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Protein Proximity Labeling Approach Enables More Precise Investigation of RNA Localization


NEW YORK (GenomeWeb) – Researchers at the National Institute of Arthritis and Musculoskeletal and Skin Disease (NIAMS) have developed a proximity labeling-based approach to study RNA-protein binding and RNA localization.

Detailed in a study published this week in Nature Methods, the technique could enable researchers to better investigate the role of RNA-binding proteins in regulating RNA and how RNA localization is linked to biological processes and disease states, said Markus Hafner, leader of the RNA Molecular Biology group at NIAMS and senior author on the paper.

The method, named Proximity-CLIP, combines two previously developed approaches — APEX protein proximity labeling and the use of UV crosslinking to irreversibly bind RNA to RNA-binding proteins — to identify RNA and RNA-binding proteins and quantify transcripts according to their location in the cell.

Hafner said the approach could help researchers better understand the processes involved in post-transcriptional gene regulation.

"Once an RNA is made, it is still subject to a lot of regulation," he said. "That regulation basically controls the maturation of the RNA, the modification of the RNA, the export of the RNA to the cytoplasm, its localization, translation, and turnover. And all of these different processes are controlled by RNA-binding proteins."

Hafner said that UV crosslinking allows researchers to effectively identify RNA-protein interactions, and, when combined with limited RNA [sequencing] can be used to identify the precise RNA element a protein interacts with.

However, he said, this still leaves the researchers without information on when and where in the cell a given RNA and protein are interacting.

Biochemical fractionation methods can help researchers obtain some localization data, Hafner said, but he noted that they aren't able to reliably isolate certain cellular compartments.

To obtain better localization data, Hafner and his colleagues turned to APEX labeling, a technique typically used for studying protein localization and protein-protein interactions. APEX labeling uses engineered ascorbic acid peroxidase (APEX) tags that are genetically inserted into proteins of interest. Upon stimulation with hydrogen peroxide, this tag releases biotin-phenoxyl radicals that tag nearby proteins in the cell, which can then be pulled out of the sample using streptavidin-based enrichment and analyzed using mass spec.

APEX tags can be inserted into proteins known to be localized to a specific cellular compartment or region of interest. Because the tagged protein biotinylates only nearby proteins, any molecules that are biotinylated and subsequently identified by mass spec are presumably localized to the same region as the APEX-tagged protein.

Combining this approach with UV crosslinking of RNA to RNA-binding proteins allowed the NIAMS team to gather data on RNA localization. And, Hafner said, because RNA is essentially never without a protein interactor, the technique allowed the researchers to quantify the levels of transcripts present in a given compartment.

"The assumption is that wherever RNA is in the cell, it is interacting with proteins," he said, adding that this assumption is "fairly well supported by the literature."

"So, when you've crosslinked RNA and RNA-binding proteins and then you isolate the [RNA-binding] proteins in that compartment, you will get, quantitatively, [a read out of] the RNA that was in that compartment," he said.

Hafner added that researchers can use limited digestion of captured RNA to isolate the portion bound to interacting proteins, which can then be sequenced to provide information on what sequence elements of RNA might be important for localization and regulation in a particular cellular compartment.

He said that while in the Nature Methods study, he and his colleagues were primarily interested in analysis of mRNA, the technique can also help researchers explore the function of non-coding RNAs like microRNA that play roles in post-transcriptional gene regulation.

Hafner said that the Nature Methods work was largely a proof-of-concept, but his lab has since begun using the technique to investigate the role of RNA localization in neurological conditions.

"It is well established that RNA localization plays an important role in neurons, that mRNAs are localized to the axons" he said. "We want to see how many mRNAs are there. Also, you have some neurological diseases that are characterized by mis-localization of these RNA-binding proteins, so we would like to see how, if you mis-localize an RNA-binding protein, that will impact the local transcriptome. We want to correlate that in the context of neurological disease."

As the authors noted, a team led by Stanford University researcher Alice Ting, developer of the APEX tagging technique, published a similar method for localization of RNA last year.

Using that approach, named APEX-RIP, Ting and her colleagues localized RNA to subcellular compartments including the mitochondrial matrix, nucleus, cytosol, and endoplasmic reticulum.

Hafner and his coauthors wrote that their Proximity-CLIP approach potentially improved upon the APEX-RIP method in that it uses UV crosslinking while the APEX-RIP approach uses formaldehyde crosslinking, which they said had the potential to crosslink distant RNAs and proteins that might not be true interactors.

Also this year, a team led by Stanford University researcher Paul Khavari published a study similarly using proximity labeling to investigate RNA-protein binding. Called RaPID (for RNA-protein interaction detection), the approach uses the biotin ligase BirA* attached not to a protein of interest but rather to an RNA molecule of interest, where it then biotinylates proteins bound to that RNA. The biotinylated proteins can then be pulled down via streptavidin capture and analyzed by mass spectrometry or other methods.

Discussing the work, Muthukumar Ramanathan, a graduate student in Khavari's lab, said that it aimed to address limitations of RNA-protein crosslinking experiments, primarily the facts that such experiments require large numbers of cells and that they are biased towards the identification of longer RNAs, which can make analysis of short RNA motifs more challenging.