RNA-binding proteins (RBPs) regulate various stages of the RNA life cycle, including splicing, cleavage, polyadenylation, stabilization, localization, editing, and translation. The interaction between RBPs and RNA is crucial for the regulation of the transcriptome and proteome. Dysfunction of RBPs can lead to physiological disorders and various diseases. Systematically identifying the dynamic changes of RBPs in cellular functions and disease states is of great significance for researchers to understand physiological functions and disease progression.
Introduction to RIP Technology
In 1979, Professor Steitz, J.A. from Yale University developed a novel method for detecting spliceosomal proteins in patients with systemic lupus erythematosus. ³²P radioactive isotope was used to label RNA, not as a fluorescent signal but detected through autoradiography, providing a vital technique for high-sensitivity detection of RNA molecules. The protein-RNA complexes were obtained using antibody-based immunoprecipitation. By extracting RNA from the complexes and performing electrophoresis on a 10% urea PAGE gel, the samples were then autoradiographed using ³²P, allowing clear visualization of RNA molecules within the protein-RNA complexes. This method, known for its high sensitivity and specificity, provided strong support for studying protein-RNA interactions.
This method was later developed into the RNP immunoprecipitation (RIP) technique. First, formaldehyde crosslinkers are used to fix ribonucleoprotein complexes (RNPs). (Include Note: UV crosslinking is an alternative, providing a reversible and often more controlled crosslinking approach.) Then, antibodies are used to immunoprecipitate the relevant RBPs, enhancing the specificity of the experiment and yielding purer RNPs. Finally, the extracted RNA is subjected to RT-qPCR to quantify RNA expression levels and directly detect interactions with RBPs. RIP has become an indispensable method for studying RNA-protein interactions, revealing key protein complexes in the RNA life cycle and their roles in specific biological processes. It also assists scientists in discovering new disease biomarkers and therapeutic targets, advancing biomedical research.
Introduction to RIP-seq Technology
In the 1990s, with rapid genomics advancements, next-generation sequencing (NGS) technology emerged to meet increasing demands for efficient and cost-effective sequencing. NGS offers significant improvements in throughput and cost reduction, crucial for large-scale genomic studies. In 2010, a team led by Professor Jeannie T. Lee from the Howard Hughes Medical Institute successfully combined RNA immunoprecipitation (RIP) with high-throughput sequencing, innovatively developing RIP-seq (RNP immunoprecipitation followed by high-throughput sequencing) technology. The main idea of this technique is to combine RIP with high-throughput sequencing, using antibodies to precipitate proteins bound to target RNA, followed by enrichment and purification, and finally sequencing the precipitated RNA using high-throughput sequencing. RIP-seq advantages include precise detection of low-abundance RNA and the powerful quantification of RNA expression profiles, refining our understanding at the intersection of RNA biology and disease.
In genomics, RIP-seq is widely used to study RNA-protein interactions and gene expression regulation mechanisms. Additionally, it has broad applications in medical research, including studying disease mechanisms, drug action mechanisms, and disease diagnosis. Therefore, RIP-seq is a valuable research tool that provides robust support for scientific investigations.
Applications
Studying RNA-protein interactions within cells.
Discovering interactions between RBPs and non-coding RNAs (e.g., LncRNA, miRNA).
Mapping genome-wide RNA-RBP interaction networks.
Technical Advantages
Genome-wide coverage: Enables the screening of protein-binding sites across the entire transcriptome with increased precision.
High sensitivity: Generates millions of sequence tags per sample, facilitating the detection of even low-frequency interactions.
High accuracy: Provides high signal-to-noise ratio data, accurately distinguishing true biological events from artifacts.
RIP-seq Experimental Workflow
Preparation:
- Provide comprehensive genomic information (including genome.fa file, gff file, pep.fa file).
- Prepare antibodies (validated by Western blot; commission validation if not available).
- Samples: Accepts animal tissues, plant tissues, tumor tissues, cells, and fungal samples.
- Controls: Utilize ‘Input’ as a positive control; set sample-specific controls where applicable.
- Antibodies: Ideally ChIP-grade; at minimum IP-grade, tag antibodies used if necessary, contingent upon successful tag expression and fusion.
Specific Steps
Main steps include careful crosslinking (consider UV for improved precision), lysate preparation, magnetic bead preparation for immunoprecipitation, immunoprecipitating protein-RNA complexes, library construction, sequencing, and bioinformatics analysis. Adapt library preparation methods based on RNA type—RNase removal critical for mRNA, LncRNA, circRNA, while small RNA libraries suit miRNA studies.
Experimental Considerations
- Rigorous sample preparation is critical in RIP experiments—ensure high-quality, high-purity cells or tissues with thorough disruption for optimal RNA extraction.
- Protein pretreatment: Essential to eliminate interference for accurate immunoprecipitation results.
- RNA extraction: Efficient, reliable methods are crucial post-immunoprecipitation for excellent analytical follow-through.
Analysis Workflow
Analysis results typically include genome alignment, read distribution across gene functional elements, PCA analysis, genome peak analysis, GO/KEGG analysis of peak-associated genes, and motif analysis of enriched regions. Inquire for a final report demo for comprehensive insights.
Multi-omics Integration and Case Studies
Technologies like RIP-seq are often paired with complementary omics, expanding research depth and scope. Integration with RNA-seq, RNA pull-down, and m6A-seq provides synergistic insights, enhancing our grasp of complex post-transcriptional networks. Profacgen’s integration services enhance these capabilities, facilitating a holistic understanding of transcriptional and post-transcriptional regulation.
By considering and addressing these aspects, Profacgen’s MeRIP-seq service not only expands the potential for discovery in RNA biology but also provides a framework for developing therapeutic applications, encompassing disease mechanism elucidation and drug action insights.
References:
He, S., Valkov, E., Cheloufi, S. et al. The nexus between RNA-binding proteins and their effectors. Nat Rev Genet 24, 276–294 (2023). https://doi.org/10.1038/s41576-022-00550-0
Lerner, Michael Rush, and Joan Argetsinger Steitz. “Antibodies to small nuclear RNAs complexed with proteins are produced by patients with systemic lupus erythematosus.” Proceedings of the National Academy of Sciences 76.11 (1979): 5495-5499.
Niranjanakumari, Somashe, et al. “Reversible cross-linking combined with immunoprecipitation to study RNA–protein interactions in vivo.” Methods 26.2 (2002): 182-190.
Zhao, Jing, et al. “Genome-wide identification of polycomb-associated RNAs by RIP-seq.” Molecular cell 40.6 (2010): 939-953.
