understanding Disease Mechanisms
This technique sheds light on disease mechanisms. It reveals how translation dysregulation contributes to diseases like viral infections, neurodegenerative disorders, and cancer. In cancer, abnormal translation may spur oncoprotein production, driving tumor growth. In neurodegenerative conditions, misfolded proteins from dysregulated translation are key factors. Understanding these links aids in disease diagnosis and treatment development.
- Virology Research
In virology, ribosome complex profiling is invaluable. It examines how viruses hijack the host's translation machinery for replication, often at the expense of normal host functions. It also explores host-cell translational responses to viral infection. By studying these, researchers can find antiviral targets and enhance the host's natural defenses against viruses.
- Biotechnology and Synthetic Biology
In biotechnology and synthetic biology, it optimizes protein production. It identifies translational bottlenecks, such as rare codons or mRNA structures, that limit synthesis efficiency. Once found, expression constructs can be refined to overcome these barriers. This leads to more efficient protein production, vital for industrial enzymes and research reagents.
Why Creative Biolabs?
Choosing Creative Biolabs for your ribosome complex profiling needs means partnering with experts dedicated to unveiling the intricacies of translational regulation. Our service is built on cutting-edge protocols, optimized for high resolution and sensitivity, ensuring you capture even subtle changes in protein synthesis. We harness advanced bioinformatics pipelines to convert raw data into valuable biological insights. Our team possesses extensive experience in diverse research areas, from basic science to drug development, allowing us to provide tailored support for your specific project goals. Published Data from numerous studies utilizing similar high-resolution ribosome profiling techniques underscore the power of this approach in making groundbreaking discoveries.
FAQs
Q: How does ribosome complex profiling differ from standard RNA sequencing (RNA-seq)?
A: While RNA-seq measures the abundance of mRNA transcripts, ribosome complex profiling specifically quantifies actively translating ribosomes on those mRNAs. This provides a more direct measure of protein synthesis activity and can reveal translational control mechanisms that are invisible to RNA-seq alone.
Q: What are the key features of using ribosome complex profiling for drug discovery?
A: It's incredibly powerful for understanding a drug's mechanism of action by revealing how it impacts the translation of specific mRNAs. It can also help identify novel drug targets by pinpointing proteins whose synthesis is dysregulated in disease.
Q: What kind of sample input is required, and are there any specific precautions for sample preparation?
A: We typically work with cell pellets or flash-frozen tissue samples. It's crucial to rapidly inhibit translation (e.g., using cycloheximide for eukaryotic cells, if appropriate for the experimental question) and freeze samples quickly to preserve the in vivo state of ribosome-mRNA complexes.
Q: Can ribosome complex profiling identify new protein-coding regions or alternative start sites?
A: Absolutely! One of the exciting applications, as listed in our "Applications" section, is the discovery of novel ORFs, including sORFs and uORFs, as well as mapping alternative translation initiation sites (TIS). This can significantly enhance genome annotation and uncover previously unknown functional peptides.
Q: How does Creative Biolabs ensure data quality and provide support for data interpretation?
A: We implement rigorous quality control checks at each stage, from sample QC to library preparation and sequencing. Our comprehensive bioinformatics analysis includes alignment, footprint quantification, and differential translation analysis.
Related Services
To further enhance your research and gain a multi-omics perspective, consider these complementary services offered by Creative Biolabs:
RNA Sequencing: Provides comprehensive transcriptome profiling, ideal for correlating with ribosome complex profiling data to calculate translational efficiency.
Proteomic Services: Directly identifies and quantifies proteins, offering a valuable validation layer for translation data and a broader view of the proteome.
Creative Biolabs is committed to providing high-quality ribosome complex profiling services that deliver deep insights into the complexities of translational regulation. Our expertise and advanced methodologies empower you to uncover novel biological mechanisms, validate therapeutic targets, and accelerate your scientific breakthroughs. Ready to advance your research with unparalleled insights into protein synthesis? Our team is here to discuss your project.
Reference
- Xiao, Zhengtao, et al. "Genome-wide assessment of differential translations with ribosome profiling data." Nature communications 7.1 (2016): 11194. Distributed under Open Access license CC BY 4.0, without modification.
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