Genome-wide transcriptome analysis shows extensive alternative RNA splicing in the zoonotic parasite Schistosoma japonicum

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Figure 1

Schematic illustration of alternative splicing. A) Exon skipping. Gene A forms two different transcripts; the first transcript has a new exon compared to the second transcript, the new exon is an inclusive exon, and the other two exons are constitutive. B) Intron retention. Gene B forms two different transcripts; the second transcript is a new exon formed from retained intron and exons on both sides. C) Alternative donor site. Gene C forms two different transcripts; the difference is one exon of an alternative 5′ splice site of the second transcript extended. D) Alternative acceptor site. Gene D forms two different transcripts; the difference is one exon of the alternative 3′ splice site of the second transcript extended. E) Mutually exclusive exon. Gene E forms two different transcripts; the different exon is an inclusive exon, the same exon is a constitutive exon, and two transcripts have different inclusive exons.

Figure 2

Proportions of sequence reads (transcripts) generated from different genetic regions in the genomes of female and male S. japonicum . More than 50% of the transcripts were generated from exons while transcripts from intron, intergenic regions, and splicing events were around 7%, 22%, and 13%, respectively.

Figure 3

Sequence mapping and verification of 5 genes with exon skipping events detected by RNA-seq by PCR and RT-PCR. The expression profiles of the same gene in female (red) and male (blue) parasites were placed under the line representing the chromosome position. The black lines represent original annotated gene structures (thick lines indicate exonic regions, and thin lines indicate intronic regions), while the active transcripts in red and blue identified from the same genes in female and male parasites are underneath. The five genes and transcripts (A, B, C, D, E) with exon skipping events were confirmed by PCR and RT-PCR (F). gE indicates PCR products amplified from genomic DNA, and cE indicates PCR products amplified from cDNA. Red arrows indicate transcripts generated by exon skipping, and green arrows indicate primer locations.

Figure 4

Sequence mapping and verification of three genes with intron retention events detected by RNA-seq by PCR and RT-PCR. The three genes and transcripts (A, B, C) with intron retention events were confirmed by PCR and RT-PCR (D). gI indicates PCR products amplified from genomic DNA, and cI indicates PCR products amplified from cDNA. Red arrows indicate transcripts generated by intron retention, and green arrows indicate primer locations.

Figure 5

Numbers of non-coding RNA transcripts identified in the transcriptomes of female and male S. japonicum .

Figure 6

GO categories of transcripts with a complete open reading frame and derived from pseudogenes. The percentages of the transcripts encoding the same proteins with similar function are indicated on the left while the numbers of the transcripts identified are indicated on the right.