CLIP-seq is a method used to study RNA molecules and their interactions with associated proteins in cells.
It is used to map RNA-protein interactions, which is useful for studying gene regulation.
This is an antibody-based method that uses specific antibodies to isolate RNA-protein complexes.
The RNA-protein complexes are crosslinked using ultraviolet (UV) light.
RNA fragments are then extracted and sequenced.
This helps identify the regulatory roles of RNA-binding proteins (RBPs) and their interactions with RNA.
RNA-Protein Interactions
Proteins interact with RNA as soon as it is made from DNA during transcription.
These proteins bind to the transcribed RNA to form ribonucleoprotein (RNP) complexes and are called RNA-binding proteins (RBPs).
RBPs help regulate RNA processes like splicing, translation, transport, and overall function.
They play an important role in regulating gene expression.
It is important to construct an accurate map of protein-RNA interactions and study these interactions to understand gene regulation.
Different methods have been developed to study protein-RNA interactions.
There are protein-centric and RNA-centric methods.
Protein-centric methods focus on a specific RBP and identify all the RNA sites it binds to, helping to understand the role of that particular protein in gene regulation.
RNA-centric methods look at all the proteins that bind to a particular RNA molecule and help understand how different proteins interact with RNA in the cell.
CLIP-seq is a protein-centric method that provides accurate information on RNA-protein interactions and helps to understand RNA regulation.
Principle of CLIP-seq
CLIP-seq works by using UV crosslinking and immunoprecipitation to covalently bind RNA molecules to associated RBPs and isolate the RNA-protein complexes.
The RNA fragments bound to RBPs are extracted and sequenced to study the RNA-protein interactions.
Initially, crosslinking is done by irradiating the cells with UV light, which forms an irreversible covalent bond between RNA and RBPs in living cells.
These linked RNA-protein complexes can be precisely isolated using immunoprecipitation.
The complexes are purified to separate proteins and associated RNA fragments.
After purification, RNA fragments are extracted, reverse transcribed, and sequenced using high-throughput methods.
This process constructs a detailed map of RNA-protein interactions.
Steps of CLIP-seq
1. UV Crosslinking
The first step in CLIP-seq is crosslinking, which involves irradiating the sample with UV light.
Exposing the cells to UV creates irreversible covalent bonds between RBPs and their target RNA molecules.
Most CLIP protocols use UV-C light (254 nm), which can crosslink without requiring any pre-treatment of cells.
Cells are usually kept on ice for a short period during crosslinking to prevent UV-induced DNA damage.
2. Cell Lysis and RNA Fragmentation
After crosslinking, the cells are lysed to release the RNA-protein complexes from the cells.
The sample is then treated with limited amounts of RNases to fragment the RNA into smaller pieces.
Fragmentation is done to generate RNA fragments of manageable size for later steps in the workflow.
3. Immunoprecipitation
immunoprecipitation is performed to isolate the RNA-protein complexes. The sample is incubated with antibodies that specifically recognize the RNA-binding protein (RBP) of interest.
These antibodies bind to the target RBP, forming RNA-protein-antibody complexes.
To capture these complexes, protein beads or other affinity-based systems are used.
After binding, the bead-antibody complexes are thoroughly washed to eliminate any non-specific proteins or RNAs, ensuring that only the RNA-protein complexes specifically associated with the target RBP remain.
4. RNA Isolation
The purified RNPs are visualized using polyacrylamide gel electrophoresis.
The resulting complexes are transferred onto a nitrocellulose membrane and RNA molecules are visualized using radioactive labels.
Then, the proteins are digested by proteinase K and the attached RNA fragments are released.
5. Adapter Ligation
Adapter ligation is necessary for reverse transcription and PCR amplification.
In the original protocol, adapters are ligated to both ends of the RNA fragments after isolating RNA.
However, this requires an additional purification step to remove the adapter.
In newer variants of CLIP-seq, only the reverse adapter is ligated to the 3′ end of the RNA fragments, and this occurs after immunoprecipitation while the RNA molecules are still in the complex.
This is called on-bead ligation and it removes the need for extra steps to remove the adapter.
Another adapter is also ligated later either during or after reverse transcription, depending on the specific method.
6. Library Preparation
The isolated RNA fragments are converted into complementary DNA (cDNA) for sequencing.
The resulting cDNA is purified to remove any unwanted byproducts.
cDNA purification can be done using gel electrophoresis, which removes excess adapter sequences or primers that could interfere with downstream processes.
However, gel electrophoresis can be time-consuming and labor-intensive.
Other recent methods for purification include using silica beads or biotinylated adapters, which are quicker and more efficient alternatives to gel electrophoresis.
Then, the purified cDNA is amplified using PCR, which creates a library ready for sequencing.
7. Sequencing
Finally, the cDNA library is sequenced using high-throughput sequencing methods.
The generated sequencing data is processed and analyzed to identify the RNA-protein interaction regions.
8. Data Analysis
CLIP-seq data analysis involves four main steps.
First, pre-processing of sequencing reads is done to remove low-quality data and trim adaptors.
Then, the cleaned reads are mapped to a reference genome.
The third step is peak calling, which identifies enriched binding sites as peaks.
These peaks represent regions where the RBP binds to the RNA.
Computational methods help to separate these peaks from background noise in the data.
Finally, post-processing analyzes these binding sites to identify motifs, gene locations, and biological functions.
Types of CLIP-seq
HITS-CLIP (High-Throughput Sequencing CLIP)
One of the original CLIP-seq versions combining CLIP with next-generation sequencing.
Uses UV crosslinking to create covalent bonds between RNA and proteins.
Protein-RNA complexes are purified and RNA is sequenced to map RBP binding sites.
Uses photoreactive nucleosides like 4-thiouridine (4SU) or 6-thioguanosine (6SG).
Cells are treated with these modified nucleotides that create stronger and more specific crosslinking under UV-A light (365 nm).
Causes mutations in sequencing data allowing easier identification of exact protein-RNA interaction sites.
Mutations from reverse transcription at crosslinking sites allow accurate mapping of binding sites.
Use of artificial nucleotides may not work well in all cell types, and 4SU can cause toxicity in some cells.
iCLIP (Individual-Nucleotide Resolution CLIP)
Developed to overcome reverse transcription stops at crosslink sites causing truncated cDNA in HITS-CLIP and PAR-CLIP.
Attaches a 3′ adapter to protein-RNA complexes and adds the 5′ adapter later during reverse transcription.
cDNA is circularized and linearized using a restriction enzyme to improve recovery of truncated fragments, preventing loss during library preparation.
eCLIP (Enhanced CLIP)
Involves two adapter ligation steps: a 3′ adapter ligated to immunoprecipitated RNA, and a second single-stranded adapter added to cDNA after reverse transcription.
Includes a size-matched input control (SMInput) to normalize background noise and reduce biases in data analysis.
irCLIP (Infrared CLIP)
Uses a 3′ adapter labeled with an infrared fluorescent dye instead of radioactive labeling.
Simplifies library preparation and improves efficiency.
Requires fewer cells and has a faster workflow.
Includes on-bead nuclease digestion for better RNA recovery and uses thermostable reverse transcriptase to minimize bias.
Advantages of CLIP-seq
CLIP-seq can accurately capture RNA-protein interactions.
Ultraviolet crosslinking in CLIP-seq allows direct identification of RBP interactions with RNA.
It does not crosslink proteins to each other, reducing background noise.
CLIP-seq has broad applications in RNA research and is useful for studying processes like RNA splicing, RNA stability, and gene expression.
The immunoprecipitation step provides high specificity in identifying RBP-RNA interactions by using specific antibodies.
This prevents non-specific binding and makes the results more reliable.
CLIP-seq is a high-throughput method that can analyze a large number of RNA-protein interactions, making it useful for large-scale studies.
Limitations of CLIP-seq
CLIP-seq involves many complex steps like crosslinking, immunoprecipitation, RNA fragmentation, and reverse transcription, requiring specialized expertise and careful handling for accurate results.
UV crosslinking has low efficiency compared to formaldehyde crosslinking, which can result in partial or inaccurate results and loss of some relevant data.
UV crosslinking can also cause mutations in DNA.
The method requires high-quality and specific antibodies for immunoprecipitation of specific RBPs.
CLIP-seq is less effective in detecting low-abundance interactions.
It may be difficult to accurately map structurally complex or highly folded RNAs.
The technique is expensive and labor-intensive, requiring specialized reagents and equipment, and takes longer time, making it less accessible for large-scale or high-throughput studies.
RNA fragmentation and library preparation steps can introduce potential biases that affect the accuracy of sequencing results.
Applications of CLIP-seq
CLIP-seq is used to study RNA-protein interactions, helping to understand how gene expression is regulated after transcription.
It helps to understand RNA processing and splicing by studying how RBPs affect RNA splicing and identifying splicing factors.
CLIP-seq is used to study RNA modifications and their functions in different cellular processes.
It has applications in disease research by identifying altered RNA-binding sites in disease states.
Many neurodegenerative diseases and cancers involve dysregulated RNA-protein interactions, and CLIP-seq helps identify disease-associated RBPs and their target RNAs.
The method is applied in plant research to study RNA regulation in plant development and identify RBPs involved in disease resistance and genetic improvements.
CLIP-seq can be used to develop RNA-targeted drugs, aiding in designing small molecules or RNA-based therapeutics for genetic disorders.
References
Bieniasz, P. D., & Kutluay, S. B. (2018). Overview of CLIP-related techniques and their use in retrovirology. Retrovirology, 15(1), 35. https://doi.org/10.1186/s12977-018-0417-2
BiologyInsights Team. (2025, March 12). Comprehensive review of CLIP-Seq and crosslinking methods. Retrieved from https://biologyinsights.com/clip-seq-a-comprehensive-overview-of-crosslinking-methods/
CD Genomics. (n.d.). Introduction to CLIP sequencing. Retrieved from https://rna.cd-genomics.com/clip-sequencing.html
University of California San Diego. (n.d.). Enhanced CLIP sequencing (eCLIP & Ribo-eCLIP) technology overview. Retrieved from https://rnacenter.ucsd.edu/technology/eclip.html
Hafner, M., Katsantoni, M., Köster, T., et al. (2021). CLIP and related complementary methods. Nature Reviews Methods Primers, 1, 20. https://doi.org/10.1038/s43586-021-00018-1
Lee, F. C., & Ule, J. (2018). Recent developments in CLIP technologies for analyzing protein-RNA interactions. Molecular Cell, 69(3), 354–369. https://doi.org/10.1016/j.molcel.2018.01.005
CD Genomics. (n.d.). Overview of RNA CLIP-Seq: principle, advantages, protocol, and applications. Retrieved from https://rna.cd-genomics.com/resource-rna-clip-seq-principle-advantages-protocol-and-applications.html
CD Genomics Blog. (2020, September 29). Comparison of RIP-Seq and CLIP-Seq: introduction, benefits, and applications. Retrieved from https://www.cd-genomics.com/blog/rip-seq-vs-clip-seq-introduction-advantages-and-applications/
