Comparative Analysis of the Circular Transcriptome in Muscle, Liver, and Testis in Three Livestock Species

Abstract

Circular RNAs have been observed in a large number of species and tissues and are now recognized as a clear component of the transcriptome. Our study takes advantage of functional datasets produced within the FAANG consortium to investigate the pervasiveness of circular RNA transcription in farm animals. We describe here the circular transcriptional landscape in pig, sheep and bovine testicular, muscular and liver tissues using total 66 RNA-seq datasets. After an exhaustive detection of circular RNAs, we propose an annotation of exonic, intronic and sub-exonic circRNAs and comparative analyses of circRNA content to evaluate the variability between individuals, tissues and species. Despite technical bias due to the various origins of the datasets, we were able to characterize some features (i) (ruminant) liver contains more exonic circRNAs than muscle (ii) in testis, the number of exonic circRNAs seems associated with the sexual maturity of the animal. (iii) a particular class of circRNAs, sub-exonic circRNAs, are produced by a large variety of multi-exonic genes (protein-coding genes, long non-coding RNAs and pseudogenes) and mono-exonic genes (protein-coding genes from mitochondrial genome and small non-coding genes). Moreover, for multi-exonic genes there seems to be a relationship between the sub-exonic circRNAs transcription level and the linear transcription level. Finally, sub-exonic circRNAs produced by mono-exonic genes (mitochondrial protein-coding genes, ribozyme, and sno) exhibit a particular behavior. Caution has to be taken regarding the interpretation of the unannotated circRNA proportion in a given tissue/species: clusters of circRNAs without annotation were characterized in genomic regions with annotation and/or assembly problems of the respective animal genomes. This study highlights the importance of improving genome annotation to better consider candidate circRNAs and to better understand the circular transcriptome. Furthermore, it emphasizes the need for considering the relative "weight" of circRNAs/parent genes for comparative analyses of several circular transcriptomes. Although there are points of agreement in the circular transcriptome of the same tissue in two species, it will be not possible to do without the characterization of it in both species.

Keywords: annotation; circular RNA; circular transcriptome; exonic circRNA; intronic circRNAs; parent genes; sub-exonic circRNA.

FIGURE 1

Number of circRNA characterized by CE2 + CIRI2. (A–G) These histograms represent the number of circRNA (per million of reads uniquely mapped) characterized jointly by CE2 and CIRI2 and which are detected by at least 4 BSJs (CIRIquant). Histograms are regrouped by SRA projects. (A) The two bovine batches produced at FBN. (B) the three ovine batches produced at Roslin Institute (C) The two batches produced by EMBL in 2017. (D) The two batches produced by EMBL in 2019. (E) The batch of 6 datasets produced from porcine pubertal testes at INRAE. (F) The two batches of bovine testes produced by Yangling University. (G) Comparison per tissue and species of the number of exonic circRNAs detected by CE2 + CIRI2 per million of reads uniquely mapped.

FIGURE 2

Comparisons circRNAs and parent genes between datasets. The three diagrams depict analyses of exonic circRNAs characterized by CE2 + CIRI2. The number of similarities (same circRNA or same parent gene) found for a comparison between two datasets was reported in a box. A-(boxes with a blue background): The expression of a circRNA in a batch has been defined as the sum of the BSJs (normalized counts) observed in the different datasets of this batch. The circRNAs were ranked on their expression to establish the Top-100 of circRNAs expressed in this batch (Lists of Top-100/circRNAs relative to these analyses were reported in Supplementary Table 1). B-(boxes with a yellow background): The circRNA expression of genes in a batch has been defined as the sum of the BSJs (normalized counts) from each circRNA produced by this gene observed in the different datasets of this batch. The parent genes were ranked on their circRNA expression to establish the Top-100 of parent genes expressed in this batch (Lists of Top-100/genes relative to these analyses were reported in Supplementary Table 2).

FIGURE 3

Number of circRNA characterized by CD. These histograms represent the number of circRNA (per million of reads uniquely mapped) which are detected by at least 5 CCRs and annotated as exonic circRNA, intronic circRNAs, sub-exonic or unannotated. Histograms are regrouped by SRA projects. (A) The two bovine batches produced at FBN. (B) the three ovine batches produced at Roslin Institute (C) The two batches produced by EMBL in 2017. (D) The two batches produced by EMBL in 2019. (E) The batch of 6 datasets produced from porcine pubertal testes at INRAE. (F) The two batches of bovine testes produced by Yangling University.

FIGURE 4

circRNAs detected by CD. (A) Number of exonic circRNA detected by CD per million of reads uniquely mapped. (B) Number of intronic circRNA detected by CD per million of reads uniquely mapped. (C) Number of sub-exonic circRNA detected by CD per million of reads uniquely mapped. (D,E) Relationship between the detection of intronic and exonic circRNAs.

FIGURE 5

Comparative analysis of the bovine and ovine transcriptomes. (A) Circular expression profiles of orthologous genes (measured as number of BSJs) were compared in liver, and in muscle. (B) Expression profiles of orthologous circRNAs (measured as number of BSJs) were compared in liver, and in muscle. Only genes/circRNAs with a substantial expression were kept for these correlation analyses (log10(BSJs) > 1.2 for each tissue and each species).