Ularcirc: visualization and enhanced analysis of circular RNAs via back and canonical forward splicing

Abstract

Circular RNAs (circRNA) are a unique class of transcripts that can only be identified from sequence alignments spanning discordant junctions, commonly referred to as backsplice junctions (BSJ). Canonical splicing is also linked with circRNA biogenesis either from the parental transcript or internal to the circRNA, and is not fully utilized in circRNA software. Here we present Ularcirc, a software tool that integrates the visualization of both BSJ and forward splicing junctions and provides downstream analysis of selected circRNA candidates. Ularcirc utilizes the output of CIRI, circExplorer, or raw chimeric output of the STAR aligner and assembles BSJ count table to allow multi-sample analysis. We used Ularcirc to identify and characterize circRNA from public and in-house generated data sets and demonstrate how it can be used to (i) discover novel splicing patterns of parental transcripts, (ii) detect internal splicing patterns of circRNA, and (iii) reveal the complexity of BSJ formation. Furthermore, we identify circRNA that have potential open reading frames longer than their linear sequence. Finally, we detected and validated the presence of a novel class of circRNA generated from ApoA4 transcripts whose BSJ derive from multiple non-canonical splicing sites within coding exons. Ularcirc is accessed via https://github.com/VCCRI/Ularcirc.

Circular RNA hsa_circ_0007364 increases cervical cancer progression through activating methionine adenosyltransferase II alpha (MAT2A) expression by restraining microRNA-101-5p

“…Of the 8 upregulated circRNAs ( Figure 1(a )), we elected to focus on the novel circRNA hsa_circ_0007364 (hsa_circRNA_100141), which is generated from exon2 and exon3 of PTP4A2 gene through back-splicing ( Figure 1(b )). Further analysis revealed that hsa_circ_0007364 reverse-transcribed using Oligo dT primers was less than from random primers ( Figure 1(c )) [ 22 ], hsa_circ_0007364 is more resistant to RNase R digestion relative to linear PTP4A2 mRNA ( Figure 1(d )), and hsa_circ_0007364 is markedly more stable than PTP4A2 mRNA following transcription inhibition with actinomycin D ( Figure 1(e )).

Figure 1.

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isoCirc catalogs full-length circular RNA isoforms in human transcriptomes

“…This shared nature complicates computational inference, especially for regions within circRNAs far from the BSJs. Indeed, although computational methods to reconstruct full-length circRNAs from short-read RNA-seq data exist [25][26][27][28][29] , these methods are unable to reconstruct long full-length circRNAs 25 . In a recent assessment of state-of-the-art reconstruction strategies, Zheng and colleagues concluded that short-read-based reconstruction methods can only be applied to short circRNAs (200-500 nt, depending on RNA-seq read length) 25 .…”

“…Similarly, inferring the protein products translated from circRNAs requires full-length circRNA sequences 11 . To fill this gap, several computational methods have been developed to reconstruct full-length circRNAs from short-read RNA-seq data [25][26][27][28][29] . However, these methods are only applicable to short circRNAs (200-500 nt, depending on RNA-seq read length), and are unable to interrogate longer full-length circRNAs 25 .…”

Comprehensive landscape and future perspectives of circular RNAs in colorectal cancer

“…The CRISPR-RfxCas13d system may serve as a useful tool for the discovery and functional study of circRNAs at both the individual and large-scale level in the near future [207]. Finally, an increasing number of online databases have been developed and improved to provide tremendous valuable information, such as Ularcirc [208], IMS-CDA [209], Lnc2Cancer 3.0 [210], LincSNP 3.0 [211], etc. These online resources will be applied for circRNA identification, characterization and functional analyses and serve as tools for investigating the interaction between circRNAs and other molecules (e.g., miRNAs and RBPs).…”