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. 2019 Mar 5;9(1):3432.
doi: 10.1038/s41598-019-39977-7.

A genome-wide map of circular RNAs in adult zebrafish

Disha Sharma  1   2 , Paras Sehgal  3   2 , Samatha Mathew  3   2 , Shamsudheen Karuthedath Vellarikkal  3   2 , Angom Ramcharan Singh  3 , Shruti Kapoor  1   2 , Rijith Jayarajan  3 , Vinod Scaria  4   5 , Sridhar Sivasubbu  6   7
Affiliations

A genome-wide map of circular RNAs in adult zebrafish

Disha Sharma et al. Sci Rep. .

Abstract

Circular RNAs (circRNAs) are transcript isoforms generated by back-splicing of exons and circularisation of the transcript. Recent genome-wide maps created for circular RNAs in humans and other model organisms have motivated us to explore the repertoire of circular RNAs in zebrafish, a popular model organism. We generated RNA-seq data for five major zebrafish tissues - Blood, Brain, Heart, Gills and Muscle. The repertoire RNA sequence reads left over after reference mapping to linear transcripts were used to identify unique back-spliced exons utilizing a split-mapping algorithm. Our analysis revealed 3,428 novel circRNAs in zebrafish. Further in-depth analysis suggested that majority of the circRNAs were derived from previously well-annotated protein-coding and long noncoding RNA gene loci. In addition, many of the circular RNAs showed extensive tissue specificity. We independently validated a subset of circRNAs using polymerase chain reaction (PCR) and divergent set of primers. Expression analysis using quantitative real time PCR recapitulate selected tissue specificity in the candidates studied. This study provides a comprehensive genome-wide map of circular RNAs in zebrafish tissues.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Schematic description of the bioinformatics analysis pipeline used for the discovery and validation of novel circular RNAs in different tissues of zebrafish.
Figure 2
Figure 2
Tissue distribution and repertoire of predicted circular RNAs (A). Circos representation for tissue-wise distribution of circular RNAs with respect to read coverage across genomic co-ordinates. (B) Illustration of the number of circular RNAs shared between tissues analysed in the present study.
Figure 3
Figure 3
Genomic distribution of the circRNAs (A). Density-wise distribution of circular RNA across chromosome length (B). Genome-wide distribution of circular RNAs (length normalized for each region) for Ensembl and RefSeq respectively.
Figure 4
Figure 4
Validation of predicted novel circRNA in different tissues. (A–E) PCR based confirmation of circRNA junctions in (A) Blood, (B) Brain, (C) Muscle, (D) Heart and (E) Gills, respectively. Divergent primers facing outwards were used to amplify cDNA (C), DNA (D) and negative control (NC). Convergent primers for actb1 gene used in every tissue as control. L- 100 bp Ladder (Fermentas, USA).
Figure 5
Figure 5
Validation of circRNA with RNAse R treatment (A). Blood (B) Brain (C) Muscle (D) Heart (E) Gills. L-ladder, +represents RNAse treated and −represents not treated with RNAse. We have used two positive controls for each tissue. One is tissue specific positive control and second is beta-actin.
Figure 6
Figure 6
Quantitative validation of putative novel circular RNAs across different tissues. qRT-PCR based relative expression analysis of candidate circRNAs in (A). Blood, (B) Brain, (C) Muscle, (D) Heart, (E) Gills. The expression levels of linear transcripts of parent gene of each candidate circRNA were also estimated. Tissue specific protein coding gene marker tal1(blood), mdka(brain), tnnt2c(muscle), vmhc(heart) and cx44.2(gills) were used as controls. Data was normalized with the expression levels of actb1. Data was collected from independent experiments and represented as relative mean fold change ± SD across tissues. circRNAs examined in a particular tissue type displayed relatively high expression in the specific tissue when compared to other tissue types.
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