Identification and characterization of microRNAs and endogenous siRNAs in Schistosoma japonicum

Abstract

Background: Small endogenous non-coding RNAs (sncRNAs) such as small interfering RNA (siRNA), microRNA and other small RNA transcripts are derived from distinct loci in the genome and play critical roles in RNA-mediated gene silencing mechanisms in plants and metazoa. They are approximately 22 nucleotides long; regulate mRNA stability through perfect or imperfect match to the targets. The biological activities of sncRNAs have been related to many biological events, from resistance to microbe infections to cellular differentiation. The development of the zoonotic parasite Schistosoma japonicum parasite includes multiple steps of morphological alterations and biological differentiations, which provide a unique model for studies on the functions of small RNAs. Characterization of the genome-wide transcription of the sncRNAs will be a major step in understanding of the parasite biology. The objective of this study is to investigate the transcriptional profile and potential function of the small non-coding RNAs in the development of S. japanicum.

Results: The endogenous siRNAs were found mainly derived from transposable elements (TE) or transposons and the natural antisense transcripts (NAT). In contrast to other organisms, the TE-derived siRNAs in S. japonicum were more predominant than other sncRNAs including microRNAs (miRNAs). Further, there were distinct length and 3'end variations in the sncRNAs, which were associated with the developmental differentiation of the parasite. Among the identified miRNA transcripts, there were 38 unique to S. japonicum and 16 that belonged to 13 miRNA families are common to other metazoan lineages. These miRNAs were either ubiquitously expressed, or they exhibited specific expression patterns related to the developmental stages or sex. Genes that encoded miRNAs are mainly located in clusters within the genome of S. japonicum. However, genes within one cluster could be differentially transcribed, which suggested that individual genes might be regulated by distinct mechanisms during parasite development.

Conclusions: Many miRNA and endogenous siRNA transcripts were identified in S. japonicum and the amount of siRNA was at least 4.4 and 1.6 times more than that of miRNA in both schistosomulum and adult worm stages respectively. SiRNAs are mainly derived from transposable elements (or transposons); while natural antisense transcripts (NAT)-derived siRNAs were much less. A majority of miRNA transcripts identified in the parasite were species-specific and the expression of certain miRNAs was found developmentally regulated. Both miRNA and siRNAs are potentially important regulators in the development of schistosomal parasites.

Figure 1

Percentage of siRNAs derived from different transposable elements (TE) in adult and schistosomulum stages. Majority of siRNAs were derived from LTR, LINE and TIR.

Figure 2

The abundance of each siRNA sequence generated from the two transposons by scanning each siRNA sequence against the two transposons (A. SACI-5 and B. TRE-1). Data represents the percentage of all matched siRNAs identified in adult (red) and schistosomulum (blue) parasites.

Figure 3

Small interfering RNAs and microRNAs identified by high-throughput sequencing in S. japonicum. A and B Length and distribution of siRNAs and miRNAs in adult and schistosomulum stages. C. 3' end variations in TE and NAT-derived siRNAs and miRNAs.

Figure 4

Repertoire of Sja-mir-36a isomers and a typical miRNA cluster. A. Sequences and the number of reads of the mature miRNA of Sja-mir-36a and the complementary miR* are represented in red and blue respectively. The predicted structure of the pre-miRNA is represented on the right side. B. The predicted secondary structure of a pre-miRNA containing a cluster of Sja-mir-2a, Sja-mir-2b, and Sja-novel-36, which form three hairpin stem-loop structures. The sequences of the mature miRNAs are shown in red and the miRNA* strands are shown in blue.

Figure 5

Sequence and transcriptional analysis of miRNAs dominantly expressed in S. japonicum. A. Alignment of sja-mir-7 sequences with homologues from other organisms. The seed sequences are shadowed in a dark colour. B. Northern-blot hybridization using probes complementary to the dominant miRNAs expressed during the adult worm stage. The 70 nt pre-miRNAs were indicated by arrows. C. Quantification analysis, relative to that in the egg stage, of miRNAs dominantly expressed in four developmental stages as indicated in different colours.