Weighted Gene Co-Expression Analyses Point to Long Non-Coding RNA Hub Genes at Different Schistosoma mansoni Life-Cycle Stages.

Long non-coding RNAs (lncRNAs) (>200 nt) are expressed at levels lower than those of the protein-coding mRNAs, and in all eukaryotic model species where they have been characterized, they are transcribed from thousands of different genomic loci. In humans, some four dozen lncRNAs have been studied in detail, and they have been shown to play important roles in transcriptional regulation, acting in conjunction with transcription factors and epigenetic marks to modulate the tissue-type specific programs of transcriptional gene activation and repression. In Schistosoma mansoni, around 10,000 lncRNAs have been identified in previous works. However, the limited number of RNA-sequencing (RNA-seq) libraries that had been previously assessed, together with the use of old and incomplete versions of the S. mansoni genome and protein-coding transcriptome annotations, have hampered the identification of all lncRNAs expressed in the parasite. Here we have used 633 publicly available S. mansoni RNA-seq libraries from whole worms at different stages (n = 121), from isolated tissues (n = 24), from cell-populations (n = 81), and from single-cells (n = 407). We have assembled a set of 16,583 lncRNA transcripts originated from 10,024 genes, of which 11,022 are novel S. mansoni lncRNA transcripts, whereas the remaining 5,561 transcripts comprise 120 lncRNAs that are identical to and 5,441 lncRNAs that have gene overlap with S. mansoni lncRNAs already reported in previous works. Most importantly, our more stringent assembly and filtering pipeline has identified and removed a set of 4,293 lncRNA transcripts from previous publications that were in fact derived from partially processed mRNAs with intron retention. We have used weighted gene co-expression network analyses and identified 15 different gene co-expression modules. Each parasite life-cycle stage has at least one highly correlated gene co-expression module, and each module is comprised of hundreds to thousands lncRNAs and mRNAs having correlated co-expression patterns at different stages. Inspection of the top most highly connected genes within the modules' networks has shown that different lncRNAs are hub genes at different life-cycle stages, being among the most promising candidate lncRNAs to be further explored for functional characterization.

Weighted gene co-expression analyses point to long non-coding RNA hub genes at different Schistosoma mansoni life-cycle stages

Long non-coding RNAs (lncRNAs) (>200 nt) are expressed at levels lower than those of the protein-coding mRNAs, and in all eukaryotic model species where they have been characterized, they are transcribed from thousands of different genomic loci. In humans, some four dozen lncRNAs have been studied in detail, and they have been shown to play important roles in transcriptional regulation, acting in conjunction with transcription factors and epigenetic marks to modulate the tissue-type specific programs of transcriptional gene activation and repression. In Schistosoma mansoni, around 10,000 lncRNAs have been identified in previous works. However, the limited number of RNA-sequencing (RNA-seq) libraries that had been previously assessed, together with the use of old and incomplete versions of the S. mansoni genome and protein-coding transcriptome annotations, have hampered the identification of all lncRNAs expressed in the parasite. Here we have used 633 publicly available S. mansoni RNA-seq libraries from whole worms at different stages (n = 121), from isolated tissues (n = 24), from cell-populations (n = 81), and from single-cells (n = 407). We have assembled a set of 16,583 lncRNA transcripts originated from 10,024 genes, of which 11,022 are novel S. mansoni lncRNA transcripts, whereas the remaining 5,561 transcripts comprise 120 lncRNAs that are identical to and 5,441 lncRNAs that have gene overlap with S. mansoni lncRNAs already reported in previous works. Most importantly, our more stringent assembly and filtering pipeline has identified and removed a set of 4,293 lncRNA transcripts from previous publications that were in fact derived from partially processed mRNAs with intron retention. We have used weighted gene co-expression network analyses and identified 15 different gene co-expression modules. Each parasite life-cycle stage has at least one highly correlated gene co-expression module, and each module is comprised of hundreds to thousands lncRNAs and mRNAs having correlated co-expression patterns at different stages. Inspection of the top most highly connected genes within the modules’ networks has shown that different lncRNAs are hub genes at different life-cycle stages, being among the most promising candidate lncRNAs to be further explored for functional characterization.

Inhibition of histone methyltransferase EZH2 in Schistosoma mansoni in vitro by GSK343 reduces egg laying and decreases the expression of genes implicated in DNA replication and noncoding RNA metabolism.

BACKGROUND: The possibility of emergence of praziquantel-resistant Schistosoma parasites and the lack of other effective drugs demand the discovery of new schistosomicidal agents. In this context the study of compounds that target histone-modifying enzymes is extremely promising. Our aim was to investigate the effect of inhibition of EZH2, a histone methyltransferase that is involved in chromatin remodeling processes and gene expression control; we tested different developmental forms of Schistosoma mansoni using GKS343, a selective inhibitor of EZH2 in human cells. METHODOLOGY/PRINCIPAL FINDINGS: Adult male and female worms and schistosomula were treated with different concentrations of GSK343 for up to two days in vitro. Western blotting showed a decrease in the H3K27me3 histone mark in all three developmental forms. Motility, mortality, pairing and egg laying were employed as schistosomicidal parameters for adult worms. Schistosomula viability was evaluated with propidium iodide staining and ATP quantification. Adult worms showed decreased motility when exposed to GSK343. Also, an approximate 40% reduction of egg laying by GSK343-treated females was observed when compared with controls (0.1% DMSO). Scanning electron microscopy showed the formation of bulges and bubbles throughout the dorsal region of GSK343-treated adult worms. In schistosomula the body was extremely contracted with the presence of numerous folds, and growth was markedly slowed. RNA-seq was applied to identify the metabolic pathways affected by GSK343 sublethal doses. GSK343-treated adult worms showed significantly altered expression of genes related to transmembrane transport, cellular homeostasis and egg development. In females, genes related to DNA replication and noncoding RNA metabolism processes were downregulated. Schistosomula showed altered expression of genes related to cell adhesion and membrane synthesis pathways. CONCLUSIONS/SIGNIFICANCE: The results indicated that GSK343 presents in vitro activities against S. mansoni, and the characterization of EZH2 as a new potential molecular target establishes EZH2 inhibitors as part of a promising new group of compounds that could be used for the development of schistosomicidal agents.

Atlas of Schistosoma mansoni long non-coding RNAs and their expression correlation to protein-coding genes.

Long non-coding RNAs (lncRNAs) have been widely discovered in several organisms with the help of high-throughput RNA sequencing. LncRNAs are over 200 nt-long transcripts that do not have protein-coding (PC) potential, having been reported in model organisms to act mainly on the overall control of PC gene expression. Little is known about the functionality of lncRNAs in evolutionarily ancient non-model metazoan organisms, like Schistosoma mansoni, the parasite that causes schistosomiasis, one of the most prevalent infectious-parasitic diseases worldwide. In a recent transcriptomics effort, we identified thousands of S. mansoni lncRNAs predicted to be functional along the course of parasite development. Here, we present an online catalog of each of the S. mansoni lncRNAs whose expression is correlated to PC genes along the parasite life-cycle, which can be conveniently browsed and downloaded through a new web resource http://verjolab.usp.br. We also provide access now to navigation on the co-expression networks disclosed in our previous publication, where we correlated mRNAs and lncRNAs transcriptional patterns across five life-cycle stages/forms, pinpointing biological processes where lncRNAs might act upon.Database URL: http://verjolab.usp.br.

The Schistosoma mansoni genome encodes thousands of long non-coding RNAs predicted to be functional at different parasite life-cycle stages.

Next Generation Sequencing (NGS) strategies, like RNA-Seq, have revealed the transcription of a wide variety of long non-coding RNAs (lncRNAs) in the genomes of several organisms. In the present work we assessed the lncRNAs complement of Schistosoma mansoni, the blood fluke that causes schistosomiasis, ranked among the most prevalent parasitic diseases worldwide. We focused on the long intergenic/intervening ncRNAs (lincRNAs), hidden within the large amount of information obtained through RNA-Seq in S. mansoni (88 libraries). Our computational pipeline identified 7029 canonically-spliced putative lincRNA genes on 2596 genomic loci (at an average 2.7 isoforms per lincRNA locus), as well as 402 spliced lncRNAs that are antisense to protein-coding (PC) genes. Hundreds of lincRNAs showed traits for being functional, such as the presence of epigenetic marks at their transcription start sites, evolutionary conservation among other schistosome species and differential expression across five different life-cycle stages of the parasite. Real-time qPCR has confirmed the differential life-cycle stage expression of a set of selected lincRNAs. We have built PC gene and lincRNA co-expression networks, unraveling key biological processes where lincRNAs might be involved during parasite development. This is the first report of a large-scale identification and structural annotation of lncRNAs in the S. mansoni genome.

Schistosoma mansoni Egg, Adult Male and Female Comparative Gene Expression Analysis and Identification of Novel Genes by RNA-Seq.

BACKGROUND: Schistosomiasis is one of the most prevalent parasitic diseases worldwide and is a public health problem. Schistosoma mansoni is the most widespread species responsible for schistosomiasis in the Americas, Middle East and Africa. Adult female worms (mated to males) release eggs in the hepatic portal vasculature and are the principal cause of morbidity. Comparative separate transcriptomes of female and male adult worms were previously assessed with using microarrays and Serial Analysis of Gene Expression (SAGE), thus limiting the possibility of finding novel genes. Moreover, the egg transcriptome was analyzed only once with limited bacterially cloned cDNA libraries. METHODOLOGY/PRINCIPAL FINDINGS: To compare the gene expression of S. mansoni eggs, females, and males, we performed RNA-Seq on these three parasite forms using 454/Roche technology and reconstructed the transcriptome using Trinity de novo assembly. The resulting contigs were mapped to the genome and were cross-referenced with predicted Smp genes and H3K4me3 ChIP-Seq public data. For the first time, we obtained separate, unbiased gene expression profiles for S. mansoni eggs and female and male adult worms, identifying enriched biological processes and specific enriched functions for each of the three parasite forms. Transcripts with no match to predicted genes were analyzed for their protein-coding potential and the presence of an encoded conserved protein domain. A set of 232 novel protein-coding genes with putative functions related to reproduction, metabolism, and cell biogenesis was detected, which contributes to the understanding of parasite biology. CONCLUSIONS/SIGNIFICANCE: Large-scale RNA-Seq analysis using de novo assembly associated with genome-wide information for histone marks in the vicinity of gene models constitutes a new approach to transcriptome analysis that has not yet been explored in schistosomes. Importantly, all data have been consolidated into a UCSC Genome Browser search- and download-tool (http://schistosoma.usp.br/). This database provides new ways to explore the schistosome genome and transcriptome and will facilitate molecular research on this important parasite.