Seeing Is Believing: Visualizing Circular RNAs

Abstract

Advancement in the RNA sequencing techniques has discovered hundreds of thousands of circular RNAs (circRNAs) in humans. However, the physiological function of most of the identified circRNAs remains unexplored. Recent studies have established that spliceosomal machinery and RNA-binding proteins modulate circRNA biogenesis. Furthermore, circRNAs have been implicated in regulating crucial cellular processes by interacting with various proteins and microRNAs. However, there are several challenges in understanding the mechanism of circRNA biogenesis, transport, and their interaction with cellular factors to regulate cellular events because of their low abundance and sequence similarity with linear RNA. Addressing these challenges requires systematic studies that directly visualize the circRNAs in cells at single-molecule resolution along with the molecular regulators. In this review, we present the design, benefits, and weaknesses of RNA imaging techniques such as single-molecule RNA fluorescence in situ hybridization and BaseScope in fixed cells and fluorescent RNA aptamers in live-cell imaging of circRNAs. Furthermore, we propose the potential use of molecular beacons, multiply labeled tetravalent RNA imaging probes, and Cas-derived systems to visualize circRNAs.

The Circ_0001367/miR-545-3p/LUZP1 Axis Regulates Cell Proliferation, Migration and Invasion in Glioma Cells

“…Therefore, it is of great significance for the clinical diagnosis and treatment of glioma to focus on the pathogenesis of glioma. CircRNAs have become a hot topic in cancer research since the recent replacement of the traditional view of circRNAs as non-functional molecules with the understanding that they influence various cellular activities (9,26). CircRNAs are not sensitive to nucleases, so it is more stable than the corresponding linear RNAs, which makes them a significant advantage as a new clinical diagnostic marker and target for therapy (27).…”

CircFISH: A Novel Method for the Simultaneous Imaging of Linear and Circular RNAs

“…High-resolution imaging also provides insights into the heterogeneous expression in clinical samples, especially in solid tumors where cells at different stages of tumorigenesis can coexist in the tumor microenvironment. Therefore, it is very important to develop novel techniques to image the distribution and associations of circRNAs at a high resolution [ 27 ].…”

“…Recently, linear DNA nanostructures were developed to image circRNAs [ 30 ]. These and other similar imaging modalities were recently reviewed extensively by Bejugam et al [ 27 ]. This review also highlighted the unmet need for sensitive and reliable methods for circRNA imaging.…”

Squaring the circle: The expression and function of axon guidance circular RNAs during neuronal development

“…In contrast to the agonistic eff ect of QKI, the RBP ADAR1 (Adenosine deaminase acting on RNA 1) inhibits the formation of circRNAs by binding double stranded RNA (Figure 2) 53,[58][59][60][61] .…”

“…There is an urgent need for the development of better genetic, biochemical and imaging tools to further elucidate circRNA expression and function. For instance i) the development of RNA-seq algorithms and databases that focus on detecting tissue-specifi c circRNAs instead of only novel circRNAs can provide better insight into circRNA spatiotemporal expression, ii) experimental validation of databases that predict function(s) of circRNAs by interaction with cellular factors are necessary, iii) improved probe design is important to enhance circRNA detection sensitivity, and v) if we can tag circRNA in live cells (systems to visualize circRNA in live cells have been proposed, but not yet described), we could follow their expression dynamics and get a better understanding of their subcellular localization 29,58 .…”