What Is circRNA and how to Study It by Sequencing?

Introduction to circRNA Sequencing

circRNA is a new type of non-coding RNA (ncRNA) produced by non-sequential exon, intron, or both backsplicing. Their covalently closed loop feature, which implies they lack 5' end caps and 3' poly-A tails, differentiates them. Because of their nuclease resistance, circRNAs are highly stable forms of ncRNAs. When circRNAs were unearthed in the 1970s, they were mistakenly believed to be viral genomes or byproducts of mis-splicing events. However, recent research has revealed that circRNAs have important biological functions and are evolutionarily conserved across plants, animals, and humans.

circRNAs have the potential to act as miRNA sponges and regulate gene expression. Surprisingly, a wide range of circRNAs is abnormally expressed in specific disease contexts, implying that they are linked to the onset and progression of human diseases. circRNAs also play a role in cellular processes as translation templates, regulators of alternative splicing and gene expression, scaffolds for protein complex assembly, and modulators of rRNA and tRNA biogenesis. Due to their involvement in immune regulation and viral infection, they can also be used to boost immune activation for antiviral therapeutic purposes.

Figure 1. The basic acknowledgment of circular RNA (circRNA). (Zhang, 2020)

The first and most essential step in understanding circRNA biogenesis and function is to identify them from high-throughput transcriptome data. In a variety of organisms, high-throughput sequencing of rRNA/linear RNA-depleted RNA combined with computational tools has resulted in the identification of thousands of new circRNAs and quantitative analysis of their linear host transcripts. Due to their size and electrophoretic mobility, circRNAs, unlike miRNAs and other small RNAs, are hard to differentiate from other RNA species. As an outcome, they are abundant in rRNA-depleted libraries and enriched in RNase R-treated libraries, which only digest linear RNA.

Identification of circRNAs

The identification of circRNAs from high-throughput transcriptome data is the first and most essential part of understanding their biogenesis and function. In a variety of organisms, high-throughput sequencing of rRNA/linear RNA-depleted RNA combined with computational tools has resulted in the identification of thousands of new circRNAs and quantitative analysis of their linear host transcripts. Due to their size and electrophoretic mobility, circRNAs, unlike miRNAs and other small RNAs, are hard to differentiate from other RNA species. As an outcome, they are abundant in rRNA-depleted libraries and enriched in RNase R-treated libraries, which only digest linear RNA. Back-splicing requires both canonical splicing signals and canonical splicing machinery, according to recent research.

Bioinformatic Analysis of circRNAs

Even though circRNAs have been studied for a long time, they are difficult to detect due to outdated technology. A growing number of circRNAs have been discovered since the introduction of next-generation RNA sequencing.

The first step is to create a cDNA library. A biotinylated specific probe is used to remove the ribosomal rRNA from the total RNA. The RNA is fragmented with temperature and an ionic environment after purification. After that, dNTPs are added to create a cDNA strand, followed by DNA polymerase I and RNase H to create double-stranded cDNA. The RNA template is removed during cDNA double-strand synthesis, and dTTP is replaced with dUTP. After purification, the ligation product of the double-stranded cDNA product is amplified, followed by the addition of the "A" base and the linker, and the final cDNA library is obtained. Finally, the sequencing library that has been created is sequenced.

The raw data generated by sequencing is then analyzed in the second step. The low-quality, joint contamination, and unknown N content of the base are filtered out in the first step. Clean reads refer to the filtered data. Clean reads are plotted to the reference genome in the second step, and circRNAs are predicted using two software programs, CIRI and find circ. After integrating the results of the two software programs, the third step is to perform quantitative and differential expression analysis of circRNAs. The functional analysis of circRNAs focuses on gene expression regulation via interactions with multiple miRNAs, which can be analyzed using databases such as analysis of common targets (ACT) and CircNet.

The circRNAs are then verified using techniques such as reverse transcription-PCR (RT-PCR), droplet digital PCR, northern blotting, and fluorescence in situ hybridization.

References:

1. Cheng D, Wang J, Dong Z, Li X. Cancer-related circular RNA: diverse biological functions. Cancer cell international. 2021 Dec;21(1):1-6.
2. Liu J, Zhang X, Yan M, Li H. Emerging role of circular RNAs in cancer. Frontiers in Oncology. 2020 May 20;10:663.
3. Zhang C, Hu J, Yu Y. CircRNA is a rising star in researches of ocular diseases. Frontiers in Cell and Developmental Biology. 2020;8.
4. Kumar-Sinha C, Tomlins SA, Chinnaiyan AM. Recurrent gene fusions in prostate cancer. Nature Reviews Cancer. 2008 Jul;8(7):497-511.