Genome-wide identification of Schistosoma japonicum microRNAs using a deep-sequencing approach

Abstract

Background: Human schistosomiasis is one of the most prevalent and serious parasitic diseases worldwide. Schistosoma japonicum is one of important pathogens of this disease. MicroRNAs (miRNAs) are a large group of non-coding RNAs that play important roles in regulating gene expression and protein translation in animals. Genome-wide identification of miRNAs in a given organism is a critical step to facilitating our understanding of genome organization, genome biology, evolution, and posttranscriptional regulation.

Methodology/principal findings: We sequenced two small RNA libraries prepared from different stages of the life cycle of S. japonicum, immature schistosomula and mature pairing adults, through a deep DNA sequencing approach, which yielded approximately 12 million high-quality short sequence reads containing a total of approximately 2 million non-redundant tags. Based on a bioinformatics pipeline, we identified 176 new S. japonicum miRNAs, of which some exhibited a differential pattern of expression between the two stages. Although 21 S. japonicum miRNAs are orthologs of known miRNAs within the metazoans, some nucleotides at many positions of Schistosoma miRNAs, such as miR-8, let-7, miR-10, miR-31, miR-92, miR-124, and miR-125, are indeed significantly distinct from other bilaterian orthologs. In addition, both miR-71 and some miR-2 family members in tandem are found to be clustered in a reversal direction model on two genomic loci, and two pairs of novel S. japonicum miRNAs were derived from sense and antisense DNA strands at the same genomic loci.

Conclusions/significance: The collection of S. japonicum miRNAs could be used as a new platform to study the genomic structure, gene regulation and networks, evolutionary processes, development, and host-parasite interactions. Some S. japonicum miRNAs and their clusters could represent the ancestral forms of the conserved orthologues and a model for the genesis of novel miRNAs.

Figure 1. The flowchart of screening and identifying S. japonicum miRNAs.

AW, mixed adult worms; SC, hepatic schistosomula.

Figure 2. Size and frequency distribution of the sequencing reads from both cDNA libraries as well as the classification of small RNAs.

(A) Length distribution of the non-redundant sequencing reads. (B) Classification of the sequenced small RNA tags from adult worms (AW) and immature hepatic schistosomula (SC), respectively.

Figure 3. Identification and properties of S. japonicum miRNAs.

(A) Length distribution of all identified S. japonicum miRNAs. The left and right Y-axes indicate the number and percentage of miRNAs, respectively. (B) Cumulative length distributions of miRNA precursors from S. japonicum and other bilaterian animals. The size distribution of S. japonicum miRNA precursors is similar to that of other bilaterian animals, including C. elegans (cel), Schmidtea mediterranea (sme), D. melanogaster (dme), Fugu rubripes (fru), mice (mmu), and humans (hsa). (C) The sequences and numbers of sequencing reads matching the sja-miR-2e hairpin. The sequence of the sja-miR-2e hairpin is displayed above the bracket-notation of its predicted secondary structure, as determined by RNAfold. Sequenced small RNAs from immature schistosomula (SC) and adult worms (AW) that map to the hairpin are aligned below, with the number of reads shown on the right. Both miR-2e-5p and the conserved miR-2e-3p were designated as reciprocal miRNA and miRNA* species and are indicated in red and in blue, respectively. (D) Relative expression levels of S. japonicum miRNAs, as indicated by fold enrichment through normalizing the frequency of sequencing reads from the AW and SC samples.

Figure 4. Genomic origin of S. japonicum miRNAs.

(A) A miRNA cluster consisting of miR-71b, miR-2f, miR-2a miR-2e-5p, and miR-2e-3p in tandem on the same genomic contig (CNUS0000011792.1). (B) A similar miRNA cluster containing miR-71a, miR-2d, miR-2b, and miR-2c was mapped in an inverted orientation to another genomic contig (CNUS0000007682.1). (C,D) Two pairs of miRNAs, sja-miR-3052 and sja-miR-3079, as well as sja-miR-3038 and sja-miR-3058 were generated from sense and antisense DNA strands within the same genomic loci (CNUS0000025876.1 and CNUS0000036392.1), respectively.

Figure 5. Evolutionary analysis of S. japonicum miRNAs.

(A) 21 S. japonicum miRNAs are orthologs of known miRNAs from other bilaterian animals. “+” indicates the high homology with the orthologs of the indicated species or phylum of animals, while “−” indicates no homology with to the orthologs. (B) Alignment of miR-8 homologues from some bilaterian animals. The nucleotides in red represent substitution that is specific to schistosomes, while blue letters indicate nucleotide substitutions found in orthologs or their paralogs in some other species and black letters represent the conserved nucleotides. Abbreviations: sja, S. japonicum; sma, S. mansoni; sme, S. mediterranea; dme, D. melanogaster; dre, D. rerio; fru, F. rubripes; gga, G. gallus; hsa, H. sapiens; mmu, M. musculus; xla, X. laevis; cel, C. elegans; csa, C. savignyi; cin, C. intestinalis; oan, O. anatinus; xtr, X. tropicalis.