Abstract:

Objective To explore the N6 adenylate methylation (m6A) modification of circular RNA (circRNA) in male and female adult worms of Schistosoma japonicum, and predict its possible role in regulating miRNA. Methods S. japonicum cercariae (100 ± 2) were used to infect mice through abdominal skin for 20 min. On 28 d post-infection, the mice were anesthetized to collect adult worms using hepatic portal vein perfusion method, of which the female and male worms were separated for extracting total RNA, respectively. The RNA samples of 0.5 μg and 1.0 μg were applied by dotting on the nylon membrane, which was transferred to a UV-crosslinker for dot hybridization through successive incubation with m6A antibody (1∶1 000) and HRP labeled secondary antibody (1∶5 000). Immunoprecipitation separation was performed for 1 µg of total RNA from both female and male worms using RNA methylation co-precipitation kit. The RNA library kit was used for library construction. After quality testing with a biological analyzer, sequencing was performed using a 150 bp double-ended mode on a high-throughput sequencer. The Cutadapt software (v1.9.3) was applied to remove joints and low-quality sequences and obtain high-quality sequences. The sequence was aligned with the Schistosoma genome (Sja_WBPS14) using Bowtie2 software, and the Find_circ software was used to identify circRNA, while MACS software was used to recognize methylation sites. Cluster analysis of differentially expressed circRNAs was conducted using Heatmap2 software, and gene ontology (GO) enrichment analysis was performed for the source genes of statistically significant differentiated m6A modified circRNA. DiffReps software was used to analyze the differences in m6A modified circRNA between male and female worms. Using RNAhybrid software, the interaction between miRNA and circRNA was predicted. The reverse-transcribed miRNA was analyzed by qPCR, and all the relative expression level of miRNA was calculated with 2^-ΔCt method. Results The dot blot hybridization results showed that there was m6A methylation modification in the total RNA of S. japonicum, and the m6A methylation signal tended to increase with the increase of RNA content. The high-throughput sequencing results showed that the proportion of high-quality sequences in S. japonicum was over 99.9%, and the RNA enrichment and library construction were effective. CircRNAs originating from overlapping regions account for 49%, followed by exons (29%) and intergenes (12%), with fewer circRNAs originating from introns (2%) and antisense chains (8%). Genes derived from highly enriched circRNAs in males may be related to cellular metabolic processes, cellular components, tissue biogenesis, and stress, while genes derived from highly enriched circRNAs in females may be involved in processes such as cell molecule binding. The number of m6A-modified circRNAs enriched in males was higher than that in females (P < 0.05, fold change ≥ 2) with 57 high abundance m6A-modified circRNAs in females and 81 in males. M6A-modified circRNAs in both male and female insects can potentially interact with multiple miRNAs, such as Sja-Bantam and Sja-miR-307, which are highly expressed in females, and Sja-miR-8-3p, Sja-miR-3504 and so on, which are highly expressed in males. Conclusion This study obtained the expression profile of m6A modified circRNA in adult worms of S. japonicum, and preliminarily revealed the miRNA that may be regulated by m6A modified circRNA.

Key words: Schistosoma japonicum, Gender, m6A modification, Circular RNA, miRNA