A surreal illustration showing RNA-protein interactions within a cell.

Unlock Your Cells' Secrets: Proximity-CLIP Reveals RNA's Hidden World

Soraya Malik in Science & Nature December 2025 • 4 min read.

"Discover how a groundbreaking technique is mapping RNA-protein interactions within cells, offering new insights into gene expression and disease."

For years, scientists have been working to understand exactly where RNA goes and what it does inside our cells. Knowing this is key to understanding how our genes work and how things can go wrong in diseases. The problem? Figuring out RNA's location and partners in the tiny, complex world of a cell is seriously difficult.

A new method called Proximity-CLIP is changing the game. It's like a super-powered microscope that can freeze a moment in time and reveal all the proteins and other molecules hanging out with a specific RNA molecule inside a cell. This isn't just about seeing where things are; it's about understanding the dynamic processes that control our genes.

This article dives into the exciting world of Proximity-CLIP, explaining how it works, what it's already revealed about RNA's behavior, and why it matters for future medical breakthroughs.

Proximity-CLIP: Capturing RNA's Interactions in High Definition

A surreal illustration showing RNA-protein interactions within a cell.

Proximity-CLIP works by combining two powerful techniques: APEX2-mediated proximity biotinylation and photoactivatable ribonucleoside-enhanced crosslinking. Basically, it's a way to tag everything near a specific protein in a cell and then use UV light to freeze those interactions in place. Here’s the breakdown:

First, researchers attach an APEX2 enzyme to a protein that hangs out in a specific location inside the cell – the nucleus, cytoplasm, or even the cell-cell interface. This APEX2 acts like a beacon, tagging nearby molecules with biotin when activated.

RNA Labeling: Next, cells are fed with 4-thiouridine (4SU), a modified building block that gets incorporated into newly made RNA.
Biotinylation: When hydrogen peroxide is added, APEX2 goes to work, biotinylating all the proteins in its immediate vicinity.
Crosslinking: UV light is then used to crosslink the 4SU-labeled RNA to any proteins it's directly interacting with. This essentially glues the RNA and proteins together.
Isolation and Analysis: Finally, the biotin tag is used to pull out all the tagged proteins and their crosslinked RNA. Scientists can then use techniques like mass spectrometry and RNA sequencing to identify exactly which proteins are binding to which RNAs.

This method is special because it doesn't require breaking the cell apart to study its contents (fractionation). It also allows researchers to study RNA processing as it's happening and to pinpoint exactly where regulatory elements on the RNA are being occupied by proteins.

What Proximity-CLIP Has Revealed So Far

Researchers have already used Proximity-CLIP to uncover some surprising details about RNA's life inside the cell. For example, they've found that transcription, the process of making RNA from DNA, sometimes continues far beyond the expected stopping point.

They've also noticed that the proteins bound to RNA molecules differ depending on whether the RNA is in the nucleus or the cytoplasm, suggesting that RNA is regulated differently in these two compartments. Furthermore, RNAs located at cell-cell interfaces, the points where cells connect, often encode regulatory proteins and contain specific sequences that bind to proteins.

Proximity-CLIP is opening up a whole new world of possibilities for understanding how our genes are controlled. By providing a detailed picture of RNA's interactions within the cell, this technique could lead to new therapies for a wide range of diseases.

About this Article -

This article was crafted using a human-AI hybrid and collaborative approach. AI assisted our team with initial drafting, research insights, identifying key questions, and image generation. Our human editors guided topic selection, defined the angle, structured the content, ensured factual accuracy and relevance, refined the tone, and conducted thorough editing to deliver helpful, high-quality information.See our About page for more information.

This article is based on research published under:

DOI-LINK: 10.1038/s41592-018-0220-y, Alternate LINK

Title: Proximity-Clip Provides A Snapshot Of Protein-Occupied Rna Elements In Subcellular Compartments

Subject: Cell Biology

Journal: Nature Methods

Publisher: Springer Science and Business Media LLC

Authors: Daniel Benhalevy, Dimitrios G. Anastasakis, Markus Hafner

Published: 2018-11-26

Everything You Need To Know

How does Proximity-CLIP capture RNA interactions in cells?

Proximity-CLIP combines APEX2-mediated proximity biotinylation and photoactivatable ribonucleoside-enhanced crosslinking. First, APEX2, attached to a protein in a specific cell location, tags nearby molecules with biotin. Cells are then fed 4-thiouridine (4SU), which gets incorporated into new RNA. Next, hydrogen peroxide activates APEX2 to biotinylate nearby proteins. UV light crosslinks the 4SU-labeled RNA to interacting proteins, essentially gluing them together. Finally, biotin tags are used to isolate the tagged proteins and their crosslinked RNA, which are then analyzed using mass spectrometry and RNA sequencing to identify the specific RNA-protein interactions. This differs from cell fractionation methods that require breaking the cell apart.

What surprising details about RNA behavior have researchers uncovered using Proximity-CLIP?

Researchers have used Proximity-CLIP to discover unexpected aspects of RNA's behavior within cells. For example, they've found that transcription, the process of making RNA from DNA, can extend beyond the anticipated stopping points. The implications of such findings suggest that gene regulation is more complex than previously understood. While the specific genes regulated were not detailed, this highlights how Proximity-CLIP can reveal new regulatory mechanisms.

What are the benefits of using Proximity-CLIP, and what are the potential limitations?

Proximity-CLIP offers advantages by allowing researchers to study RNA processing in real-time and identify where regulatory elements on the RNA are occupied by proteins, all without breaking the cell apart. However, the technique's limitations weren't discussed, such as potential biases in identifying interactions due to the proximity of APEX2 or the efficiency of crosslinking. Addressing these limitations will refine our understanding of RNA dynamics in cell biology.

What is the role of 4-thiouridine (4SU) in Proximity-CLIP?

4-thiouridine (4SU) is a modified building block of RNA that is used in the Proximity-CLIP method. Cells are fed 4SU, which gets incorporated into newly made RNA. When UV light is applied, the 4SU-labeled RNA crosslinks to any proteins it is directly interacting with. This crosslinking step is essential for 'gluing' the RNA and proteins together so they can be isolated and identified later. Without the use of 4SU, the crosslinking of RNA to proteins would not be possible, and the interacting molecules could not be isolated.

What is the purpose of APEX2 in the Proximity-CLIP process?

APEX2 is an enzyme used in the Proximity-CLIP method. Researchers attach APEX2 to a protein that resides in a specific location inside the cell, and APEX2 acts like a beacon, tagging nearby molecules with biotin when activated by hydrogen peroxide. This biotinylation of nearby proteins is crucial for isolating and analyzing RNA-protein interactions. The tagged molecules can then be pulled out and examined using techniques like mass spectrometry and RNA sequencing. Without APEX2, the method would be unable to identify the molecules that are in close proximity to the protein being studied.