A quantitative view of the transcriptome of Schistosoma mansoni adult-worms using SAGE

Parasites, mRNA extraction and SAGE

Pooled (male and female) adult worms from BH isolate of S. mansoni were maintained in the laboratory by routine passage through mice and snails and recovered from the porto-mesenteric system by perfusion, after 7 to 8 weeks of infection. Worms were washed in saline solution and stored at -20°C in RNAlater (Ambion) prior to mRNA extraction. Poly-A mRNA was isolated with MACS kit (Miltenyi Biotec Auburn, CA, USA), eluted in 200 μL of DEPC-treated water and treated twice with Promega RQ1 RNAse-free DNAse (1 U/10 μL) for 30 min at 37°C. DNAse was inactivated at 65°C for 10 min. mRNA purity and integrity were checked by RT-PCR using appropriate primer pairs of known genes and also negative controls as described in Verjovski-Almeida et al. [5]. Ninety nanograms of poly-A+ mRNA were used for the construction of a SAGE library, according to the standard I-SAGE Kit protocol (Invitrogen, USA). After size-selection, concatamers were cloned into pZERO-1 and sequenced using standard dye terminator techniques.

Bioinformatic analysis

Sequences from cloning vectors were trimmed and tags were extracted from high-quality segments using Phred [39]. Sequences with Phred-scores bellow 20, as well as identical ditags (which are likely to be the result of amplification or cloning artifacts) were excluded from further analysis. The remaining tags were ordered in a list according to their frequency.

A second list, containing putative SAGE tags of S. mansoni genes was generated in silico after mapping the Nla III restriction sites (CATG) to the complete set of full-length cDNA sequences from S. mansoni available from GenBank, from the TIGR tentative consensus and the complete set of clusters and singlets generated by our group as part of the S. mansoni transcriptome project [5]. Sequences from the three above-mentioned databases were merged to eliminate the redundancy of transcripts. The downstream 10 nt sequence that was adjacent to each Nla III restriction site in the transcripts dataset was extracted, thus generating a list of putative S. mansoni tags. These tags were annotated according to the information available for the transcripts from which they were derived. Top priority annotation was given to full-length genes, followed by TIGR consensus and our S. mansoni transcriptome project [5]. These tags were then cross-referenced with the tag list derived from our SAGE library, enabling the definition of the most abundant genes in adult worms.

Full length S. mansoni transcripts were also screened for putative alternative poly-adenylation sites using SAGE data. For this purpose, the list containing all putative SAGE-tags (adjacent to NlaIII sites) from S. mansoni full-length genes available in GenBank, was cross-referenced with the tag list and the putative tags and ranked according to their position in relation to the 3' end. The most 3' tags, that are more likely to be bona fide tags for the canonical transcripts, were ranked as zero and the remaining tags were organized in ascending order from 3' to 5'. Tags that have rank > 0, a number of counts > 1, and were not followed by a putative site of internal binding of an oligo-dT primer (at least 8 adenines in a window of 10 bases) [28] were considered as indicative of putative poly-adenylation.

Evaluation of positional distribution of SAGE tags and ESTs over S. mansoni full-length cDNAs was carried over a set of 208 genes that were tagged by at least two SAGE tags. Blast analyses showed 26,888 ESTs and 9,589 SAGE tags mapping to these genes, allowing the identification of gene regions covered by these sequences. The mapped coordinates were normalized in terms of relative position of EST over the mRNA and relative coverage over all genes was calculated. This positional distribution was plotted together the distribution of the SAGE tags over the same gene set, where the 0% and 100% are equivalent to 5' and 3' positions of mRNAs, respectively.

Functional classification of S. mansoni transcripts was undertaken using the Gene Ontology database. For this, blast analyses of the genes mapped by our SAGE tags were performed against 2,413,334 protein sequences available from Gene Ontology database (02/2007). All ontologies associated to the first hit matched by the query sequence were recovered and then was assumed that S. mansoni gene would have the same functional annotation. Evaluations of function were performed for 3 different classes of abundance including: abundant (represented by more than 500 tags), intermediate (499 to 100 tags) and less abundant (lower than 100 tags).

Comparing SAGE and EST data

To establish how the transcriptome derived from SAGE and ESTs can be compared to each other, we evaluated the relative distribution of SAGE and EST sequences over a set of 208 worm full-length mRNA sequences available in GenBank. The 208 full-length transcripts are covered by 26,888 ESTs and 9,589 SAGE tags. As expected, 42% of the SAGE tags that map to the set of 208 full-length genes are positioned in the last 20% of the transcripts. On the other hand, only 17% of the ESTs mapped to these genes cover this same 3' portion of the transcripts (see Additional file 3). This clearly results from the biased distribution of the ESTs that were produced using the ORESTES technique (94,308/110,328 ESTs available at the time of preparation of this manuscript) and shows the necessity of generating further S. mansoni ESTs from the 3' end of the transcripts, for a more complete knowledge of the schistosome transcriptome. This also points to a reduced overlap of the SAGE and available EST data, which will result in a poor coverage of low expressed genes by non-normalized 3' UTR ESTs and in the failure of SAGE-to-transcript assignment.

Indeed, from the total of 6,263 tags with frequency higher than one, 2,916 (46.6%) found no matches on the transcript databases used. As expected, this failure in finding the correspondent gene for a specific tag was found to be directly related to the low expression of the corresponding transcript, and its reduced coverage by ESTs. In fact this can be used as an indirect measurement of correlation of SAGE and EST coverage. Whereas 96% of the 50 most frequent tags or 92% of top 100 tags could be identified in a transcript, only 53% of all ditags (6,263 top) or only 40% of all 15,655 tags could be assigned to its correspondent gene. As the S. mansoni SAGE tags are usually located at 242 nt upstream from the 3' end of the transcripts (average position of the CATG tags in full length transcripts), this data clearly demonstrates that more 3' sequences from normalized cDNA libraries are required for deciphering the transcriptome of this parasite.

Putative poly-adenylation in S. mansoni

While the same tag can be mapped to many transcripts (indicating a conservation of a nucleotide motif), we also see that a single transcript might sometimes generate various different tags. This parallels to what happens in proteomic studies when the same protein sometimes generates different spots in a gel. The occurrence of multiple tags deriving from the same transcript could occur by methodological problems (such as an incomplete digestion by the anchoring enzyme or the presence of false-polyA tails) or due to biological features such as splicing variants in the transcript region containing the most 3' tag or as the result of the use of multiple poly-adenylation sites. Whereas the use of SAGE tags to evaluate alternative-splicing is more difficult, the occurrence of alternative poly-adenylation events could be evaluated with less assumptions. In order to reduce the impact of methodological aspects over the determination of alternative poly-adenylation events, we have not considered tags sequenced only once, ambiguous tags (those that could be mapped to different transcripts) or internal tags that appeared before long stretches of A's in the transcript, which could have been used as false polyA tails during the cDNA synthesis step [28].

After using the above described filters, consistent events of multiple tags in a single transcript were identified in 13 full length genes. Poly-adenylation events cause a reduction in the transcript size, blocking the transcription of portions of its 3' region, together with the most 3' restriction site of the enzyme used for constructing the SAGE library. The reduction of the 3' UTR observed here, caused by the alternative poly-adenylation was usually accompanied by a removal of a significant portion of the putative ARE transcript repertoire (Adenosine and Uridine-Rich Elements) [29]. AREs are elements that can target host mRNAs towards rapid degradation (by a mechanism dependent on deadenylation), can repress their translation or can increase their stability [reviewed in [30]], dependent on the ligation of ARE binding proteins (ARE-BPs). The putative removal of AREs (observed in 11 out of the 16 putative poly-adenylation events), and the identification of ARE-BPs (such as hnRNPs, CUG-BP and nucleolin) in the transcriptome of S. mansoni, suggests that this parasite employs this mechanism for regulating mRNA stability. We should note that the occurrence of partial digestion with NlaIII seems to be rare here, as in our list of 15,655 distinct tags, not a single CATG (the restriction site for NlaIII) could be found.

Comparing transcriptome and proteome data

Some reports of proteomic analysis of different developmental stages of S. mansoni became recently available. Curwen et al. [31], presented an analysis of the four commonly used schistosome-soluble protein preparations (derived from cercariae, lung-stage, adults and eggs), finding 32 distinct proteins among the most expressed. In adult worms, 26 of the 40 most abundant spots were identified, and corresponded to 22 different proteins. According to Curwen et al. [31], the top 40 most abundant soluble proteins in adult worms, accounted for 27.4% of the total protein content of this stage. In our SAGE analysis, we reached a similar value as the 40 top genes were tagged by 12,364 tags or 21% of the total tags. When the top 10 most abundant adult-worm soluble proteins identified by Curwen et al. [31] are compared to our expression rank based on SAGE, we see that 5/10 proteins are ranked among our top 20 most abundant transcripts (14-3-3 homolog, GST28, FABP, fructose 1,6 bisphosphate aldolase and GAPDH). The remaining proteins vary in our ranking from 21^st to 253^th (see Additional file 2), suggesting higher stability and/or higher translation rates of these less transcribed genes, when pos-transcriptional events are acting as a second mechanism in the regulation of protein abundance.

RNA analysis by SAGE enabled the evaluation of genes coding for proteins whose physical-chemical properties impaired their analysis by 2D gel electrophoresis. An example is the determination of transcript abundance of priority vaccine candidates of the World Health Organization (such as Sm23 the 793^th transcript with 13 tags and paramyosin the 1456^th with 7 tags) that could not be evaluated by proteomic analysis [31] due to technical limitations, such as protein size or solubility, imposed by 2D gels.

Functional classification

The analysis of SAGE tags as to their mapping to genes coding for proteins classified into Gene Ontology functional categories, provides a general view of the parasite functions in terms of their relative frequency. From the data generated it is clear that in the adult stage, the parasite still undergoes intense cellular activity, possibly due to its accelerated membrane turnover as well as metabolic activities possibly involved with immune response evasion and the intense egg-laying activity. Furthermore, the large proportion of proteins potentially involved in defense mechanisms, suggests a dynamic interaction with host and its immune defense system.

The use of SAGE to interrogate the S. mansoni transcriptome

The most abundant tag identified here is 'ACTATTCGGG', a sequence tag that matches diverse isoforms of the gene encoding SmP14, or F10 eggshell protein family. The frequency of this tag strongly suggests that this is the most abundant mRNA species found in adult worms. This abundance is highly significant, especially if we consider the larger biomass of male worms as well as the male bias found in the sex ratio of S. mansoni infections [32]. Indeed, among the top 5% most abundant transcripts of adult worms, we can find other eggshell-related genes such as P40 (146^th most abundant transcript, with 56 tags), P19 (202^nd with 42 tags) and P48 (356^th with 26 tags), which advocates their importance in the early-stages of eggshell formation. We should observe that no tags could be identified for egg-secreted proteins (such as ESP3-6 and ESP15), suggesting their expression only in later stages of the eggshell development. The high expression of actin and myosin (heavy and light chains) was also observed, with the identification of their respective genes and gene-paralogs among the top 100 transcripts, reflecting the musculature as one of the major worm tissues. Among the 50 top transcripts, as expected, we observe the high abundance of 12 ribosomal-protein genes as well as genes that encode proteins involved in protein and carbohydrate metabolism. It is also interesting to note the high abundance of the gene that codes for a protein similar to thymosin beta (17^th most abundant transcript in adult worms), especially due to its involvement with wound healing [33], its anti-inflammatory properties [34–36] and its possible involvement in the escape from the host immune system in malaria [37].