Identification and characterization of argonaute protein, Ago2 and its associated small RNAs in Schistosoma japonicum

Abstract

Background: The complex life cycle of the genus Schistosoma drives the parasites to employ subtle developmentally dependent gene regulatory machineries. Small non-coding RNAs (sncRNAs) are essential gene regulatory factors that, through their impact on mRNA and genome stability, control stage-specific gene expression. Abundant sncRNAs have been identified in this genus. However, their functionally associated partners, Argonaute family proteins, which are the key components of the RNA-induced silencing complex (RISC), have not yet been fully explored.

Methodology/principal findings: Two monoclonal antibodies (mAbs) specific to Schistosoma japonicum Argonaute protein Ago2 (SjAgo2), but not SjAgo1 and SjAgo3, were generated. Soluble adult worm antigen preparation (SWAP) was subjected to immunoprecipitation with the mAbs and the captured SjAgo2 protein was subsequently confirmed by Western blot and mass spectrometry (MS) analysis. The small RNA population associated with native SjAgo2 in adult parasites was extracted from the immunoprecipitated complex and subjected to library construction. High-through-put sequencing of these libraries yielded a total of ≈50 million high-quality reads. Classification of these small RNAs showed that endogenous siRNAs (endo-siRNAs) generated from transposable elements (TEs), especially from the subclasses of LINE and LTR, were prominent. Further bioinformatics analysis revealed that siRNAs derived from ten types of well-defined retrotransposons were dramatically enriched in the SjAgo2-specific libraries compared to small RNA libraries constructed with total small RNAs from separated adult worms. These results suggest that a key function of SjAgo2 is to maintain genome stability through suppressing the activities of retrotransposons.

Conclusions/significance: In this study, we identified and characterized one of the three S. japonicum Argonautes, SjAgo2, and its associated small RNAs were found to be predominantly derived from particular classes of retrotransposons. Thus, a major function of SjAgo2 appears to associate with the maintenance of genome stability via suppression of retroelements. The data advance our understanding of the gene regulatory mechanisms in the blood fluke.

Figure 1. Trnascriptional analysis of three S. japonicum Argonaute genes.

Relative levels of SjAgo1 (A), SjAgo2 (B), and SjAgo3 (C) transcripts at different life stages (C, cercariae; S, hepatic schistosomula; M, male adult worms; F, female adult worms; E, eggs) were detected by qRT-PCR performed with three technical replicates. Transcriptional levels were calibrated based on the comparative 2^−ΔΔCt method using the housekeeping gene SjPSMD4 as an endogenous control, and normalized to the expression in the cercarial stage. Error bars represent the standard deviations of the mean from the three technical replicates. The Student's t-test was employed to analyze the differential expression of SjAgos between male and female adult worms. *, p<0.05; ***, p<0.001. Here only the significance in difference between male and female parasite is indicated.

Figure 2. The expression of SjAgo proteins in eukaryotic cells and specific recognition by mAbs 11E8 and 27A9 raised against SjAgo2.

A. The expression of Flag-tagged S. japonicum Argonaute proteins in 293T cells was detected by Western blot analysis using anti-Flag mAb M2. Lane 1, tSjAgo1 (aa198-1009); lane 2, SjAgo3; lane 3, SjAgo2; lane 4, Mock: Cells transfected with the empty vector. B. Over-expression of SjAgo2 was recognized by mAbs. Lane 1, Mock; lane 2–4, SjAgo2 was recognized by anti-Flag mAb M2, 11E8, and 27A9, respectively. C. Differential recognition of over-expressed SjAgos by mAb 11E8 or 27A9. Left panel, over-expressed SjAgos detected by Western blot with mAb 11E8. Right panel, over-expressed SjAgos detected by Western blot with mAb 27A9. M, prestained protein ladder SM0671.

Figure 3. Detection of the native SjAgo2 protein by Western blot. A. Detection of the native SjAgo2 protein in different SWAP immunoprecipitates.

Lanes 1 and 5, no antibody added; lanes 2 and 6, immunoprecipitated by normal mouse IgG; lanes 3 and 7, immunoprecipitated by mAb 11E8; lanes 4 and 8, immunoprecipitated by mAb 27A9. Left panel, immunoprecipitates were detected by Western blot with mAb 11E8. Right panel, immunoprecipitates were detected by Western blot with mAb 27A9. HRP-conjugated goat anti-mouse IgG was used as a secondary antibody. The arrows indicate the specific recognition of SjAgo2 by mAbs; the asterisks indicate the band recognized by normal murine IgG. B. SWAP was resolved by 12% SDS-PAGE and detected by Western blot with mAb 27A9. The arrows indicate the two bands recognized by mAb 27A9. M, prestained protein ladder SM0671.

Figure 4. Length distribution of the small RNAs perfectly matched the draft of S. japonicum genome.

A. Length distribution of reads from the SP1 and B. SP2 libraries. Both at unique and total levels, the 20-nt reads were predominant in the two libraries, followed by the 21-nt reads.

Figure 5. Classification and percentage of small non-coding RNAs in different libraries.

A. Classification of small RNAs in the SP1 and B. SP2 libraries using the bioinformatic pipeline described in . C. Small RNA classification of the SjM and D. SjF libraries using the data from our previous study . E. Small RNA classification of the SP1 and F. SP2 libraries using an alternative bioinformatic pipeline as described in the Materials and Methods.

Figure 6. The distribution and abundance of retrotransposon-derived siRNAs in different small RNA libraries from adult worms.

Bars with different colors were created to indicate the abundance (reflected as TPM value) of retrotransposon-derived siRNAs in different libraries. A. Endo-siRNAs mapped to the LTR retrotransposons, SjCHGCS11, SjCHGCS13, and SjCHGCS14. Both sense and antisense siRNAs generated from these LTR elements were enriched in the SjAgo2-specific libraries SP1 and SP2. B. Endo-siRNAs mapped to Penelope-like retrotransposon Sj-penelope1. Both sense and antisense siRNAs generated from the retrotransposon were dramatically accumulated in the SP1 and SP2 libraries. C. Endo-siRNAs mapped to the LINEs, SjCHGCS21, SjR1, SjR2, and Sjpido. Sense siRNAs originated from SjCHGCS21 were enriched in the SjAgo2-specific libraries. However, siRNAs generated from SjR1 and Sjpido were at a low level in the SP1 and SP2 libraries. SjR2-derived siRNAs were hardly detected in these two libraries. The percentage of total siRNAs derived from a particular TE among the four libraries was calculated by using the sum of TPM value of each siRNA and was displayed in the pie charts. The upper panel in each chart represents sense siRNAs; the lower panel in each chart represents antisense siRNAs.