CLIP-seq is a method used to study RNA molecules and their interactions with associated proteins in cells. This method is used to map RNA-protein interactions which is useful for studying gene regulation.
It is an antibody-based method that uses specific antibodies to isolate RNA-protein complexes that are crosslinked using ultraviolet (UV) light. The RNA fragments are extracted and sequenced to 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. So, 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 the 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. This helps 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 and purified to separate proteins and associated RNA fragments. After purification, RNA fragments are extracted, reverse transcribed, and sequenced using high-throughput methods to construct 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
Then, immunoprecipitation is used to isolate the RNA-protein complexes. The sample is incubated with antibodies specific to the RBP of interest. Then, the antibody-bound RNPs are captured using protein beads or other affinity-based methods. The bead-antibody complexes are then washed to remove unbound or non-specific proteins and RNAs which leaves only the RNA-protein complexes or RNPs specifically bound to the RBP of interest.
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 when 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.
- 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 and reduces background noise.
- It 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 which is useful for large-scale studies.
- CLIP-seq involves many complex steps like crosslinking, immunoprecipitation, RNA fragmentation, and reverse transcription. This requires specialized expertise and careful handling for accurate results.
- UV crosslinking has low efficiency compared to formaldehyde crosslinking. This can result in partial or inaccurate results leading to the loss of some relevant data. It can also cause mutations in DNA.
- It requires high-quality and specific antibodies for immunoprecipitation of specific RBPs.
- It is less effective in detecting low-abundance interactions.
- It may be difficult to accurately map structurally complex or highly folded RNAs.
- CLIP-seq is expensive and labor-intensive as it requires specialized reagents and equipment. It also takes a longer time. This makes it less accessible for large-scale or high-throughput studies.
- RNA fragmentation and library preparation steps can introduce potential biases that can affect the accuracy of the sequencing results.
- CLIP-seq is used to study RNA-protein interactions which helps to understand how gene expression is regulated after transcription.
- It also helps to understand RNA processing and splicing. It can be used to study how RBPs affect RNA splicing and identify splicing factors.
- It is also used to study RNA modifications and their functions in different cellular processes.
- It has applications in disease research. It can be used to identify altered RNA-binding sites in disease states. Many neurodegenerative diseases and cancers involve dysregulated RNA-protein interactions. CLIP-seq helps identify disease-associated RBPs and their target RNAs.
- It also has applications in plant research. It can be used to study RNA regulation in plant development and identify RBPs involved in disease resistance and genetic improvements.
- It can be used to develop RNA-targeted drugs. It helps in designing small molecules or RNA-based therapeutics for genetic disorders.
- Bieniasz, P. D., & Kutluay, S. B. (2018). CLIP-related methodologies and their application to retrovirology. Retrovirology, 15(1), 35. https://doi.org/10.1186/s12977-018-0417-2
- BiologyInsights Team. (2025, March 12). CLIP-Seq: A Comprehensive Overview of Crosslinking Methods – BiologyInsights. Retrieved from https://biologyinsights.com/clip-seq-a-comprehensive-overview-of-crosslinking-methods/
- CLIP Sequencing – CD Genomics. (n.d.). Retrieved from https://rna.cd-genomics.com/clip-sequencing.html
- Enhanced CLIP sequencing (eCLIP & Ribo-eCLIP). (n.d.). Retrieved from https://rnacenter.ucsd.edu/technology/eclip.html
- Hafner, M., Katsantoni, M., Köster, T. et al. (2021). CLIP and complementary methods. Nature Reviews Methods Primers, 1, 20. https://doi.org/10.1038/s43586-021-00018-1
- Lee, F. C., & Ule, J. (2018). Advances in CLIP technologies for studies of Protein-RNA Interactions. Molecular Cell, 69(3), 354–369. https://doi.org/10.1016/j.molcel.2018.01.005
- Overview of RNA CLIP-Seq – CD genomics. (n.d.). Retrieved from https://rna.cd-genomics.com/resource-rna-clip-seq-principle-advantages-protocol-and-applications.html
- RIP-Seq vs. CLIP-Seq: Introduction, Advantages, and Applications | CD Genomics Blog. (2020, September 29). Retrieved from https://www.cd-genomics.com/blog/rip-seq-vs-clip-seq-introduction-advantages-and-applications/
Types of CLIP-seq
HITS-CLIP (High-Throughput Sequencing CLIP)
This is one of the original versions of CLIP-seq that combines CLIP with next-generation sequencing. This method uses UV crosslinking to create covalent bonds between RNA and proteins. The protein-RNA complexes are purified and the RNA is sequenced to map binding sites of RBPs.
PAR-CLIP (Photoactivatable Ribonucleoside-Enhanced CLIP)
This method uses photoreactive nucleosides like 4-thiouridine (4SU) or 6-thioguanosine (6SG). Cells are treated with these modified nucleotides which create stronger and more specific crosslinking when exposed to UV-A light (365 nm). This leads to mutations in the sequencing data which makes it easier to identify exact sites where proteins interact with RNA. The mutations caused by reverse transcription at the crosslinking site allow accurate mapping of RNA binding sites. However, using artificial nucleotides may not work well in all cell types and 4SU treatment can cause toxicity in some cells.
iCLIP (Individual-Nucleotide Resolution CLIP)
In HITS-CLIP and PAR-CLIP, reverse transcription (RT) often stops at the crosslinking sites causing truncation and loss of the starting material. To address this problem, iCLIP was developed. iCLIP involves attaching a 3ʹ adapter to the protein-RNA complexes and adding the 5′ adapter later during RT. The cDNA is circularized and then linearized using a restriction enzyme which improves the recovery of truncated cDNA fragments by preventing loss during library preparation.
eCLIP (Enhanced CLIP)
This method also involves two different adapter ligation steps. First, a 3ʹ adapter is ligated to the immunoprecipitated RNA then the second ligation occurs after reverse transcription where a single-stranded adapter is added to cDNA molecules. This method also includes a size-matched input control (SMInput) which helps normalize background noise and reduce biases in data analysis.
irCLIP (Infrared CLIP)
This method uses a 3ʹ adapter labeled with an infrared fluorescent dye instead of radioactive labeling like in other standard CLIP methods. This simplifies library preparation and improves efficiency. It also requires fewer cells and has a faster workflow. irCLIP also involves on-bead nuclease digestion for better RNA recovery and the use of thermostable reverse transcriptase which minimizes bias.
Advantages of CLIP-seq
Limitations of CLIP-seq
Applications of CLIP-seq
References
