Inquiry Basket
What is circRNA-Protein Interaction?
Currently, circRNA has emerged as a hot topic in RNA research due to its diverse biological functions, ranging from gene expression regulation to protein encoding and mRNA competition. As a covalently closed circular molecule, circRNA exhibits greater stability compared to other RNA, a crucial trait that may prove beneficial in its future development as a biological marker. CircRNA has also been shown to serve as a useful molecule for targeting multiple diseases, including diabetes, neurological disorders, cardiovascular diseases, chronic inflammatory diseases, and cancer. Its primary function involves acting as a miRNA sponge, with its secondary significant function being mediated through circRNA-protein interactions. Among the proteins most renowned for interacting with RNA molecules is RBP. RBPs are a class of proteins involved in RNA metabolism processes such as maturation, transport, localization, and translation, with some even participating in the formation of ribonucleoprotein complexes. Although the enrichment of predicted RBP binding sites in circRNA sequences is minimal, many circRNAs are expected to interact with RBPs through specific binding sites. However, RNA-RBP interactions are significantly influenced by the three-dimensional structure of RNA molecules. Hence, the unique tertiary structure of circRNA may impact its protein-binding ability, with the choice of binding mode likely depending on specific circumstances. The interaction between circRNA and proteins exhibits bidirectional effects. RNA-protein interactions can affect protein expression and function, while also regulating the synthesis and degradation of circRNA. CircRNA can act as a protein sponge or decoy to influence cellular functions, thereby regulating processes such as gene transcription, inhibition of cell cycle progression, promotion of cardiac aging, induction of cell apoptosis, stimulation of proliferation, and cell survival.
Schematic diagram of RNA-Protein Interactions.
*Related Services from BOC RNA
| Services | Price |
| Custom Circular Oligonucleotide Synthesis | Inquiry |
| Custom RNA Oligonucleotides Synthesis | Inquiry |
Studying CircRNA-Protein Interactions with a Focus on Proteins
RNA Immunoprecipitation (RIP)
RNA Immunoprecipitation (RIP) has evolved as one of the most commonly used standard techniques for detecting and analyzing the association between RNA-binding proteins (RBPs) and their target RNAs, such as circular RNAs (circRNAs). In RIP, the desired RBP is extracted from cell lysates under antibody mediation. The RBP-RNA complex is then washed to remove nonspecific binding molecules while preserving the native complex. Subsequently, the captured RNA is purified and subjected to RNA identification and analysis, such as through RT-qPCR or sequencing. Crosslinking of proteins to RNA before cell lysis, facilitated by formaldehyde or UV irradiation, is optional and enhances the capture of weak or transient interactions. RIP experiments analyzed by RT-qPCR have been widely employed in the circRNA field. Additionally, RIP-seq offers the advantage of detecting relevant circular RNAs at the transcriptome level. Given the typically low abundance of most circRNAs, deep sequencing is required to obtain reads spanning the circular structure. Some RIP-seq experiments have revealed specific protein bindings to circRNAs.
Crosslinking and Immunoprecipitation (CLIP)
Crosslinking and Immunoprecipitation (CLIP) is a widely used global method for detecting protein-RNA interactions in vivo. Different versions of this technique have been developed in recent years, such as iCLIP, HITS-CLIP, PAR-CLIP, and eCLIP. Prior to cell lysis, all these methods share a UV crosslinking step in cells, which covalently links amino acid residues closely to RNA bases. Subsequently, the relevant proteins are pulled down by a specific antibody, while non-crosslinked RNA and other proteins are removed through stringent washing. CLIP technology has also been employed to identify circRNA-protein interactions, although to a lesser extent compared to RIP experiments. The first application of CLIP in the circRNA field was the AGO-CLIP experiment.
Studying CircRNA-Protein Interactions with a Focus on CircRNA
Antisense circRNP Pulldown Purification
The principle of antisense pulldown relies on specific base pairing between biotinylated antisense probes and their target RNA sequences, such as circRNA, which must be accessible within RNPs. Following cell lysis and probe hybridization, the target circular RNA is captured using streptavidin-coated beads. Through rigorous washing steps, nonspecific binders can be removed, allowing further analysis of the complex, such as through immunoblotting or mass spectrometry, to identify associated proteins.
Affinity Purification of CircRNA Using Tagged CircRNA
An alternative method for purifying circular RNA involves using internally labeled circRNA. This can be divided into two general strategies: affinity purification of in vitro labeled circRNA and affinity purification of adapter-tagged and overexpressed circRNA. The method of affinity purification of in vitro labeled circRNA relies on using internally modified circular RNA, which can be transfected into cell cultures (in vivo) or incubated with cell extracts (in vitro), allowing purification of the circular RNA through affinity matrix. The labeling of in vitro circular RNA is achieved by incorporating modified or modifiable nucleotides during transcription. The most commonly used chemical moiety is biotin, as the addition of multiple biotin residues can result in a relatively large molecule, which may significantly impair subsequent circularization efficiency. Although labeling efficiency can be fine-tuned by altering the ratio of labeled to unlabeled nucleotides during transcription, the advantage of modifiable nucleotides is that they can be biotinylated post-circularization through click chemistry.
RNase Protection
The binding of RBPs to RNA can locally protect RNA from RNase degradation, thereby defining direct interaction sites. This method for locating RBP binding sites typically focuses on specific, radiolabeled RNAs incubated with recombinant proteins or cell extracts but can also be applied in a transcriptome-wide manner. However, the fundamental principle of RNase protection was initially used for gene quantitation or identification of transcription start sites, through antisense probe hybridization and subsequent single-stranded RNAse treatment. It is noteworthy that RNase protection assays have been employed to validate the circular structure of smn-derived circRNA isoforms.
Gradient Sedimentation Analysis
A powerful and universal tool for describing circRNA-protein interactions is the analysis of crude cell extracts through density gradient ultracentrifugation. Depending on the concentration range used and the gradient material employed, resolution can be adjusted to study RNP complexes within a broad size range. In principle, two gradients can be distinguished: glycerol gradients (between 5% and 30%) for medium to small molecular weight complexes (e.g., snRNPs, circRNPs) and gradients for analyzing high molecular weight complexes (e.g., mRNPs and ribosomes).
Fluorescence In Situ Hybridization (FISH) and Immunofluorescence (IF)
The expression status of many circRNAs may vary greatly between cell types or tissues, and most circRNAs have relatively low copy numbers per cell. The low abundance of circular RNA, along with the need to distinguish it from its linear counterpart, makes imaging methods like fluorescence in situ hybridization (FISH) particularly challenging to apply. Thus, most ISH schemes for circRNA detection rely on hybridization of single labeled probes targeting the circ junction. However, recent advances in probe design and signal amplification have made single molecule detection possible. In principle, two successful single molecule FISH (smFISH) methods have been used to detect circRNA:
- Detection of circRNA based on a set of oligonucleotides labeled with fluorescent molecules at the 3' end. As this set of probes can also detect the corresponding linear mRNA, this method is particularly suitable for circRNA without abundant linear counterparts or treated with RNAse R.
- Amplified visualization of circRNA (ViewRNA, RNAscope, BaseScope) using double z-probes on the circ-junction. In this case, hybridization of the two probes of the double z pair results in signal generation and amplification, achieving specificity for circRNA.
Prospects of circRNA-Protein Interactions Analysis
CircRNA, a large class of independent non-coding RNAs, was only discovered in the past decade, and their functions are just beginning to be understood. The methods used to discover circRNA-protein interactions have made significant contributions to our understanding of circRNA function. Experimentally, most protein-centric methods rely on appropriate, specific antibodies, while some circRNA-centric methods depend on the introduction of RNA modifications. The latter is particularly challenging for circRNA because conventional end-labeling techniques are not applicable, and modifications should not hinder RNA circularization or disrupt RNA structure. Therefore, to date, only a few fluorescence-based methods have been adopted among the many available techniques. So far, fluorescent probes have been inserted into circRNAs for visualization, relying on tRNA splicing-associated methods; theoretically, other circRNA labeling methods could be used. The application of visualizing circRNA in CRISPR-Cas-engineered cell lines expressing fluorescently labeled proteins holds future potential for studying circRNA-protein interactions.
* Only for research. Not suitable for any diagnostic or therapeutic use.
