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

Abstract

Circular RNAs (circRNAs) are regulatory RNAs which have recently been shown to have clinical significance in several diseases, including, but not limited to, various cancers, neurological diseases and cardiovascular diseases. The function of such regulatory RNAs is largely dependent on their subcellular localization. Several circRNAs have been shown to conduct antagonistic roles compared to the products of the linear isoforms, and thus need to be characterized distinctly from the linear RNAs. However, conventional fluorescent in situ hybridization (FISH) techniques cannot be employed directly to distinguish the signals from linear and circular isoforms because most circRNAs share the same sequence with the linear RNAs. In order to address this unmet need, we adapted the well-established method of single-molecule FISH by designing two sets of probes to differentiate the linear and circular RNA isoforms by virtue of signal colocalization. We call this method ‘circular fluorescent in situ hybridization’ (circFISH). Linear and circular RNAs were successfully visualized and quantified at a single-molecule resolution in fixed cells. RNase R treatment during the circFISH reduced the levels of linear RNAs while the circRNA levels remain unaltered. Furthermore, cells with shRNAs specific to circRNA showed the loss of circRNA levels, whereas the linear RNA levels were unaffected. The optimization of the in-situ RNase R treatment allowed the multiplexing of circFISH to combine it with organelle staining. CircFISH was found to be compatible with multiple sample types, including cultured cells and fresh-frozen and formalin-fixed tissue sections. Thus, we present circFISH as a versatile method for the simultaneous visualization and quantification of the distribution and localization of linear and circular RNA in fixed cells and tissue samples.

Regulation of microRNA by circular RNA

“…The circFISH and ddPCR can be instrumental in assessing the actual copy number of circRNA, miRNA, and mRNA in the regulatory axis (A. Das et al, 2022;Koppula et al, 2022). It is also important to acknowledge that hundreds of other transcripts with the same MRE expressed in any given condition are also part of the ceRNA crosstalk (Tay et al, 2014).…”

“…Comprehensive analysis of the actual copy number of miRNA in question, MREs in target circRNA, and mRNAs in a specific condition may help identify genuine miRNA sponges that regulate downstream target genes. The circFISH and ddPCR can be instrumental in assessing the actual copy number of circRNA, miRNA, and mRNA in the regulatory axis (A. Das et al, 2022; Koppula et al, 2022). It is also important to acknowledge that hundreds of other transcripts with the same MRE expressed in any given condition are also part of the ceRNA crosstalk (Tay et al, 2014).…”

“…This method relies on Cy3‐labeled circRNA and FAM‐labeled or Alexa‐labeled miRNA subjected to hybridization followed by confocal microscopy (Figure 3e; Lv et al, 2023; Y. Wang et al, 2023). Moreover, a newer single‐molecule resolution‐based method called circFISH has been recently developed to enhance the specificity of circRNA probes, which can be utilized in conjugation with miRNA‐FISH to support the interaction of circRNA–miRNA in vivo (Koppula et al, 2022).…”

Identification of Novel Circular RNAs of the Human Protein Arginine Methyltransferase 1 (PRMT1) Gene, Expressed in Breast Cancer Cells

“…Another interesting approach would be to examine their expression in exosomes, given that exosomal secretion of various circRNAs is reported to affect breast cancer aggressiveness, mostly through aberrant signaling [ 64 ], as well as therapy resistance [ 65 ]. Moreover, the possible functions of these circRNAs could be validated by visualizing their distribution and cellular localization by circFISH [ 66 ], or by verifying in which compartments are the circRNAs localized by circRNA expression profiling among the subcellular fractions [ 67 ]. In addition, the circRNA–miRNA interactions could be further examined by tagging a novel PRMT1 circRNA for affinity pulldown assays using a biotinylated antisense oligonucleotide that targets the back-splice junction and can be pulled down specifically with streptavidin-coated beads [ 68 ].…”

Novel circular RNAs of the apoptosis‐related BAX and BCL2L12 genes identified in a chronic lymphocytic leukemia cell line using nanopore sequencing

“…A set of probes was designed to target the regions of BAX and BCL2L12 found in each respective circRNA (Table S3 ), as previously described [ 21 ]. Briefly, the probes were synthesized with a terminal ‐NH 2 modification by LGC Biosearch Technologies Inc (Berlin, Germany).…”