The interaction between RNA and protein is the core of many key processes in life activities, such as gene expression regulation, RNA processing, transportation, and translation. An in-depth analysis of these interactions is of great significance for understanding the mysteries of life, revealing the pathogenesis of diseases, and developing new treatments. With the continuous development of molecular biology technology, the research methods for studying RNA-protein interactions are also constantly evolving. From the initial cross-linking and immunoprecipitation (CLIP) technology to the current Enhanced Cross-Linking and Immunoprecipitation followed by Sequencing (eCLIP-seq) technology, every technological breakthrough brings new opportunities and progress to the research in this field.

This article explores the technological evolution from CLIP to eCLIP-seq in mapping RNA-protein interactions, covering their variants, improvements, performance comparisons, impacts, and future directions.

The Need for RNA-Protein Interaction Studies

RNA and protein are two important biological macromolecules in cells, and their dynamic interaction runs through the whole life cycle of cells. In the process of gene expression:

• Transcription factors combine with RNA polymerase to regulate the initiation of transcription.
• Various RNA-binding proteins participate in the splicing, tail-adding, transportation, and translation of mRNA, ensuring the accurate and orderly transmission of gene information.
• Some RNA-binding proteins determine their location in cells by recognizing specific sequences on mRNA, thus affecting the synthesis position and function of the protein.

In addition, the abnormality of RNA-protein interaction is closely related to the occurrence of many diseases.

• In cancer, the expression level or activity of some RNA-binding proteins changes, which leads to abnormal stability or translation efficiency of target gene mRNA, and then promotes abnormal cell proliferation and metastasis.
• Neurodegenerative diseases, such as Alzheimer's disease, have also been found to be related to aggregates formed by the wrong interaction between RNA and protein.

Therefore, the systematic study of RNA-protein interaction can not only deepen our understanding of the basic laws of life activities, but also provide new targets and ideas for the diagnosis and treatment of diseases.

RNA-centric methods for the purification and identification of RNA-binding proteins (McHugh et al., 2014)

Major CLIP Variants and Their Innovations

CLIP technology is an important means to study RNA-protein interaction. Its basic principle is to cross-link RNA with bound protein by ultraviolet rays, then immunoprecipitate RNA-protein complex by using specific antibodies, and finally separate, identify, and analyze the precipitated RNA. With the deepening of research, CLIP technology has been continuously improved and optimized, resulting in a variety of variants, each of which has made innovations in different aspects.

PAR-CLIP

Photomovable ribonuclease-enhanced cross-linking and immunoprecipitation (PAR-CLIP) is one of the important variants. By adding photoactive nucleoside analogues (such as 4-thiouridine) in cell culture, these analogues can cross-link with bound protein more efficiently when irradiated by ultraviolet rays, thus improving the cross-linking efficiency and specificity. At the same time, PAR-CLIP can lead to specific base mutations during reverse transcription, which can be used as a marker of RNA binding to protein and help to locate the binding site more accurately.

iCLIP

Individual-nucleotide resolution cross-linking and immunoprecipitations (iCLIP) realizes the identification of RNA binding sites with single-nucleotide resolution. In the process of cDNA amplification, the traditional CLIP technology may lose some information because of the incomplete connection of RNA fragments. iCLIP, by terminal labeling and processing the immunoprecipitated RNA, combined with PCR amplification and high-throughput sequencing, can more accurately determine the position of single-nucleotide binding to protein on RNA, which greatly improves the resolution and accuracy of the data.

HITS-CLIP

High-Throughput Sequencing of RNA Isolated by Cross-Linking and Immunoprecipitation (HITS-CLIP) combines high-throughput sequencing technology with CLIP, so that a large number of RNA binding sites can be identified simultaneously in one experiment. It overcomes the limitation of low throughput of RNA identification by traditional CLIP technology, realizes large-scale and systematic analysis of RNA-protein interaction groups, and provides a powerful tool for comprehensively understanding the target RNA of specific RNA-binding proteins.

Protein-centric methods for detecting RNA-protein interactions (McHugh et al., 2014)

The Breakthrough: eCLIP-seq

Although CLIP and its variants have played an important role in the study of RNA-protein interaction, they still have some shortcomings in the complexity of experimental procedures, data repeatability, and efficiency. The emergence of eCLIP-Seq technology has achieved remarkable breakthroughs in these areas.

Key Improvement of eCLIP

• A. Input comparison of size matching
  • a) In the previous CLIP-related technologies, various deviations are easy to occur in the process of PCR amplification and sequencing, which may interfere with the accuracy of experimental results. ECLIP introduces the input comparison of size matching, which is of great significance. It can effectively correct the amplification deviation caused by the difference of fragment size in the PCR amplification process and the possible preference in the sequencing process by setting the input sample that matches the immunoprecipitation sample in RNA fragment size. In this way, the final sequencing data can truly reflect the combination of RNA and protein, which greatly improves the reliability of the experimental results.
• B. Optimized joint connection
  • a) Linker connection is a key step in the construction of an RNA library, and its efficiency and specificity directly affect the quality of the library. ECLIP optimized them all. By improving the design of the linker and the conditions of the ligation reaction, the ligation efficiency of the RNA fragment and linker was improved, and the occurrence of nonspecific reactions such as self-ligation and multi-ligation of the linker was reduced. This not only increases the complexity of the library, so that more RNA fragments can be successfully captured and sequenced, but also improves the detection ability of low-abundance RNA binding events, so that experiments can capture more abundant RNA-protein interaction information.
• C. Simplified experimental process
  • a) The traditional CLIP technology has complicated experimental steps and complicated operation, which is not only time-consuming and laborious, but also easy to lead to poor repeatability of experimental results due to differences in operating processes, which also limits its application in large-scale research. ECLIP greatly simplifies the experimental process, reduces unnecessary operation steps, and reduces the difficulty and complexity of experimental operation.
  • b) This improvement significantly improves the reproducibility of the experiment and makes the experimental results of different laboratories and batches more comparable. At the same time, the simplified process also enhanced the scalability of the technology, which enabled it to be applied to large-scale RNA binding protein research more efficiently, and laid the foundation for Qualcomm's quantitative and systematic analysis.

Evaluation of RBPNet on iCLlP and miCLlP data (Horlacher et al., 2023)

Performance Comparison: eCLIP, iCLIP and PAR-CLIP

In the study of RNA-protein interaction, eCLIP, iCLIP, and PAR-CLIP are commonly used variants of CLIP technology. They have their characteristics in principle and application. By comparing the key properties such as sensitivity, specificity, and ease of use, they can present their respective advantages and limitations, provide important reference for researchers to choose appropriate technologies, and promote the research in this field to be carried out more efficiently.

Sensitivity

• eCLIP is excellent in sensitivity. Due to the improvement of its optimized linker connection and size-matching input control, it can capture low-abundance RNA-protein complexes more effectively and detect weak binding signals better.
• Although iCLIP has achieved single-nucleotide resolution, its sensitivity is slightly lower than eCLIP, and it may be missed for some binding events with extremely low abundance.
• PAR-CLIP improves the cross-linking efficiency through photoactive nucleoside analogues and enhances the sensitivity to some extent, but in contrast, eCLIP has more advantages in the overall signal detection ability.

Specificity

• Specificity is the key to ensuring the accuracy of experimental results. The size-matching input control introduced by eCLIP can effectively reduce the background noise, improve the specificity of detection, and make the identified RNA binding sites more reliable.
• With the resolution of single nucleotide, iCLIP has high specificity in the location of binding sites, which can be accurate to a single nucleotide.
• Due to the use of photoactive nucleoside analogues, the cross-linking reaction of PAR-CLIP has a certain sequence preference, which may lead to the detection of some nonspecific binding, and its specificity is slightly lower than that of eCLIP and iCLIP.

Usability

• In terms of ease of use, eCLIP has obvious advantages because of its simplified experimental process, relatively simple operation, lower technical requirements for experimenters, and easier popularization and application in different laboratories.
• The experimental steps of iCLIP are complicated, especially in the process of realizing single-nucleotide resolution, which requires more detailed operation and control and higher skills of experimenters.
• PAR-CLIP needs to add photoactive nucleoside analogues in cell culture, and the cross-linking conditions are also strictly controlled; the complexity of operation is between eCLIP and iCLIP.

Scoring of 232 splicing mutations from MutSpliceDB along with 6087 control mutationsfrom gnomAD taken in their vicinity, using 40 splicing-related RBPNet models (Horlacher et al., 2023)

Impact and Future Directions of eCLIP-seq

The emergence of eCLI-seq technology has brought many important biological discoveries to the research field of RNA-protein interaction, and also promoted the development of related emerging technologies. However, there are still some unresolved challenges in this field.

Biological Discoveries Enabled by eCLIP-seq

With its high sensitivity, high specificity, and high efficiency, eCLIP has successfully mapped the whole genome of RNA-binding proteins (RBP). These maps clearly show the binding position and distribution characteristics of different RBPs on the genome, which enables researchers to systematically understand the interaction mode between RBPs and RNA, and provides a solid foundation for further exploring the functions of RBPs in life processes such as gene expression regulation.

In disease research, eCLIP technology helps to reveal RNA networks related to many diseases. In cancer research, eCLIP analysis shows that the abnormal binding of some RBPs will lead to changes in RNA processing and stability of oncogenes or tumor suppressor genes, thus promoting the occurrence and development of cancer. In neurodegenerative diseases, eCLIP technology helps to identify abnormal RNA-protein interactions related to the disease, which may be involved in pathological processes such as protein aggregation and neuronal dysfunction, providing a new perspective for understanding the disease mechanism.

RBPNet prediction performance on ENCODE eCLIP datasets (Horlacher et al., 2023)

Emerging technologies beyond eCLIP

Although eCLIP technology has greatly promoted the study of RNA-protein interaction, the deepening of scientific exploration has spawned more emerging technologies. They provide a brand-new tool for analyzing more complex RNA regulatory networks and revealing instantaneous or low-abundance interactions.

Single-cell CLIP

The traditional eCLIP technology usually analyzes a large number of cells, and the results obtained are the average level of the population, which cannot reflect the differences between individual cells. Single-cell CLIP (scCLIP) technology can study the interaction between RBP and RNA at the single-cell level, which effectively solves the problem of heterogeneity of RBP. It can reveal the differences of RBP binding patterns in different cells, which is of great significance for understanding cell differentiation, cell response to environmental stimuli, and cell heterogeneity in disease microenvironment.

Long-read eCLIP

Conventional eCLIP technology mainly analyzes short RNA fragments, and it is difficult to fully understand the binding of full-length RNA isomers. The appearance of long reading and long eCLIP technology has enabled the analysis of the binding of full-length RNA isomers. It can capture longer RNA fragments, thus more accurately determining the binding sites of RBP on different RNA isomers, which is helpful to further study the functions of RNA isomers and the regulation mechanism of RBP.

Existing Challenges

Although eCLIP and related technologies have made great progress, there are still many key problems to be solved urgently in this field.

• Firstly, the availability of antibodies seriously restricts the application scope of eCLIP technology. Many RBPs lack antibodies with high specificity and affinity, which makes it difficult to carry out in-depth research on these RBPs.
• Secondly, the scarcity of samples of rare cell groups brings great challenges to the eCLIP experiment. Due to the limited number of cells, it is difficult to meet the sample size requirements of the eCLIP experiment, which greatly hinders the research process of RNA-protein interaction in these cells.
• In addition, RNA-protein interaction is highly dynamic, which is regulated by many complex factors such as intracellular homeostasis and external stimuli. At present, capturing these dynamic interactions in real time and accurately is still a major scientific problem in this field.

RBPNet feature attribution maps and binding motif discovery (Horlacher et al., 2023)

Conclusion

The evolution from CLIP technology to eCLIP-seq technology is the embodiment of the continuous development and progress in the research field of RNA-protein interaction. Each technology has made an important contribution to the research in this field at a specific historical stage, and eCLIP-seq technology has become an important means to study RNA-protein interaction by optimizing the experimental process and improving the quality and efficiency of data.

The development of these technologies not only deepens our understanding of the interaction mechanism between RNA and protein in life activities, but also provides new ideas and methods for disease research and treatment. With the continuous innovation and improvement of technology, I believe that in the future, we will be able to analyze the mystery of RNA-protein interaction more deeply and comprehensively, and make greater contributions to the development of life science and human health.

References

1. McHugh C.A, Russell P, Guttman M. Methods for comprehensive experimental identification of RNA-protein interactions. Genome Biol. 2014 15: 203.
2. Horlacher M, Wagner N, Moyon L, et al. Towards in silico CLIP-seq: predicting protein-RNA interaction via sequence-to-signal learning. Genome Biol. 2023 24(1): 180.