New CLIP method maps where RNA-binding proteins work inside living cells

Inside every cell, hundreds of RNA-binding proteins patrol the RNA landscape, guiding molecules through splicing, translation, decay, and localization. Yet a persistent blind spot has frustrated biologists for years: most of these proteins act in several compartments at once, and conventional techniques cannot tell where in the cell a protein touched a particular RNA. Now, a team reporting in Genome Biology has unveiled RBProximity-CLIP, a method that resolves this ambiguity by capturing RNA-binding protein interactions separately in each subcellular neighborhood while preserving nucleotide-level detail of exactly where each protein bound.

The technique, developed by Iwona Nowak, Ahsan H. Polash, Hang T. Huynh, Mahekdeep Kaur and colleagues in the laboratories of Aishe A. Sarshad, Markus Hafner, and Daniel Benhalevy, fuses two powerful approaches that had never been married at this scale. The first is APEX2-based proximity labeling, an enzymatic tagging system that marks proteins and nucleic acids within a narrow radius of a genetically targeted protein. The second is 4-thiouridine-enhanced ultraviolet crosslinking, which covalently locks RNA-binding proteins onto their RNA targets and allows precise identification of crosslinked nucleotides. By combining these with sequential affinity purifications, first for the RNA-binding protein of interest and then for the biotin tag, the researchers built a pipeline that isolates only those protein-RNA contacts that occurred in the marked compartment.

The logic of the method is elegant in its layering. A protein of interest, such as the RNA-binding factor being studied, is expressed as a fusion with APEX2, a peroxidase that in the presence of biotin-phenol and hydrogen peroxide converts nearby molecules into biotin-tagged derivatives. Cells are first treated with 4-thiouridine, which is incorporated into newly synthesized RNA and dramatically strengthens ultraviolet-induced crosslinking between RNA and any protein touching it, a strategy borrowed from fPAR-CLIP protocols. When ultraviolet light fires, RNA-binding proteins are frozen onto their targets with nucleotide precision. APEX2 then labels everything in its immediate vicinity with biotin, creating a spatial signature.

What follows is a double selection. The researchers purify the RNA-binding protein and its crosslinked RNA fragments through an initial affinity step, recovering crosslinked RNA with mutations that reveal the exact binding sites, the hallmark of enhanced crosslinking-and-immunoprecipitation approaches. They then recover only those complexes that also carry the APEX2-deposited biotin tag in a second purification. A transcript must pass both filters to be counted: it must have been physically touched by the protein and must have been within the proximity-labeling radius, effectively bracketing the interaction in space and time. The result is a map of protein-RNA contacts that is simultaneously compartment-specific and nucleotide-resolved.

Control experiments were central to validating the approach. The team confirmed that APEX2 fusion proteins localized correctly to their intended compartments and that proximity-dependent biotinylation was restricted to the expected neighborhoods, presenting this evidence across multiple supplementary figures alongside imaging and immunoblot analyses. Without accurate spatial confinement, any apparent compartment specificity could simply reflect leakage or overexpression artifacts, so establishing the fidelity of the labeling step was essential before biological conclusions could be drawn.

With the method proven, the researchers turned it loose on three of the most intensively studied RNA-binding proteins: AGO2, the central effector of the microRNA silencing machinery; YBX1, a multifunctional nucleic acid-binding protein involved in mRNA stability and translation; and ELAVL1, better known in some circles as HuR, a regulator of mRNA stability and stress responses. All three proteins are known to operate in multiple cellular compartments, making them ideal test cases for a technology designed to disentangle location-dependent behavior.

The findings were striking. Each of the three proteins displayed distinct, compartment-specific RNA-binding patterns, targeting different sets of transcripts and even different sequence contexts depending on where in the cell it was acting. Importantly, the researchers found that these spatial differences arose through canonical motif recognition: the same proteins continued to recognize their characteristic sequence motifs in each compartment, but the repertoire of available targets, and thus the functional consequences of binding, shifted dramatically with location. In other words, the proteins did not change their binding preferences so much as their access to substrates changed, partitioning the accessible RNA targets and reshaping each protein’s regulome across the cell.

This partitioning concept has broad implications. For AGO2, compartment-specific mapping opens a window onto how microRNA targeting might differ between the cytoplasm, where canonical silencing occurs, and other locales where AGO2 has been implicated in less conventional roles. For YBX1 and ELAVL1, both implicated in cancer biology and stress physiology, knowing which transcripts are bound in which compartments could clarify how these proteins execute distinct functions from seemingly identical biochemical activities. The method essentially converts a single flat binding profile into a set of spatially annotated maps, revealing that what looked like one interactome is actually several overlapping ones.

Technically, RBProximity-CLIP also demonstrates the value of the 4-thiouridine enhancement inherited from fPAR-CLIP. Enhanced crosslinking increases crosslinking efficiency and yields the characteristic signature of crosslink-induced mutations, allowing binding sites to be pinpointed to individual nucleotides. Combined with proximity tagging, this delivers a rare combination of spatial and sequence resolution in a single experiment, something neither classical CLIP nor proximity labeling alone can provide. The authors provide detailed reagent lists, including antibodies, detection reagents for imaging and immunoblotting, and oligonucleotide sequences for library preparation, alongside full-length uncropped gel and blot images, underscoring the reproducibility infrastructure that accompanies methodological advances of this kind.

The work was a genuinely international effort, jointly led from the University of Gothenburg’s Wallenberg Centre for Molecular and Translational Medicine, the RNA Molecular Biology Laboratory at the National Institute of Arthritis and Musculoskeletal and Skin Diseases in Bethesda, and Tel Aviv University, with support from the Swedish Research Council, the Knut and Alice Wallenberg Foundation, the Swedish Society for Medical Research, the National Institutes of Health Intramural Research Program, the Israel Science Foundation, and the Hellenic Foundation for Research and Innovation. Published as an open-access article in Genome Biology, the study arrives at a moment when the field is increasingly aware that spatial context is not a luxury but a fundamental dimension of gene regulation. As RBProximity-CLIP is adopted and extended, it promises to turn the cell from a featureless bag of molecules into a mapped territory, where every protein-RNA encounter has an address, and where the grammar of gene expression can finally be read with both sequence and location in view.

Subject of Research: A subcellular-resolution RNA-binding protein mapping method combining proximity labeling and enhanced crosslinking

Article Title: RBProximity-CLIP enables subcellular mapping of RNA-binding protein interactions at nucleotide resolution

Article References: Nowak, I., Polash, A. H., Huynh, H. T., Kaur, M., Lobo, V., Scutenaire, J., Fong, M., Alluhaibi, G., Anastasakis, D. G., Hafner, M., Benhalevy, D., & Sarshad, A. A. (2026). RBProximity-CLIP enables subcellular mapping of RNA-binding protein interactions at nucleotide resolution. Genome Biology. https://doi.org/10.1186/s13059-026-04278-6

Image Credits: AI Generated

DOI: 10.1186/s13059-026-04278-6

Keywords: RNA-binding proteins, RBProximity-CLIP, proximity labeling, APEX2, CLIP-seq, AGO2, YBX1, ELAVL1, 4-thiouridine, subcellular resolution, protein-RNA interactions, Genome Biology