Why is Circular RNA(circRNA) a Hot Topic in Gene Regulation?

What is Circular RNA?

Over the last few decades, there has been a great change in the biological role of RNA. It was previously considered as merely an intermediary between DNA and proteins, serving merely as a passive carrier of genetic information. However, the modern view of RNA extends its functions far beyond such a basic role. The identification of circRNA greatly increases the depth of RNA biology. These RNA molecules are generated by a special splicing mode, resembling molecular origami, and form closed circular structures that distinguish them from the traditional linear RNAs in biological functions. CircRNA was initially considered a splicing byproduct, but with the development of research, it has been found to be widely expressed in many organisms and plays an important role in cancer, cardiovascular diseases, Alzheimer's disease, and other pathological processes. Moreover, circRNAs' stability and regulatory capacity also made them promising candidates both for therapeutic agents and biomarkers.

Schematic representation of the function and mechanism of cyclic RNA. (Liu, X.; et al, 2013)

The formation of circRNA originates from a unique back-splicing mechanism, which is entirely different from the conventional precursor mRNA splicing that forms linear mRNAs through intron removal and exon joining. Back-splicing causes the 3' end of an exon to reverse and connect to the 5' end of an upstream exon, forming a circular structure. This distinct structure not only gives circRNA non-linear characteristics but also imparts a variety of functions. CircRNAs have been found to act as regulatory factors within the cell and interact with various RNA binding proteins (RBPs) and other RNA molecules, playing crucial roles in gene expression regulation, cell differentiation, and disease progression.

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Circular RNA Functions

Although circRNAs are relatively rare in cells, comprising only about 0.1% of non-ribosomal RNA, they exhibit a wide range of functions, significantly influencing cellular processes. The formation of circRNA involves a special back-splicing event (back-splice junction), making its structure fundamentally different from linear RNA. The unique structure of circRNA endows it with diverse functions, primarily including:

Interaction with MicroRNA (miRNA)

CircRNAs can act as "sponges" for miRNAs, binding to them and thereby modulating their inhibition of target mRNAs. This mechanism, known as the "miRNA sponge effect," reduces the activity of miRNAs, indirectly increasing the expression of downstream genes. For example, circRNA Cdr1as can bind to miR-7, regulating the release of the neurotransmitter glutamate in neurons, thereby affecting neural functions. Studies have shown that Cdr1as binds to over 70 miR-7 binding sites, making it highly effective at absorbing miR-7 and playing a crucial regulatory role in the nervous system.

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Interaction with RNA Binding Proteins (RBPs)

CircRNAs can regulate the activity of RNA-binding proteins by interacting with them or serve as scaffolds for protein-protein interactions. Specifically, some circRNAs enhance or support protein interactions as scaffolds, influencing signaling pathways and gene expression regulation. For instance, circRNA circ-Foxo3 has been found to bind with cell cycle regulatory proteins, inhibiting cell cycle progression and negatively regulating cell proliferation. Additionally, circZKSCAN1 interacts with RBPs to suppress liver cancer cell metastasis and invasion, indicating that circRNAs play a key role in cancer development.

Coding Functional Peptides

While most circRNAs lack coding ability, under certain conditions, they can be translated into biologically functional peptides. A 2023 study demonstrated that some circRNAs could translate into functional peptides that play significant roles in cellular processes. For example, circ-SHPRH encodes a peptide, SHPRH-146aa, which suppresses malignant proliferation in gliomas. Additionally, circFBXW7 encodes a peptide (FBXW7-185aa) related to cell cycle regulation, revealing the potential therapeutic roles of circRNAs in cancer and other diseases.

Important Role in Gene Regulation

CircRNAs can also directly interact with DNA, affecting gene transcription. Some circRNAs bind to specific genomic loci, possibly promoting gene rearrangements, a mechanism that plays a significant role in the development of cancers such as leukemia. For example, circSMARCA5 has been shown to bind to a specific gene promoter region, thereby regulating gene transcription. These circRNAs have important roles in chromatin remodeling, gene expression regulation, and cell fate determination.

circRNA Detection

The low abundance of circRNAs and their similarity to linear RNAs make their detection challenging. The unique characteristic of circRNA is its closed circular structure, which makes it difficult to distinguish and detect. Conventional RNA sequencing methods often overlook circRNAs because standard RNA library construction relies on the poly(A) tail of mRNAs, a feature that circRNAs lack.

• To effectively identify circRNAs, researchers have focused on detecting "back-splice junctions," the location where the 3′ end of an RNA chain is connected to the upstream 5′ end. The diversity of these splice patterns and the presence of introns increase the complexity of detection. To overcome these challenges, researchers have developed various specific tools, such as CirComPara2 and circTools, to improve detection accuracy and reliability. A 2023 comparison of 16 different tools found that the number of circRNAs detected in the same cell line ranged from 1,372 to 58,032, demonstrating significant differences in sensitivity and specificity between algorithms. Common validation methods include quantitative PCR (qPCR) and Northern blotting, which help confirm the true existence and specificity of circRNAs.
• Another effective method for circRNA detection involves removing linear RNA using RNase R, an exonuclease that degrades linear RNA without affecting circRNA, thereby enriching circRNA for subsequent analysis. However, some linear RNAs with complex secondary structures, such as G-quadruplexes, may resist RNase R, so careful control of enzyme concentration and reaction conditions is required. Additionally, high-throughput sequencing technologies, such as Illumina short-read sequencing and PacBio long-read sequencing, have enabled comprehensive identification and quantification of circRNAs.

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How to Study Circular RNA?

Validating the function of circRNAs is a critical part of exploring their biological significance.

• Researchers employ various methods to confirm the functions of these molecules, including gene knockdown and overexpression experiments. For example, RNA interference (RNAi) technology can target the back-splice junction of circRNAs to reduce their expression and investigate their roles in specific physiological or pathological states.
• In recent years, the CRISPR-Cas system has also been applied to circRNA research. By using the Cas13 enzyme to target specific sites within circRNAs, researchers can efficiently knock down circRNAs without affecting other parts of the genome. This approach has knockdown efficiency of 80-90%, significantly higher than traditional RNA interference methods. The CRISPR-Cas13 system is highly specific, targeting RNA levels without causing permanent changes to the genome, making it a powerful tool for studying circRNA function. Additionally, CRISPR-Cas9 targeting the genomic loci of circRNA formation can indirectly study its function by observing the effects of circRNA deletion on cells or organisms.
• The functional validation of circRNAs also depends on the construction of cell and animal models. For example, by overexpressing circRNAs in specific cell lines, researchers can assess their impact on cell proliferation, apoptosis, migration, and other biological processes. In vivo studies using mouse models can reveal the specific functions of circRNAs under physiological and pathological conditions. Rajewsky's team recently employed a combination of chemical and electrical stimulation, single-cell RNA imaging, and microRNA knockout to investigate the role of circRNA Cdr1as in neurons, discovering its crucial interaction with miR-7 in neurotransmitter release.

Applications of Circular RNA: From Basic Research to Clinical Use

The unique structure and function of circRNA have led to broad applications in medicine.

Circular RNA as Biomarker

circRNA has enormous potential as a biomarker. Due to its relative stability in cells and significant expression changes in specific disease states, circRNA can be used for early disease diagnosis and prognosis assessment. For example, the levels of certain circRNAs are significantly elevated in the blood of cancer patients, offering new possibilities for non-invasive cancer diagnosis. Additionally, the differential expression of circRNAs in cardiovascular diseases, neurodegenerative diseases, and other conditions makes them promising candidates for broad-spectrum diagnostic and prognostic markers.

Circular RNA Therapeutics

• circRNAs can serve as therapeutic targets or therapeutic agents. By targeting specific circRNAs, their interactions with miRNAs or proteins can be modulated, influencing downstream signaling pathways and gene expression. For instance, targeting circRNA Cdr1as can regulate miR-7 activity, producing important therapeutic effects in the nervous system.
• circRNAs can be synthesized ex vivo and introduced into the body to act as therapeutic agents directly. The stability of circRNAs makes them less prone to degradation within cells, making them an ideal molecular foundation for drug development. Research has already shown that ex vivo synthesized circRNAs can regulate immune responses and exhibit anti-cancer activity, providing a theoretical basis for their use as nucleic acid drugs or circular RNA vaccines.

Circular RNA in Gene Editing

Circular RNA (circRNA) has emerged as an exciting tool in the field of gene editing, particularly when combined with advanced technologies like the CRISPR/Cas9 system. Due to its unique structure and biological properties, circRNA can play several important roles in gene editing, enhancing the specificity and efficiency of gene manipulation processes.

Circular RNA in Cell Therapy

Moreover, the biological functions of circRNAs make them highly applicable in cell therapy. For example, circRNAs can be used as regulatory switches for gene expression, enhancing the specificity and efficacy of CAR-T cell therapies, reducing side effects, and improving therapeutic outcomes.

CircRNA research is rapidly advancing and demonstrating a wide range of biological functions and significant medical applications. As detection technologies and functional research methods continue to improve, we are likely to uncover more roles of circRNAs in cells and diseases, with the potential for clinical diagnosis and treatment applications. Further investigations will likely uncover how circRNAs can contribute to overcoming challenges in diagnostics, drug development, and disease treatment, further elevating their status as critical players in cellular biology and medicine.

Why is Circular RNA(circRNA) a Hot Topic in Gene Regulation?

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