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. 2010 Sep 17:3:90.
doi: 10.1186/1756-3305-3-90.

Analysis and characterization of the genes encoding the Dicer and Argonaute proteins of Schistosoma japonicum

Rong Luo  1 , Xiangyang Xue, Zhangxun Wang, Jun Sun, Ying Zou, Weiqing Pan
Affiliations

Analysis and characterization of the genes encoding the Dicer and Argonaute proteins of Schistosoma japonicum

Rong Luo et al. Parasit Vectors. .

Abstract

Background: The Dicer and Argonaute(AGO) proteins within the small RNA regulatory pathways (SRRPs) play an indispensable role in regulation of gene expression. In this study, we analyzed two genes, Dicer and Argonaute, from Schistosoma japonicum, along with their expression through a combination of bioinformatics and experimental approaches.

Results: Our results indicate that one Dicer and four Argonaute genes exist in Schistosoma japonicum, termed SjDicer and SjAGO1, 2, 3, and 4, respectively. SjDicer encodes 2590 amino acid residues that contains 5 conserved domains, including one amino-terminal helicase domain, one PAZ (Piwi-Argonaut-Zwille) domain, two RNAse III domains, and one dsRNA-binding domain. SjAGO1, 2, and 3 encode 1009, 945, and 904 amino acid residues, respectively, all of which contain PAZ and PIWI domains. In addition, we analyzed the expression profiles of SjDicer and SjAGO1 genes by qRT-PCR in eggs, miracidium, cercariae, schistosomula, and adult worms. Results showed consistent expression of both SjDicer and SjAGO1 in different stages; however, their expression levels were stage-dependent, with the highest being in the miracidium stage.

Conclusions: This study provided the sequence of the Dicer and Ago genes of S. japonicum and their expression profiles which are essential for further investigation of functions of miRNA in Schistosoma japonicum.

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Figures

Figure 1
Figure 1
Diagrammatic representation of SjDicer. The SjDicer gene is approximately 60,000 bp in length and located on contig CCON0000000957. The complete mRNA of the SjDicer gene is 7,796 nt and contains 28 exons, which encode a 2590 aa protein, including an amino-terminal helicase domain, a DUF283(Domain of unknown function), a PAZ(Piwi-Argonaut-Zwille) domain, two tandom RNAse III domains, and an dsRBD(double strand RNA-binding domain).
Figure 2
Figure 2
Phylogenetic trees of Argonaute and Dicer proteins. Multiple alignments were performed using ClustalX 2.0 and Mega 4.0 with bootstrap analysis. Bootstrap percentages are indicated at each branch. Phylogenetic tree of Argonaute proteins from plants, animals, and fungi. Accession numbers of sequences for Argonaute and Dicer proteins: Hs.AGO1 (NP_036331), Hs.AGO2 (NP_036286), Hs.AGO3 (NP_079128), Hs.AGO4 (NP_060099), Dm.AGO1 (NP_725341), Ce.ALG1 (NP_510322.2), Ce.ALG2 (NP_493837.1), Ce.TAG76 (NP_499192.1), At.AGO1 (gbAAB91987.1), At.AGO4 (NP_565633.1), At.AGO7 (NP_177103.1), At.AGO9 (CAD66636.1), Ce.ERGO1 (NP_503362.2), SmAGO1 (Smp_140010), SmAGO2 (Smp_179320), SmAGO3 (Smp_102690.2), SmAGO4 (Smp_102690.3), Dm.Dicer1 (NP_524453.1), Dm.Dicer2 (ABB54751.1), Dy.Dicer2 (ABB54764.1), Aa.Dicer1 (XP_001659747.1), Aa.Dicer2 (AAW48725.1), Hs.Dicer1 (NP_085124.2), Am.Dicer1 (XP_624510.2), Ce.DCR1 (NP_498761.1), SmDicer (smp_169750).
Figure 3
Figure 3
Diagrammatic representation of the conserved domains of the SjAGO protein family. They all contain PAZ and PIWI conserved domains. In addition, the SjAGO1 gene has a DUF1785 domain. SjAGO1, 2, and 3 proteins are 987 aa, 924 aa, and 904 aa in size, respectively
Figure 4
Figure 4
Developmental expression analysis of S. japonicum (a) SjDicer and (b) SjAGO1. The mRNA expression levels were measured, based on three replicates, in the life stages: eggs, miracidium, cercariae, schistosomula, and adult worms, using quantitative RT-PCR. Expression levels were calibrated according to the comparative 2-ΔΔCt method, using the constitutively expressed SjGAPDH gene as an endogenous control, and were normalized relative to the egg stage.
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