Genome-wide transcriptome analysis shows extensive alternative RNA splicing in the zoonotic parasite Schistosoma japonicum
© Piao et al.; licensee BioMed Central Ltd. 2014
Received: 22 July 2014
Accepted: 19 August 2014
Published: 26 August 2014
Schistosoma japonicum is a pathogen of the phylum Platyhelminthes that causes zoonotic schistosomiasis in China and Southeast Asian countries where a lack of efficient measures has hampered disease control. The development of tools for diagnosis of acute and chronic infection and for novel antiparasite reagents relies on understanding the biological mechanisms that the parasite exploits.
In this study, the polyadenylated transcripts from the male and female S. japonicum were sequenced using a high-throughput RNA-seq technique. Bioinformatic and experimental analyses focused on post-transcriptional RNA processing, which revealed extensive alternative splicing events in the adult stage of the parasite. The numbers of protein-coding sequences identified in the transcriptomes of the female and male S. japonicum were 15,939 and 19,501 respectively, which is more than predicted from the annotated genome sequence. Further, we identified four types of post-transcriptional processing, or alternative splicing, in both female and male worms of S. japonicum: exon skipping, intron retention, and alternative donor and acceptor sites. Unlike mammalian organisms, in S. japonicum, the alternative donor and acceptor sites were more common than the other two types of post-transcriptional processing. In total, respectively 13,438 and 16,507 alternative splicing events were predicted in the transcriptomes of female and male S. japonicum.
By using RNA-seq technology, we obtained the global transcriptomes of male and female S. japonicum. These results further provide a comprehensive view of the global transcriptome of S. japonicum. The findings of a substantial level of alternative splicing events dynamically occurring in S. japonicum parasitization of mammalian hosts suggest complicated transcriptional and post-transcriptional regulation mechanisms employed by the parasite. These data should not only significantly improve the re-annotation of the genome sequences but also should provide new information about the biology of the parasite.
Human schistosomiasis, which is second only to malaria in terms of morbidity and mortality, is a chronic debilitating disease caused by infections of Schistosoma species that vary depending on the endemic region of the parasites . Three principal Schistosoma species can infect humans and cause severe diseases: Schistosoma japonicum, Schistosoma mansoni, and Schistosoma haematobium. S. japonicum is the causative agent of zoonotic schistosomiasis, affecting millions of people in several East and Southeast Asian countries. Despite the availability of a highly effective chemotherapeutic drug (Praziquantel), the high re-infection rates in humans and animals plus the requirement of frequent administration of the agent still limits the overall success of chemotherapy and disease control efforts. Novel targets for drug and vaccine development remain to be defined for optimal treatment and disease prevention; however, the lack of knowledge about this parasite’s biology remains a hurdle. Schistosoma parasites can persist in a mammalian host for decades in the presence of the host immune system, and current knowledge about the mechanism of parasitization is still fragmented. What is known is that the successful host-evasion mechanisms of the parasite involve the inert tegument that covers the surface in most developmental stages, the recruitment of host components to the surface, and the expression of various antigens and immune-regulating factors [2–5].
Schistosoma parasites have a complicated developmental and biological cycle. They are among the few platyhelminth parasites to adopt a dioecious lifestyle and possess heteromorphic sex chromosomes. The genome of S. japonicum contains eight pairs of chromosomes comprising seven pairs of autosomes and one pair of sexual chromosomes, with an estimated 397 Mb containing primarily 13,469 protein-coding sequences [6, 7] that account for 4% of the genome. The decoding and availability of the genome sequences of the three most pathogenic parasites, S. mansoni, S. japonicum, and S. haematobium, has proved pivotal for the systematic dissection of the parasite biology [7–10].
Parasites and RNA purification
Schistosoma japonicum–infected Oncomelania hupensis were purchased from Jiangxi Institute of Parasitic Disease, Nanchang, China. Cercariae were freshly shed from the infected snails. One New Zealand white female rabbit was percutaneously infected with ~1,500 S. japonicum cercariae, as described previously . Mature adult parasites were isolated at 6 weeks post-infection from the rabbit by flushing the blood vessels with phosphate-buffered saline, as described previously [5, 22–24]. Male and female parasites were manually separated with the aid of a light microscope. Total RNA from the parasites was purified with Trizol reagent (Invitrogen, CA, USA), and contaminating genomic DNA was removed using the RNase-Free DNase Set (Qiagen, Germany). RNA quantification and quality were examined with a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA) and standard agarose gel electrophoresis. All RNA samples were stored at -80°C until use.
Library preparation and sequencing
Polyadenylated RNA samples from adult male and female S. japonicum parasites were isolated from total RNA using oligo-(dT) conjugated magnetic beads (Dynabeads®, Invitrogen, CA, USA). The mRNA was interrupted into short fragments by adding the fragmentation buffer provided by the manufacturer (Illumina RNA-seq kit, part no. 1004898). With these short fragments as templates, random hexamer primers were used to synthesize the first-strand cDNA. The second-strand cDNA was synthesized using buffer, dNTPs, RNase H, and DNA polymerase I, respectively. Short fragments were purified following instructions accompanying the kit (QiaQuick PCR Purification Kit, Qiagen, Germany), and double-stranded cDNAs were end-repaired according to manufacturer-recommended protocols, followed by connection with Illumina adapters (Illumina RNA-seq kit, part no. 1004898). The fragments were first amplified by PCR. Purified cDNA fragments were pooled and indexed and loaded onto one lane of an Illumina GA IIX flow cell. A total of 75 pair-end sequencing cycles were carried out. Cluster formation, primer hybridization, and pair-end sequencing were performed according to the provided protocols .
Low-quality reads (more than half of the bases had a quality value less than 5), reads in which unknown bases represented more than 10%, and adapter sequences were removed from the reads, and the clean reads were mapped onto the S. japonicum genome of SGST, (http://lifecenter.sgst.cn/schistosoma/en/schdownload.do) by TopHat (version v2.0.4; default parameters were used) , then assembled with Cufflinks (version v2.0.2)  to construct unique transcript sequences using the parameter: -g –b –u –o (-g/–GTF-guide: use reference transcript annotation to guide assembly; –b/–frag-bias-correct: use bias correction-reference fasta required; –u/–multi-read-correct: use ‘rescue method’ for multi-reads; –o/–output-dir: write all output files to this directory). The Cufflinks assembler is freely available at http://cufflinks.cbcb.umd.edu/. Cuffcompare  was used to compare the assembled transcripts of each library to the referenced annotated genes and build a non-redundant transcript dataset among the libraries. Then, Cuffdiff was used to find significant changes in gene expression level . We used FDR to correct P values and obtained Q values; for Q value ≤5%, we considered the genes to be differentially expressed (Additional file 1). Several Perl scripts were written to summarize the splicing forms in each library. The following algorithms were used to detect alternative splicing events. First, junction sites, which give information about boundaries and combinations of different exons in a transcript, were detected by TopHat (with all default parameters). Then, all junction sites of the same gene were used to distinguish the type of alternative splicing event  (Additional file 2: Figure S1 and Figure 1).
Functional annotation and classification
Transcripts were first compared using the Kyoto Encyclopedia of Genes and Genomes database (KEGG, release 58)  with BLASTX  at E values ≤ 1e-10. A Perl script was used to retrieve KO (KEGG Ontology) information from the Blast result and establish pathway associations between UniGene and the database.
InterPro  domains were annotated by InterProScan  (Release 27.0), and functional assignments were mapped onto Gene Ontology (GO) . WEGO  was employed to do GO classification and draw the GO tree. The significance analysis of functional pathways was performed using IDEG6 .
To identify pseudogenes in the S. japonicum genome, we used PseudoPipe . The assembled transcripts that fell into or included the predicted position of pseudogenes were designated as pseudogenes. WEGO was used for the GO classification.
Non-coding RNA annotation
Rfam  (Release 10.1) databases were used to annotate the non-coding transcripts. The assembled novel transcripts were compared to Rfam by Blast at E values ≤ 1e-10.
Verification of alternative splicing transcripts by RT-PCR and sequencing
Genomic DNA of S. japonicum (adult male and female worms) was purified with the DNeasy Blood & Tissue Kit (Qiagen, Germany) according to the manufacturer’s instructions. Total RNA was prepared using TRIzol reagent (Invitrogen), as previously described , and contaminating genomic DNA was removed with the RNase-Free DNase Set (Ambion). PCR was conducted in triplicate, and each reaction involved 35 amplification cycles on an Applied Biosystems 9700 PCR system (Applied Biosystems, Foster City, CA, USA). The 20 μl reaction system contained 50 ng of total RNA (50 ng RNA was used for the first-strand synthesis step) or 80 ng DNA, 0.5 μM of each primer, and 10 μl of Premix Ex Taq (version 2.0, TaKaRa). The reaction conditions were as follows: 94°C for 3 min; 35 cycles of 94°C, 30 s; 55°C, 30 s; and 72°C, 90 s; and then 10 min at 72°C. An 8 μl aliquot of each PCR sample was then subjected to electrophoresis in a 1.5% agarose gel. The RT-PCR primer sequences are listed in Additional file 3: Table S1.
Results and discussion
Identification of a large number of novel transcripts from un-annotated genome loci following deep sequencing of the S. japonicumtranscriptome
Summary data of the transcriptome analysis
Number of paired reads
Total length (bp)
Predicted genes (loci)
Transcripts per locus
Alternative splicing in S. japonicum
Statistics for alternative splicing events
Alternative splicing class
Sj-F vs reference*
Sj-M vs reference*
Sj-F vs Sj-M
# of loci with alternative splicing
Total alternative splicing events
Alternative donor site
Alternative acceptor site
Functional category of alternatively spliced genes in S. japonicum
GO classification statistics of alternatively processed genes that were differentially expressed in female and male S. japonicum
Total AS & Diff
Auxiliary transport protein activity
Electron carrier activity
Enzyme regulator activity
Molecular transducer activity
Obsolete molecular function
Proteasome regulator activity
Structural molecule activity
Transcription regulator activity
Translation regulator activity
Anatomical structure formation
Cellular component biogenesis
Cellular component organization
Establishment of localization
Immune system process
Multicellular organismal process
Obsolete biological process
Response to stimulus
Identification of novel transcripts from intergenic regions and previously determined pseudogenes
In summary, by using RNA-seq technology, we obtained the global transcriptomes of male and female S. japonicum. Approximately 80% of the total reference genes (http://lifecenter.sgst.cn/schistosoma/en/schdownload.do) were expressed in the adult stage of the parasite, representing the majority of the transcriptomes. These results further provide a comprehensive view of the global transcriptome of S. japonicum. The findings of a substantial level of alternative splicing events dynamically occurring in the parasitization in the mammalian hosts of the S. japonicum suggest complicated transcriptional and post-transcriptional regulatory mechanisms employed by the parasite. The data should not only significantly improve the re-annotation of the genome sequences but also should provide new information about the biology of the parasite.
We appreciate very much the bioinformatic support of Dr. Haibo Sun at MininGene Biotechnology and the efforts of technicians at Shenzhen BGI for Solexa sequencing. We also thank the Schistosoma japonicum Genome Sequencing and Functional Analysis Consortium for making the S. japonicum genome available on line publicly.
This study was supported by the National Natural Science Foundation of China (#81270026), the intramural grant from Institute of Pathogen Biology, CAMS (2012IPB207), the National S & T Major Program (Grant No. 2012ZX10004-220) and the Program for Changjiang Scholars and Innovative Research Team in University(IRT13007).
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