Understanding RNA-Protein Interactions

Abstract

In the traditional view of cellular information processing, RNA is something of an afterthought. RNA, in this view, is literally just the messenger—the molecule that ferries genomic instructions from the nucleus to the cytoplasm or that performs the mundane task of translating that message into protein.

Today, researchers understand that RNA biology is rich with subtlety and nuance, with noncoding molecules carrying out crucial gene regulatory and epigenetic tasks. Yet RNA does not perform such feats on its own; it often acts in concert with protein partners. Researchers are understandably keen to dissect such interactions, and the set of available tools for doing so is growing rapidly. Here, we review some of the latest options available to researchers.

Protein-centric methods

Researchers interested in mapping specific protein-DNA interactions at the genome level typically use chromatin immunoprecipitation (ChIP) techniques. Similarly, researchers studying protein-RNA interactions can use RNA immunoprecipitation (RIP).

Commercialized by Active Motif, EMD Millipore and Sigma-Aldrich, among others, RIP is a straightforward translation of the ChIP process to RNA. With Sigma’s Imprint® RNA Immunoprecipitation Kit, for instance, protein-nucleic acid contacts are stabilized using a reversible formaldehyde crosslinking step. Following cell lysis, the RNAs associated with a specific RNA-binding protein are isolated by immunoprecipitation using an antibody to the desired protein, released by reversing the crosslink and then detected using either qRT-PCR or cDNA sequencing.

When applied to whole cells, the Sigma kit can detect any RNA-protein complex in the cell, whether in the nucleus or cytoplasm. But some long noncoding RNAs (lncRNAs) tend to be confined to the nuclear compartment; Active Motif and EMD Millipore have commercialized versions of this method that specifically target them.

Active Motif’s RNA-ChIP IT® kit, for instance, crosslinks RNA to chromatin to map lncRNAs that are associated with genomic DNA while eliminating those interactions that occur beyond the chromatin surface. “We are specifically looking at chromatin associations, as opposed to just nuclear RNA-protein associations,” explains product manager Kyle Hondorp.

Similarly, EMD Millipore offers kits for isolation of both native and crosslinked RNA-protein nuclear complexes, the former of which can be used to characterize high-affinity interactions, says John Rosenfeld, external innovation manager at EMD Millipore.

Jernej Ule, professor of molecular neuroscience at the University College London Institute of Neurology, studies the dynamics of RNA-protein complexes during cell differentiation using a home-brewed method he devised called UV crosslinking and immunoprecipitation (CLIP) [1].

Like RIP, CLIP uses antibodies to purify protein-RNA interactions. What makes the method unique, Ule says, is its use of ultraviolet light for crosslinking protein and RNA. Unlike formaldehyde, UV crosslinking is permanent, he explains, making it possible to subject complexes to harsher treatments and purification strategies, including SDS-PAGE. Moreover, it allows the RNA to be fragmented into short pieces, which narrows down the likely position of protein-RNA interactions.

Ule’s team has developed two variants of CLIP, including iCLIP (individual nucleotide resolution CLIP), which reveals the location of protein binding to RNA with nucleotide resolution [2]. Following crosslinking, RNA fragmentation and immunoprecipitation, the RNA is radio-labeled and coupled at the 3’ end to a linker. The protein-RNA complex is then resolved on a gel, and appropriately sized complexes are purified to eliminate nonspecific RNAs. Then the RNA is reverse transcribed, using a process that enables amplification even of cDNAs that have truncated at the crosslink sites. After sequencing, analysis of these truncated cDNAs enables a nucleotide-resolution view of protein-RNA crosslinking across the genome.

Recently, Ule’s team developed a second variant, hiCLIP (hybrid iCLIP), which reveals long-range RNA topology genome-wide, similar in concept to the chromosomal-topology method called Hi-C [3]. “It can tell you that two distal regions of an RNA are forming a duplex, or that two RNAs are contacting each other,” Ule explains.

RNA-centric methods

According to Hondorp, chromatin-associated RIP and related methods provide one piece of a larger epigenetic puzzle. Using ChIP, researchers can determine where on the chromatin a given RNA-binding protein is located; with RIP, they can trace what Hondorp calls “the next layer of complexity”—the RNAs to which that protein is bound.

But to determine which RNAs are associated at a given chromosomal location, researchers need to adopt a different strategy. Several methods have been devised to provide this information, including RAP (RNA antisense purification), CHART (capture hybridization analysis of RNA targets) and ChIRP (chromatin isolation by RNA purification). The latter of these recently was commercialized by EMD Millipore as the EZ-Magna ChIRP RNA Interactome Kit.

ChIRP reveals the locations at which an RNA is bound across the genome as well as the proteins that may be mediating those interactions. Developed in Howard Chang’s laboratory at Stanford University School of Medicine [4], ChIRP is akin to ChIP, except “it’s more like reverse-ChIP, in the sense that you’re not going after protein but nucleic acid,” Rosenfeld explains.

As in ChIP, chromatin complexes are fixed with a reversible crosslink, isolated and fragmented. Biotinylated oligonucleotides complementary to and tiling across a particular lncRNA are then hybridized to the RNA and used to pull down complexes of interest using streptavidin beads. At this point, researchers can sequence either the associated genomic DNA or proteins. The result, says Rosenfeld, is “a functional map of what genes are potentially being regulated by presence of this lncRNA.”

A more recent variant called domain-specific ChIRP (dChIRP) uses oligonucleotide pools targeting discrete RNA structural domains to detail protein-RNA interactions with greater resolution [5].

Though it has not commercialized the method per se, Sigma-Aldrich sells most of the required reagents for ChIRP and offers a detailed protocol on its website, says Vikas Palhan, the senior scientist at the company who worked out the method. 

According to Palhan, the method requires two to three days and is relatively complicated (involving, for instance, the formulation of six separate buffers and reagents). He advises researchers new to the method to first establish an appropriate cell line for testing—i.e., one that expresses the desired lncRNA to high levels—as well as effective strategies for over- and under-expressing it. You’ll need about one 20-mer biotinylated probe per hundred bases of RNA, he says, which can be designed at SingleMoleculeFISH.com. And pay particular attention to the crosslinking and sonication steps—with glutaraldehyde, “do not [crosslink for] over 10 minutes, and sonicate [the DNA] to only 400 base pairs.”

For those more broadly interested in the proteins bound by a specific lncRNA, there’s RNA EMSA (the Electrophoretic Mobility Shift Assay). Alternatively, the Thermo Fisher Scientific Pierce Magnetic RNA-Protein Pull-Down Kit could provide the answer. According to Peter Bell, senior director of research and development for protein biology products at Thermo Fisher Scientific, the kit uses a purified biotinylated RNA—either a synthetic nucleic acid, the product of in vitro transcription or a purified endogenous RNA—to pull down protein partners from cellular extracts, which then can be analyzed using Western blotting or mass spectrometry.

According to Bell, this system provides complementary information to RIP. Given a known protein, RIP identifies associated transcripts; the Pull-Down Kit, in contrast, isolates binding partners given a known RNA.

Whichever method you choose, detecting an interaction is actually the easy part; the trick is determining the significance of the observed interaction, a substantially more challenging proposition. But for those on the cutting edge of RNA biology, that’s par for the course.

* As this article was in editing, a new method for RNA-protein analysis was described in Nature Methods. Developed by Marvin Wickens of the University of Wisconsin, Madison, and colleagues, RNA Tagging uses a fusion of an RNA-binding protein of interest to a C. elegans poly(U)-polymerase to tag bound transcripts with runs of uridine residues in vivo, which then identified via sequencing. [Nat Methods, Nov. 2, 2015, doi:10.1038/nmeth.3651]

References

[1] König, J, et al., “Protein-RNA interactions: New genomic technologies and perspectives,” Nat Rev Genet, 13:77-83, 2012. [PMID: 22251872]

[2] Huppertz, I, et al., “iCLIP: Protein-RNA interactions at nucleotide resolution,” Methods, 65:274-87, 2014. [PMID: 24184352]

[3] Sugimoto, Y, et al., “hiCLIP reveals the in vitro atlas of mRNA secondary structures recognized by Staufen 1,” Nature, 519:491-4, 2015. [PMID: 25799984]

[4] Chu, C, et al., “Genomic maps of long noncoding RNA occupancy reveal principles of RNA-chromatin interactions,” Mol Cell, 44:667-78, 2011. [PMID: 21963238]

[5] Quinn, JJ, et al., “Revealing long noncoding RNA architecture and functions using domain-specific chromatin isolation by RNA purification,” Nat Biotechnol, 32:933-40, 2014. [PMID: 24997788]