Genome-Wide Identification of CircRNAs of Infective Larvae and Adult Worms of Parasitic Nematode, Haemonchus contortus

Abstract

CircRNAs, a novel class of ncRNA family, are endogenous transcriptional products involved in various biological and physiological processes in plants and animals. However, almost no information is available for circRNAs of parasitic helminths. In the present study, the circRNAs repertoire was comprehensively explored in Haemonchus contortus, a blood-sucking parasitic nematode of ruminants. In total, 20073 circRNAs were identified and annotated from three key developmental stages/genders of H. contortus including the free-living infective third-stage larvae (L3, 18883), parasitic adult female (Af, 3491), and male worms (Am, 2550) via deep-sequencing technology and bioinformatic analysis. Among these identified circRNAs, 71% were derived from exonic regions of protein-coding genes. The number of circRNAs transcribed from the X chromosome (4704) was higher than that from Chromosome I-V (3143, 3273, 3041, 3030, 2882). The amount of highly expressed circRNAs in third-stage larvae was significantly more abundant than that in adult stage. 15948 and 16847 circRNAs were differentially expressed between Af and L3s and between Am and L3, respectively. Among them, 13409 circRNAs existed in both comparisons. Furthermore, 1119 circRNAs were differentially expressed between Af_and_Am. GO enrichment analysis indicated that source genes of circRNAs differentially expressed between Am and L3 as well as between Af and L3 were significantly enriched in many biological processes, primarily including signaling, signal transduction and cell communication terms. KEGG analysis revealed that parental genes of differentially expressed circRNAs were mainly related to metabolism (pyruvate metabolism, glycerophospholipid metabolism, and carbon metabolism), MAPK signaling pathway, and phosphatidylinositol signaling system. Moreover, many circRNAs contained one or more miRNA potential binding sites, suggesting that they could regulate gene expression at the post-transcriptional level. Furthermore, the correctness of head-to-tail back splicing site and alternative circularization events were verified by Sanger sequencing using both divergent and convergent primers. Finally, the reliability of RNA-Seq data and the resistance of circRNAs to RNase R digestion were confirmed by quantitative RT-PCR. Taken together, our findings provide a foundation for elucidating the regulatory mechanisms of circRNAs in H. contortus, which will advance the understanding of circRNAs in parasitic nematodes.

Expression and secretion of circular RNAs in the parasitic nematode,Ascaris suum

“…A total of 1,686 exonic circRNAs have been identified in various C. elegans life stages (Ivanov et al, 2015; Memczack et al, 2013; Cortés-López et al, 2018). In comparison, we found a similar number of exonic circRNAs (1,178) in adult female Ascaris suum whilst 14,251 exonic circRNA were discovered in H. contortus (Zhou et al, 2021) across three life stages (the infective third stage larvae, adult male and adult female worms). The number of exonic circRNAs is comparable between Ascaris and C. elegans but significantly higher in Haemonchus , with the greatest number being expressed in the third stage larvae of that species.…”

“…Small interfering RNAs (siRNAs) have been used to good effect for downregulating circRNAs in cultured cells (Legnini et al, 2017) and may have some potential for translation to parasitic nematodes as some of these worms are susceptible to RNAi (Song et al, 2010; Verma et al, 2017). Lastly, by targeting back-splice junction sites, a CRISPR/Cas13 approach has been used to successfully screen for circRNA function (Li S. et al, 2021). This strategy may be possible if DNA transformation of parasitic nematodes becomes more feasible.…”

“…While circRNAs have been described in the free-living nematode Caenorhabditis elegans (Memczak et al, 2013; Ivanov et al, 2015) and now a more thorough description from Haemonchus contortus (Zhou et al, 2021), a parasitic nematode of small ruminants, our understanding of circRNA expression and function in nematodes is lacking. A summary of circRNA biogenesis is presented in Figure 1 and the identification of exonic, intergenic and intronic circRNAs in in this study and others supports this model in nematodes.…”

“…al, 2013), insects (Westholm et al, 2014), plants (Zhang et al, 2020), fungi (Shao et al 2019), and germane to the current study, the model nematode Caenorhabditis elegans (Memczak et al, 2013; Ivanov et al, 2015; Cortés-López et al, 2018). The recognition that circRNAs are expressed in C. elegans has recently seeded their discovery in parasitic nematodes, specifically, the small ruminant gastrointestinal parasitic nematode, Haemonchus contortus (Zhou et al, 2021).…”

“…As regulators of biological processes, a description of circRNA complement and function in parasites is warranted as they may have value from a control perspective. circRNAs have been identified in C. elegans (Memczak et al, 2013; Ivanov et al, 2015; Cortés-López et al, 2018) with the first descriptive study of a nematode circRNA complement based on H. contortus recently emerging (Zhou et al, 2021). These manuscripts focus on the presence of circRNAs in these two clade V nematode species but do not give much insight into the functional significance of circRNAs in worms.…”