CLIP-seq: Identifying RNA-binding Protein Binding Sites

What is CLIP-seq?

RNA-binding proteins (RBPs) play an important role in the regulation of gene expression, mRNA stability, splicing, and translation. Identification of RBP binding sites on RNA molecules is essential to understand their functional roles in various cellular processes. Cross-linking immunoprecipitation (CLIP) followed by high-throughput sequencing (CLIP-seq) is a powerful technique used to identify RBP binding sites on RNA molecules.

The CLIP-seq technique involves cross-linking of RBPs to their target RNA molecules, followed by immunoprecipitation of the RBP-RNA complex using an antibody specific to the RBP. The cross-linking is then reversed, and the RNA molecules bound to the RBP are isolated and subjected to high-throughput sequencing. The resulting sequencing data is then analyzed to identify RBP binding sites on RNA molecules.

Schematic overview of the core steps common to most variants of the cross-linking and immunoprecipitation (CLIP) protocol (Hafner et al., 2021).

CLIP-seq analysis: A step-by-step guide

• CLIP-seq analysis involves several steps, including library preparation, sequencing, quality control, read mapping, peak calling, and data visualization. Here is a step-by-step guide for CLIP-seq analysis:
• Library preparation: RNA molecules bound to the RBP of interest are cross-linked and immunoprecipitated using an antibody specific to the RBP. The cross-linking is then reversed, and the RNA molecules are fragmented and converted into cDNA libraries using reverse transcription and PCR amplification.
• Sequencing: The cDNA libraries are then subjected to high-throughput sequencing using a next-generation sequencing platform such as Illumina.
• Quality control: The sequencing data is subjected to quality control analysis to assess the quality of the sequencing reads, including read length distribution, base quality distribution, and error rates.
• Read mapping: The sequencing reads are then mapped to the reference genome or transcriptome using a read mapping software such as Bowtie or BWA.
• Peak calling: The mapped reads are then analyzed to identify RBP binding sites on RNA molecules using a peak calling software such as MACS or HOMER.
• Data visualization: The peak calling results are then visualized using genome browsers such as UCSC Genome Browser or IGV.

CLIP-seq vs RIP-seq: What's the difference?

RIP-seq (RNA immunoprecipitation sequencing) is a technique used to identify RNA molecules bound to RBPs. RIP-seq involves immunoprecipitation of the RBP-RNA complex using an antibody specific to the RBP, followed by high-throughput sequencing of the isolated RNA molecules. The main difference between CLIP-seq and RIP-seq is that CLIP-seq involves cross-linking of RBPs to RNA molecules prior to immunoprecipitation, while RIP-seq does not involve cross-linking.

The cross-linking step in CLIP-seq allows for the identification of direct RBP-RNA interactions, while RIP-seq only identifies RNA molecules that are indirectly associated with RBPs. However, CLIP-seq is more technically challenging than RIP-seq and requires more stringent quality control measures to ensure that the identified binding sites are not artifacts of the cross-linking process.

Simplified biological principles of RIP-seq (Li et al., 2013)

CLIP-seq vs ChIP-seq: Which technique is right for you?

ChIP-seq (chromatin immunoprecipitation sequencing) is a technique used to identify DNA sequences bound to DNA-binding proteins such as transcription factors. ChIP-seq involves cross-linking of the protein-DNA complex, followed by immunoprecipitation of the protein-DNA complex using an antibody specific to the protein. The isolated DNA fragments are then subjected to high-throughput sequencing.

CLIP-seq and ChIP-seq are both immunoprecipitation techniques, but they differ in the type of molecule that is being studied. CLIP-seq is used to identify RNA molecules bound to RBPs, while ChIP-seq is used to identify DNA sequences bound to DNA-binding proteins. Therefore, the choice between CLIP-seq and ChIP-seq depends on the specific research question and the type of molecule that is being studied.

Simplified biological principles of ChIP-seq (Li et al., 2013)

CLIP-seq vs PAR-CLIP: What are the key differences?

PAR-CLIP (photoactivatable-ribonucleoside-enhanced CLIP) is a modified CLIP-seq technique that uses a photoactivatable nucleoside analog to enhance the efficiency of cross-linking. The photoactivatable nucleoside analog is incorporated into newly synthesized RNA molecules, and upon exposure to UV light, it induces cross-linking between RBPs and RNA molecules at higher efficiency than traditional CLIP-seq.

The key difference between CLIP-seq and PAR-CLIP is the efficiency of cross-linking. PAR-CLIP allows for more efficient cross-linking, resulting in higher quality sequencing data and more precise identification of RBP binding sites on RNA molecules. However, PAR-CLIP also requires additional experimental steps compared to traditional CLIP-seq, such as incorporation of the photoactivatable nucleoside analog and UV irradiation.

PAR-CLIP vs Ago CLIP-seq: Which technique is better?

Ago CLIP-seq is a modified CLIP-seq technique that specifically targets RNA molecules bound to the Argonaute (Ago) protein, which is involved in RNA interference and microRNA-mediated gene regulation. Ago CLIP-seq involves cross-linking of Ago protein to RNA molecules, followed by immunoprecipitation of the Ago-RNA complex using an antibody specific to Ago. The isolated RNA fragments are then subjected to high-throughput sequencing.

The choice between PAR-CLIP and Ago CLIP-seq depends on the specific research question and the RBPs of interest. PAR-CLIP is a more general technique that can be used to study any RBP, while Ago CLIP-seq is specific to RNA molecules bound to the Ago protein. Therefore, if the research question specifically focuses on Ago-mediated gene regulation, Ago CLIP-seq would be a better choice. However, if the research question is more general and involves studying RBPs other than Ago, PAR-CLIP would be a better choice.

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