Mapping RNA-Protein Interactions with RBP-eCLIP: Powering Discovery in RNA Biology

Guest author Dr Vince Harjono is an Application Scientist Manager at Eclipsebio. CST and Eclipsebio have partnered to deliver rigorously validated antibodies to help jump-start your RNA biology research.

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RNA-binding proteins (RBPs) are central players in gene regulation, shaping the fate of RNAs from transcription until decay. As the study of RNA biology enters a new era, fueled by high-throughput sequencing and transcriptome-wide analyses, tools that allow precise mapping of RBP-RNA interactions have become essential. One technology has changed the way researchers explore these interactions: Enhanced Crosslinking and Immunoprecipitation, or RBP-eCLIP.

In the years since its introduction, RBP-eCLIP has become a cornerstone of post-transcriptional gene regulation studies, allowing researchers to investigate fundamental mechanisms, disease processes, and therapeutic targets with unprecedented resolution. 

This blog explores the scientific impact and recent applications of RBP-eCLIP, and why antibody quality is crucial to the method’s success. Read on to learn more, or explore pre-validated eCLIP antibodies in the CST product catalog:

The Expanding Relevance of RNA Binding Protein Biology

RBPs govern nearly every aspect of RNA metabolism, including splicing, export, stability, localization, and translation. As a class of proteins, they are also heavily implicated in disease: dysfunction in RBPs like TDP-43, FUS, or UPF1 has been linked to conditions ranging from ALS and Fragile X syndrome to various cancers and immunological disorders. 

Enhanced cross-linking and immunoprecipitation (eCLIP) was performed with RNA from K-562 cells and recombinant monoclonal antibody FUS/TLS (E3O8I) Rabbit mAb #67840 using a protocol based on the RBP-eCLIP method from Eclipsebio. The figure shows binding across the FUS transcript. Data is kindly provided by the laboratory of Dr. Gene Yeo and used with permission.

While over 1,500 human proteins are predicted to bind RNA, only a fraction of these have been functionally annotated, and the study of RNA regulation and translational control remains a key area of focus. Techniques like RBP-eCLIP have proven indispensable in addressing this gap, enabling researchers to pinpoint binding sites transcriptome-wide and uncover the regulatory logic underlying RNA-protein networks.

The Impact of RBP-eCLIP

Initially developed in Professor Gene Yeo’s lab at UCSD and refined for broader use by Eclipsebio, RBP-eCLIP has empowered the research community by producing large-scale, reproducible datasets. Its integration into the ENCODE project brought standardization and public access, offering researchers a robust reference atlas of RBP binding landscapes. 

View the RNA Lifecycle Diagram to explore the role of RNA-binding proteins and related CST antibodies

But RBP-eCLIP is not just a method—it’s become a framework for hypothesis-driven discovery. In neuroscience, cancer biology, and RNA therapeutic development, the method is pushing boundaries: 

• In neuroscience, RBP-eCLIP has revealed how localized RNA regulation contributes to neuronal identity and plasticity. 
• In oncology, RBP-eCLIP has shown how cancer cells repurpose RNA regulation to support proliferation, immune evasion, and drug resistance. 
• In developmental biology, RBP-eCLIP has been used to discover the tightly choreographed roles of RBPs during cell fate transitions. 

The study of RBPs and their influence also intersects with the field of epigenetics, where modifications to RNA and associated proteins can impact gene expression without altering the underlying DNA sequence.

High-Impact Applications of RNA Biology Studies

Numerous recent studies have leveraged RBP-eCLIP and related methods to drive insights in diverse fields. Here are a few that stand out: 

	Decoding Alternative Splicing Regulation  A large-scale analysis using RBP-eCLIP across over 100 RBPs was recently published in Nature Biotechnology,1 revealing how individual RBPs coordinate to regulate exon inclusion or exclusion. The study mapped direct RBP-RNA contacts and linked them to splicing outcomes across ENCODE datasets, highlighting both well-known and novel splicing regulators.
	Motif Discovery & Functional Annotation  Schwarzl et al.2 introduced DEWSeq, a bioinformatics package tailored to RBP-eCLIP datasets, significantly increasing detection of sequence motifs in binding peaks. Applying this across over 100 RBPs, the authors found that incorporating size-matched input controls and replicate-aware analysis strategies dramatically improved biological relevance and motif enrichment.
	Mapping RBP Networks in Neurons  Work from Han et al. published in Molecular Cell 3 used iCLIP to chart the developmental dynamics of splicing regulators in the mouse brain, demonstrating how sequential changes in RBP expression are associated with shifts in exon usage during neurogenesis. These findings suggest a mechanistic basis for how splicing misregulation might lead to neurological disease.
	Therapeutic Discovery & RNA Drugs  In pharmaceutical research, RBP-eCLIP can be used to assess the on-target and off-target effects of antisense oligonucleotides (ASOs) and siRNAs. By overlaying binding maps of key RBPs like TIAL1 or ELAVL1 with drug-modified transcriptomes, researchers can identify unintended shifts in RNA-protein interaction networks—a key consideration for RNA-targeted drug development.

Making RBP-eCLIP More Accessible: Validated Antibodies & Standards

One barrier to entry for RBP-eCLIP has been the variability of antibody performance in RBP-eCLIP protocols. Unlike standard immunoprecipitation, RBP-eCLIP requires robust antibodies that retain high affinity under stringent wash and crosslinking conditions. This has historically limited the scope of RBPs that could be reliably profiled.

To solve this, Eclipsebio and CST have partnered to pre-validate antibodies specifically for RBP-eCLIP. Their antibody validation process assesses both protein pulldown (Western blot) and RNA yield (biotin blot), ensuring that only high-performing antibodies are recommended for use in RBP-eCLIP.

Enhanced cross-linking and immunoprecipitation (eCLIP) was performed with RNA from K-562 cells and recombinant monoclonal antibody QKI (E7O4A) Rabbit mAb #23065 using a protocol based on the RBP-eCLIP method from Eclipsebio. The figure shows binding across the QKI transcript. Data is kindly provided by the laboratory of Dr Gene Yeo and used with permission.

Researchers using eCLIP validated CST antibodies—like those targeting QKI, FUS, or TIAR—report clear enrichment peaks, reproducible datasets across replicates, and strong overlap with published ENCODE data. These rigorously tested tools dramatically reduce experimental variability and accelerate discovery.

Download the research poster from CST and Eclipsebio, Accelerating eCLIP Studies: Importance of Antibody Validation and Pre-Validated Antibodies for RNA Binding Proteins, to explore the validation data.

Pushing the Frontier in Neuronal and Disease Biology

RBP-eCLIP is especially well-suited to complex, cell-type-specific systems like the nervous system, where post-transcriptional regulation plays a central role. Neurons depend on localized translation in dendrites and axons, making the spatial and temporal binding patterns of RBPs uniquely important. RBP-eCLIP has been used to explore: 

• Subcellular localization of RBP-RNA complexes, such as QKI’s role in myelination. 
  Developmental dynamics, where RBPs exhibit shifting target sets during neural differentiation.
• Neurodegeneration, helping to define direct RNA targets of TDP-43 or FUS and how their mislocalization alters gene expression.
• In parallel, cancer studies use RBP-eCLIP to chart how RBPs like PTBP1 or hnRNPA1 are reprogrammed to stabilize oncogenic transcripts or repress immune regulators, providing insight into how RNA regulation is co-opted in malignancy. 

Looking Ahead: The RBP Atlas is Still in Progress

From basic mechanistic insight to translational applications, RBP-eCLIP has become an indispensable technology in RNA biology. However, much remains unexplored. Estimates suggest that more than half of all human RBPs have not yet been profiled using RBP-eCLIP due to a lack of validated antibodies or contextual datasets. But with the continued development of new tools and expanding validation partnerships, the pace is accelerating.

RNA-binding proteins are at the center of the post-transcriptional regulatory universe. By offering resolution, reproducibility, and scalability, researchers have the means to ask more ambitious and precise questions about RNA regulation. RBP-eCLIP has opened the door to systematically and rigorously exploring their RNA targets, transforming our understanding of gene regulation in development and disease.

Jump-start your RNA biology research today with pre-validated RBP-eCLIP reagents from CST:

Learn More: RNA Biology Blogs

• tRNA Modification by m7G MTase: Understanding its Role in Cancer and Developmental Disorders by guest author Richard I. Gregory, PhD, Professor in the Department of Biological Chemistry and Molecular Pharmacology at Harvard Medical School

• M6A: A Cell’s Hidden Signal of Gene Regulation Through RNA by guest author Gina Lee, PhD, Assistant Professor of Microbiology and Molecular Genetics at the University of California Irvine and Chao Family Comprehensive Cancer Center

Select References

1. Schwarzl T, Sahadevan S, Lang B, et al. Improved discovery of RNA-binding protein binding sites in eCLIP data using DEWSeq. Nucleic Acids Res. 2023;51(1):e1. doi:10.1093/nar/gkad998 
2. Schmok JC, Jain M, Street LA, et al. Large-scale evaluation of the ability of RNA-binding proteins to activate exon inclusion. Nat Biotechnol. 2024;42:1429–1441. doi:10.1038/s41587-023-02014-0
3. Han H, Best AJ, Braunschweig U, et al. Systematic exploration of dynamic splicing networks reveals conserved multistage regulators of neurogenesis.