Decoding Circular RNAs: The Next Big Thing in Biomedical Research

I. Introduction

Circular RNAs (circRNAs) represent a fascinating category of RNA molecules, distinguished by their unique properties and varied cellular functions. Unlike conventional linear RNAs, circRNAs are characterized by closed-loop structures formed through precursor mRNA back-splicing, which renders them resistant to exonuclease degradation. This distinctive nature has garnered considerable attention in the scientific community, prompting extensive research into their genesis, roles, and potential clinical uses. As of the end of 2023, there have been 7,385 studies on circRNAs, with a notable surge in research output and citations since 2015. Bibliometric Analysis of Global Research on Circular RNA: Current Status and Future Directions - Molecular Biotechnology.

II. Unveiling the Biogenesis of circRNAs

Biogenesis refers to the biological process through which living organisms produce complex molecules, such as RNA or protein, within their cells. A well-known example is the expression of a gene leading to the creation of messenger RNA (mRNA), a topic widely discussed during the development of COVID-19 vaccines. Canonical splicing, the most prevalent RNA transcript processing method in cells, involves precise cutting of the RNA at designated sites. In this process, the exons, or RNA segments, are linearly joined, while the introns, the non-coding sections, are discarded. This results in a linear RNA strand capable of being translated into a protein. The formation of circular RNAs (circRNAs) involves a unique process called back-splicing. This mechanism connects the 3' end of a downstream exon to the 5' end of an upstream exon, contrary to canonical splicing that linearly joins consecutive exons. Back-splicing competes with canonical splicing, leading to the production of circRNAs. While the precise regulatory mechanisms of circRNA biogenesis are still under investigation, it is acknowledged that certain elements, such as complementary intronic sequences, facilitate the formation of circRNAs. Numerous studies have been devoted to uncovering these regulatory mechanisms. Key factors influencing circRNA biogenesis include:

Spliceosomal Machinery: The spliceosome, a complex that mediates splicing, plays a critical role in the efficiency and selection of back-splicing.
Intronic Sequences: Complementary sequences found in introns are vital for exon circularization, aiding in the back-splicing process.
RNA Binding Proteins: These proteins, by binding to specific RNA sequences, can alter the splicing process and impact circRNA formation.
Transcriptional Pacing: The rate at which RNA polymerase transcribes can influence the splice site availability for back-splicing.
Exon-Intron Architecture: The length and composition of a gene's exons and introns can affect the likelihood of its pre-mRNA forming circRNAs.
Alternative Splicing: The presence of various splicing options, including alternative back-splicing, adds to the diversity of circRNA production. The expanding regulatory mechanisms and cellular functions of Circular RN.

An RNA molecule with a 5' cap (left) and a 3' poly(A) (right) tail (Image by DALL-E).

III. The Unique Characteristics of circRNAs

circRNAs are distinguished from linear RNAs by their unique features. Their closed-loop structure, lacking both a 5' cap and a 3' poly(A) tail, imparts remarkable stability by rendering them resistant to exonuclease degradation. This degradation process involves enzymes, known as exonucleases, which sequentially remove nucleotides from the end of nucleic acid molecules like DNA or RNA. In addition to their stability, circRNAs are characterized by their widespread abundance and conservation across various species, often exhibiting specific expression patterns tied to certain tissues or developmental stages. For instance, an analysis by circAtlas identified 413,657 circRNAs in humans, 169,618 in macaques, 175,273 in mice, 80,158 in rats, 75,953 in pigs, and 92,428 in chickens. Interestingly, most circRNAs (approximately 61.7%) were species-specific, with only 797 circRNAs being common across all six analyzed species. These attributes position circRNAs as promising biomarkers for a range of diseases, including cancer. CircAtlas: an integrated resource of one million highly accurate circular RNAs from 1070 vertebrate transcriptomes - Genome Biology

IV. Exploring the Functions of circRNAs

circRNAs exhibit a range of functions within cells, utilizing multiple mechanisms to exert their influence. A prominent function of circRNAs is their role as microRNA (miRNA) sponges. This involves sequestering miRNAs, thereby preventing them from regulating target genes. miRNAs are short, non-coding RNA molecules that attach to complementary sequences on messenger RNAs (mRNAs). This binding typically leads to gene silencing, achieved through translational repression or target mRNA degradation. By competing for miRNA binding, circRNAs regulate the availability of miRNAs for their target mRNAs, indirectly affecting gene expression. Such regulation can significantly impact various cellular processes, including differentiation, proliferation, and apoptosis. Furthermore, circRNAs contribute to cellular functionality by influencing gene splicing and transcription. They also act as decoys for proteins and RNA-binding proteins. These diverse roles of circRNAs highlight their potential implications in various diseases, notably cancer. Natural RNA circles function as efficient microRNA sponges - PubMed.

V. The Role of circRNAs in Cancer

Cancer, a complex and heterogeneous disease, is marked by the dysregulation of various cellular processes. Recent research has illuminated the role of circRNAs in the development and progression of cancer. These RNAs are implicated in critical aspects of cancer such as tumor growth, migration, invasion, and the epithelial-mesenchymal transition (EMT). EMT is a biological process wherein cells transition from an epithelial to a mesenchymal state. In their epithelial form, cells are tightly packed, forming barriers with specific functions, as seen in cells lining organ interiors. Conversely, in the mesenchymal state, cells adopt a looser, more mobile arrangement typical of connective tissues. In the context of cancer, EMT facilitates the detaching of cancer cells from the primary tumor and invading other tissues, a key factor in metastasis. The dysregulation of circRNAs in cancer cells underscores their potential as indicators for prognosis and targets for therapy. The emerging roles of circRNAs in cancer and oncology.

VI. The Clinical Relevance of circRNAs

The potential of circRNAs as biomarkers and therapeutic targets has brought them to the forefront of tumor pathology research. The advent of circRNA research has catalyzed the development of numerous databases (such as circAtlas) and specialized methodologies for circRNA analysis. These advancements have significantly aided in identifying and characterizing circRNAs, offering critical insights into their functions in cancer and other diseases. Ongoing research is actively exploring the clinical utility of circRNAs in managing and treating cancer patients.

VII. Extracellular Transport and Degradation of circRNAs

Contrary to initial beliefs that circRNAs were limited to the intracellular domain, recent findings have revealed their presence in extracellular spaces. CircRNAs have been identified in various bodily fluids, including plasma, saliva, and urine, underscoring their potential as non-invasive diagnostic tools. The mechanisms governing circRNA extracellular transport and degradation are currently under intense study. Understanding these processes is essential for shedding light on circRNA-mediated intercellular communication. Intercellular communication via circRNAs plays a crucial role in numerous cellular functions and holds significance in both physiological and pathological contexts. For instance, exosomal circRNAs can mediate interactions within and between cancerous and non-cancerous cells, influencing tumor growth and aggressiveness. Due to their stability and detectability in bodily fluids, circRNAs are promising candidates for cancer diagnosis and prognosis. Additionally, the role of exosomal circRNAs in modulating immune cell interactions with cancer cells is an area of particular interest. These interactions may either promote cancer development by creating immunosuppressive environments or bolster anti-cancer immune responses. Circular RNAs in body fluids as cancer biomarkers: the new frontier of liquid biopsies - Molecular Cancer;Novel insights into exosomal circular RNAs: Redefining intercellular communication in cancer biology;Recent advances of exosomal circRNAs in cancer and their potential clinical applications - Journal of Translational Medicine;Roles of exosomal circRNAs in tumour immunity and cancer progression.

VIII. Future Perspectives for circRNAs in Cancer Management

The recognition of circRNAs as potential biomarkers and therapeutic targets heralds new possibilities in cancer management. The development of advanced diagnostic tools utilizing circRNA profiles could significantly improve early detection and foster personalized treatment approaches. Moreover, focusing on particular circRNAs that contribute to tumor progression, metastasis, and drug resistance presents opportunities for innovative therapeutic strategies. Nonetheless, a deeper understanding of circRNA biology is essential, and ongoing research is crucial to translate these insights into clinical applications effectively.

IX. Final thoughts

The exploration of circRNAs represents an exciting and dynamic field in scientific research. Characterized by their unique biogenesis and versatile cellular functions, circRNAs have increasingly intrigued researchers, particularly regarding their implications in disease diagnosis, prognosis, and therapy. This interest is notably evident in cancer research, where the expanding knowledge of circRNAs offers promising avenues for novel strategies in battling this formidable illness. As research progresses, the potential of circRNAs to transform cancer management appears increasingly feasible.

The discovery of circular RNAs marks a significant milestone in RNA biology, unveiling new dimensions in gene regulation and cellular mechanisms. Continued research in this arena promises to deepen our understanding of diseases like cancer and foster groundbreaking diagnostic and therapeutic innovations.