Characterizing the circular RNA circPCNX through BaseScope RNA FISH

In eukaryotic cells, a transcribed pre‐mRNA is spliced by the spliceosome, a catalytic machinery comprising proteins and noncoding RNAs. Splicing typically removes introns, but it can also generate circular RNAs (circRNAs). The body of the circRNA is fully present in the parent pre‐mRNA; the only unique sequence is found where the ends of the linear RNA are ligated—the junction. Initially, circRNAs were thought to be inert byproducts of this splicing process. However, in recent years, it has been discovered that circRNAs may have important functions within the cell, including acting as a microRNA “sponge” that limits the functions of the miRNA. Through this and other functions, circRNAs have been implicated in many diseases including cancer. The goal of this study is to visualize a particular circRNA in the cell. We have adopted the BaseScope technology (Advanced Cell Diagnostics, Newark, CA) to locate and visualize a circRNA via in situ hybridization. This technology utilizes a “ZZ”‐shaped probe to target specific sequences in RNA and greatly amplify a chromogenic signal. This method allows for probe hybridization to small RNA sequences, which is essential for visualization of circRNA, as the probe is specific to the circRNA junction sequence and will not hybridize to the pre‐mRNA. We have performed this method in HeLa cells to identify the circRNA circPCNX , a circRNA that associates with the cancer‐related RNA‐binding protein AUF1. We have also visualized PCNX mRNA, the linear counterpart of circPCNX. Ongoing BaseScope analysis reveals punctate dots, each representing a single instance of circPCNX , which allows for quantification of RNA copy numbers as well as visualization of their subcellular localization. Preliminary findings suggest that circPCNX exists in low abundancy in HeLa cells, while PCNX mRNA is found to be more abundant, and exists in both the cytoplasm and nucleus of HeLa cells. This method provides both descriptive and quantitative analyses of circPCNX that complement computational strategies to investigate circPCNX levels and localization in HeLa cells. Efforts to visualize circPCNX and other circRNAs shed light on the dynamics and functions of circRNAs and allow us to harness their therapeutic potential. Support or Funding Information This work was supported by the National Institute on Aging Intramural Research Program of the NIH.