Profiles of Small Non-Coding RNAs in Schistosoma japonicum during Development
Citation: Cai P, Hou N, Piao X, Liu S, Liu H, et al. (
Profiles of Small Non-Coding RNAs in Schistosoma japonicum during Development
Pengfei Cai 0
Nan Hou 0
Xianyu Piao 0
Shuai Liu 0
Haiying Liu 0
Fan Yang 0
Jianwei Wang 0
Qi Jin 0
Qijun Chen 0
Malcolm K. Jones, University of Queensland, Australia
0 1 Laboratory of Parasitology, Institute of Pathogen Biology/Institute of Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College , Beijing , China , 2 Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, Jilin University , Changchun , China
Background: The gene regulation mechanism along the life cycle of the genus Schistosoma is complex. Small non-coding RNAs (sncRNAs) are essential post transcriptional gene regulation elements that affect gene expression and mRNA stability. Preliminary studies indicated that sncRNAs in schistosomal parasites are generated through different pathways, which are developmentally regulated. However, the data of sncRNAs of schistosomal parasites are still fragmental and a complete expression profile of sncRNAs during the parasite development requires a deep investigation. Methodology/Principal Findings: We employed high-throughput genome-wide transcriptome analytic techniques to explore the dynamic expression of microRNAs (miRNAs) and endogenous siRNAs (endo-siRNAs) of Schistosoma japonicum covering the free-living cercarial stage and all stages in the definitive host. This led us to analyze over 70 million clean reads represented both high and low abundance of the small RNA population. Patterns of differential expression of miRNAs and endo-siRNAs were observed. MiRNAs was twice more than endo-siRNAs in cercariae, but gradually decreased along with the development of the parasite. Both small RNA types were presented in equal aboudance in lung-stage schistosomula, while endo-siRNAs accumulated to 6 times more than miRNAs in adult female worms and hepatic eggs. Further, miRNAs were found mainly derived from genes located in the intergenic regions, while endo-siRNAs were mainly generated from transposable elements (TEs). The expression pattern of TE-siRNAs, as well as the pseudogene-derived siRNAs clustered in mRNAs of cytoskeletal proteins, stress proteins, enzymes related to energy metabolism also revealed distinction throughout different developmental stages. Natural antisense transcripts (NATs)-related siRNAs accounted for minor proportion of the endo-siRNAs which were dominantly expressed in cercariae. Conclusions/Significance: Our results represented a comprehensive expression profile of sncRNAs in various developmental stages of S. japonicum with high accuracy and coverage. The data would facilitate a deep understanding of the parasite biology and potential discovery of novel targets for the design of anti-parasite drugs.
Funding: This study was supported by the National Natural Science Foundation of China (#30901254), the intramural grant from Institute of Pathogen Biology,
CAMS (2008IPB204), and the National Science and Technology Specific Projects (2008ZX10004-011) and the Grant for Young Distinguished Scientist to QC (NSFC,
30625029). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Schistosomiasis is a chronic debilitating disease that afflicts more
than 200 million individuals in the tropics and sub-tropics regions
. The agents of this disease, parasitic flatworms of the genus
Schistosoma, have a complex developmental life cycle characterized
by a distinct parasitic phase in mammalian and molluscan hosts
and a free-living phase in freshwater. There are at least seven
discrete developmental stages of the parasite within the definitive
(lung-stage schistosomula, juvenile, adult male and female worms,
and eggs) and intermediate (sporocysts) hosts as well as the aquatic,
free-swimming miracidia and cercariae, with dramatically
morphological changes . And they are among the few platyhelminth
parasites to adopt a dioecious lifestyle and possess heteromorphic
sex chromosomes, which are arrayed in 7 pairs of autosomal
chromosomes and one pair of sexual chromosomes (Z, W),
homozygous (ZZ) for male and heterozygous (ZW) for female
[3,4]. Previous investigations on Schistosoma japonicum had revealed
that a complex gene regulation pattern was deployed by this genus
of parasites  and its haploid genome, which is about 270 Mb in
size, has been recently decoded as a valuable entity for
identification of small regulatory RNAs of this parasite .
Small non-coding RNA transcripts, approximately 1830
nucleotides in length, are critical regulators in silencing of target
genes in fungi, plants, and metazoans . Three major
categories of sncRNAs, siRNAs, miRNAs, and Piwi-interacting
RNAs (piRNAs) have been well established and extensively studied
. SncRNAs exert their regulatory functions in chromatin
architecture modelling, post-transcriptional repression and mRNA
destabilization, mobile genetic elements suppression, and virus
defence, usually through guiding the RNA-induced silencing
complex (RISC) to their target genes [7,1113]. In Drosophila,
Schistosomiasis, a debilitating disease, caused by agents of
the genus Schistosoma afflicts more than 200 million
people worldwide. Schistosomes could serve as an
interesting model to explore gene regulation due to its
evolutional position, complex life cycle and sexual
dimorphism. We previously indicated that sncRNA profile
in the parasite S. japonicum was developmentally
regulated in hepatic and adult stages. In this study, we
systematically investigated mircoRNA (miRNA) and
endogenous siRNA (endo-siRNA) profile in this parasite in more
detailed developmental stages (cercariae, lung-stage
schistosomula, separated adult worms, and liver
tissuetrapped eggs) using high-throughput RNA sequencing
technology. We observed that the ratio of miRNAs to
endo-siRNAs was dynamically changed throughout
different developmental stages of the parasite. MiRNAs were
expressed dominantly in cercariae, while endo-siRNAs
accumulated in adult female worms and hepatic eggs.
We demonstrated that miRNAs were mostly derived from
intergenic regions whereas siRNAs were mostly derived
from transposable elements. We also annotated miRNAs
and siRNAs with stage- and gender- biased expression. Our
findings would facilitate to understand the gene
regulation mechanism of this parasite and discover novel targets
for anti-parasite drugs.
sncRNAs are generated through Dicer-dependent or independent
pathways . Dicer-1 generates miRNAs whereas Dicer-2
creates endo-siRNAs. Recently, it was found that the Argonaute
protein family, which include the ubiquitous AGO (AGO1 and
AGO2) and the germline-specific Piwi (AGO3) were devoted to
different small RNA-mediated regulatory pathways . AGO1
functions primarily in the miRNA-dependent pathway that
silences messenger RNA, whereas AGO2 has been involved in
RNAi-mediated silencing directed by exogenous and endogenous
siRNAs. Further study in Drosophila somatic cells revealed that
there were two classes of endo-siRNAs, one was generated from
TEs and involved in retrotransposon repression; the other was
produced in a Dicer-2-dependent manner from distinctive
genomic loci, through refolding of RNA transcripts. The function
of the second class of endo-siRNAs was likely to regulate mRNA
stability in somatic cells .
Recently, several groups have endeavored to identify and
characterize sncRNAs of schistosome with conventional cloning
method and the deep-sequencing technique, mainly focused on
juvenile and mixed adult worms, the two relatively closed
developmental stages of the parasite . A repertoire of
miRNA transcripts unique to S. japonicum or those conserved to
other metazoan lineages was identified. Differential expression of
certain miRNAs was observed between the two developmental
stages of S. japonicum (hepatic schistosomula and adult worms) and
S. mansoni (7d schistosomula and adult worms), suggesting that
miRNAs play a distinct role in modulating development,
maturation, and reproduction of the parasite [1719,21].
Importantly, miRNA genes within one cluster could be
differentially expressed, which emphasized that individual transcript might
be developmentally regulated by distinct mechanisms [17,19].
Meanwhile, a set of endo-siRNAs derived mainly from
transposable elements (TEs) and the natural antisense transcripts (NATs) of
S. japonicum has also been defined [17,19]. Interestingly, the distinct
length and 39 end heterogeneity of endo-siRNAs derived from
both TEs and NATs were also associated with the developmental
differentiation of the parasite .
Though the knowledge regarding sncRNA biology within the
juvenile and mixed adult worms of S. japonicum is expanding, it is
indispensable to systematically profile the repertoire of sncRNAs
in other stages, especially the cercariae, which is the only infectious
stage to penetrate its mammalian hosts; the lung-stage
schistosomula, that is viewed as the most susceptible stage for
intervention [22,23]; the tissue trapped eggs, which is the critical
mediator for severe pathology in schistosomiasis, and the
difference between the two sexes of adult worms. In this study,
the expression profile of sncRNAs in the four critical
developmental stages of S. japonicum was systematically investigated. The
data, for the first time, provide a broader view of small non-coding
RNAs in the parasite.
Materials and Methods
Parasites and animals
The freshly released cercariae of S. japonicum were harvested
from parasite-infected Oncomelania hupensis purchased from Jiangxi
Institute of Parasitic Diseases, Nanchang, China. The lung-stage
schistosomula (3 days post infection) were isolated from lung
tissues of infected Kunming strain mice as previously described
. Adult worms were obtained by hepatic-portal perfusion of
New Zealand White rabbits or BALB/c mice 7-weeks post
infection. Male and female worms were manually separated with
the aid of a light microscope. Liver tissues deposited with
schistosome eggs were obtained from BALB/c mice at day 30
and 45 post infection, respectively. All procedures performed on
animals within this study were conducted following animal
husbandry guidelines of the Chinese Academy of Medical Sciences
and with permission from the Experimental Animal Committee.
All animal work have been conducted according to Chinese and
Total RNAs isolation
Total RNAs of S. japonicum at different developmental stages
(cercariae, lung-stage schistosomula, adult male and female worms
perfused from infected rabbits) and liver total RNAs of BALB/c
mice 30d and 45d post infection were extracted using Trizol
reagent (Invitrogen, CA, USA). RNA quantification and quality
were evaluated by Nanodrop ND-1000 spectrophotometer
(Nanodrop Technologies, Wilmington, DE) and Agilent 2100
Bioanalyzer (Agilent Technologies, Palo Alto, CA).
Small RNA libraries construction and sequencing
Construction of small RNA libraries was carried out as
described previously. Briefly, RNAs between 1530 nucleotides
(nt) were excised from a 15% TBE urea polyacrylamide gel
electrophoresis (PAGE). RNA samples were purified and ligated to
Illuminas proprietary 59 and 39 adaptors, and further converted
into single-stranded cDNA with Superscript II reverse
transcriptase (Invitrogen, CA, USA) and Illuminas small RNA RT-Primer.
The cDNA was amplified with high fidelity Phusion DNA
polymerase (Finnzymes Oy, Finland) in 18 PCR cycles using
Illuminas small RNA primer set. The purified PCR products were
sequenced by an Illumina Genome Analyzer at the BGI (Beijing
Genomics Institute, Shenzhen, China).
Mapping sequence reads to the reference genome
Raw datasets produced by deep sequencing from the libraries
(cercariae, lung-stage schistosomula, adult male and female
worms, and infected liver tissues) were pooled. Clean reads were
obtained after removing of the low quality reads, adaptor null
reads, insert null reads, 59 adaptor contaminants, and reads with
ployA tail. Adapter sequences were then trimmed from both ends
of clean reads. All identical sequences were counted and merged as
unique sequences, herein referred to as sequence tags. The unique
reads along with associated read counts were mapped to the S.
japonicum genome sequences
(http://lifecenter.sgst.cn/schistosoma/cn/schdownload.do) using the program SOAP . As for
the liver libraries, the unique reads were mapped to the genome of
with SOAP, and those perfectly matched ones were removed prior
to mapping to the S. japonicum genome.
Bioinformatic analysis of S. japonicum small RNAs
Briefly, the perfectly matched reads were first BLAST-searched
against the 78 known mature miRNAs of S. japonicum deposited in
Sanger miRBase [26,27] (Release 15) using the program Patscan
. The remains were then BLAST-searched against Metazoa
other than S. japonicum miRNAs allowing two mismatches to
identify homologs of known Metazoa miRNAs. These homologs,
as well as non-conserved reads (with rRNA, tRNA, snoRNA and
high repetitive reads being filtered out ) were considered as
potential miRNAs. To avoid repeated prediction and reduce the
calculation redundancy, we then searched against the genome of S.
japonicum and combined candidate unique reads located in close
proximity in the reference genome with less than 150 bp and we
called the joint genomic fragment as a block. For each block,
150 nt upstream and 150 nt downstream sequence were extracted
for secondary structure analysis. We used software Einverted of
Emboss  to find the inverted repeats (step loops or hairpin
structure), with the parameters threshold = 30, match score = 3,
mismatch score = 3, gap penalty = 6, and maximum repeat
length = 240 as described . Each inverted repeat was extended
10 nt on each side, the secondary structure of the inverted repeat
was folded using RNAfold  and evaluated by mirCheck using
default parameters . MiRNA candidates passed mirCheck
were Blast-searched against Metazoa miRNAs except those of S.
japonicum using the program Patscan again and labeled with
conserved and non-conserved (novel) miRNAs, respectively. The
novel unique reads that sequenced less than 2 times were removed.
Finally, miRNA precursors that passed MirCheck were inspected
manually in order to remove the false prediction. We employed
IDEG6 to identify miRNAs showing statistically significant
difference in relative abundance (as reflected by TPM value)
between any two small RNA libraries . The general Chi262
test was applied to determine whether one particular miRNA was
significantly differentially expressed between any two samples (P
value , = 0.01). Hierarchical clustering of the known S. japonicum
miRNAs was constructed based on the transformed data of log10
of TPM value.
The transposable elements in the S. japonicum genome were
predicted by using REPET (http://urgi.versailles.inra.fr/index.
php/urgi/Tools/REPET). TE-derived siRNAs were identified as
previously described . Figures were constructed to reflect the
relative abundance of sense and antisense of TE-derived siRNAs
during the parasite development. Briefly, the expression value of
each base on TE was the sum of the expression of siRNAs that
mapped to the position. After a proper bin (2050 bases) was
selected based on the length of TE sequences, the average
expression value was calculated for each bin, and the expression
level for four stages was marked by different colors. The natural
antisense transcripts of S. japonicum were annotated and
NATderived siRNAs were confirmed as described . The small
RNAs that failed to pass mirCheck were aligned to S. japonicum
predicted mRNA sequences of SGST (http://lifecenter.sgst.cn/
schistosoma/cn/schdownload.do) using the program SOAP, and
perfectly matched reads were retained. Then a Perl script was
wrote to scan the predicted mRNAs, if the region continuous
covered by small RNAs is longer than 100 bp, the region was
deemed as a siRNA-Cluster.
Quantitative RT-PCR analysis of sex-biased miRNAs
Stem-loop qRT-PCR was performed to quantitate the
sexbiased expressed miRNAs [20,34]. Stem-loop RT primers were
designed to reverse-transcribe target miRNAs into cDNAs using
total RNAs isolated from male and female adult worms,
respectively (from the same smaples used for Solexa sequecing).
The 20 ml reaction system contained 1 mg of total RNA, 50 nM of
each individual stem-loop RT primer, 16RT buffer, 0.5 mM
dNTPs (Takara), 0.01 M DTT (Invitrogen), 0.25 ml Superscript
III reverse transcriptase (200 U/ml, Invitrogen, CA, USA), and
0.1 ml RNaseOUT inhibitor (40 U/ml, Invitrogen). cDNA was
synthesized by incubation for 30 min at 16uC, 30 min at 42uC,
15 min at 70uC. Real-time quantification was carried out using an
Applied Biosystems StepOne Plus system. PCR reactions were set
up by combining 0.5 mM miRNA-specific forward primer, 0.5 mM
common reverse primer, 2 ml of RT product (1:1 dilution), 10 ml of
Power SYBR Green PCR Master Mix (ABI, CA, USA), and
adjusted to a final volume of 20 ml with DEPC-treated water in
triplicates. For endogenous control, 1 mg of male or female total
RNA was converted to cDNA with oligo(dT). The forward primer:
59-CCTTCATGGTAGACAACGAAGCT-39 and reverse
primer: 59-TGTAGGTTGGACGCTCTATGTCC-39, were used to
amplify the a-tubulin gene as an endogenous control. The reaction
conditions were as follows: 95uC for 5 min, followed by 40 cycles
of 95uC for 5 sec and 60uC for 30 sec. The quantification of each
miRNA relative to a-tubulin mRNA was calculated using the
equation: N = 22DCt, DCt = CtmiRNA - Cta-tubulin . All primers
used are listed in Table S1.
59-DIG-labeled miRCURY LNA probes were ordered from
Exiqon (Vedbaek, Denmark) (Http://www.exiqon.com). Northern
blot analysis was performed mianly by a method described in a
previous study . Total RNAs were isolated from male adult
female adult worms perfused from BALB/c mice 7-weeks post
infection. 10 mg total RNA of each smaple was resolved by 15%
denaturing (7 M urea) PAGE and were blotted by capillary
transfer to neutral Nylon Membranes (Hybond-NX, GE) with
206SSC. RNAs were further cross-linked to the membrane by
EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) method
. Blots were pre-hybridized by incubation with DIG Easy
Granule (Roche) at 37uC for 3 h. And hybridization were carried
out in the same buffer containing 1 nM DIG-labeled LNA probe
at temperature recommended by manufacturer (RNA Tm - 30uC)
overnight. Blots were washed twice in a low stringently buffer
(26SSC, 0.1% w/v SDS), and four times in a high stringently
buffer (0.16SSC, 0.1% w/v SDS), for 30 min each, both at
hybridization temperature. The membrane was rinsed in washing
buffer, and incubated in blocking solution at room temperature for
at least 2 h (DIG washing buffer and blocking solution Set,
Roche). Subsequently, blots were incubated with a 10,000-fold
dilution of anti-DIG-AP (Roche) in blocking solution at room
temperature for 30 min, washed 5 times for 15 min each in
washing buffer. After rinsing in detection buffer for 5 min, the
blots were detected using CDP-star chemiluminescent substrate for
alkaline phosphatase (Roche). Blots were stripped by boiling for
1 min at 100uC in 10 mM Tris-HCl, pH 8.0, 5 mM EDTA, and
0.1% SDS and probed up to three times.
Results and Discussion
Small RNA distribution in libraries from various
Six small RNA libraries were generated by high-throughput
RNA sequencing (see Materials and Methods and Table S2). Four
libraries, SjC, SjL, SjM, and SjF, were constructed from sequences
that were directly derived from the cercariae, lung schistosomula,
adult male, and female worms, respectively. The two remaining
libraries, SjE30 and SjE45, were egg libraries derived from the
hepatic tissues of BALB/c mice 30 and 45 days post-infection,
respectively. The reads that aligned to the mouse genome were
filtered before they were mapped to the genome of S. japonicum. In
total, 65,630,916 clean reads were obtained from libraries SjC,
SjL, SjM, and SjF, which were merged into 6,989,949 unique tags,
thus resulted in an average redundancy as high as 89.3
(Redundancy = 100-(Total Unique Tags/Total Clean Reads
6100)). Among these unique tags, 1,593,604 (22.80%) could be
aligned to the genome of S. japonicum (Table S3). The match rate
was varied among different libraries, from the lowest of 20.46%
(SjM) to the highest of 31.95% (SjF), this phenomenon may related
with the change of ratio of different small RNAs during
development and/or between sexes. The low match ratio to the
genome may be caused by either genome variation of different
parasite isolates or due to less sequence information of the
intergenic regions where most of the miRNAs were generated.
The phenomenon was also observed in similar studies by others,
and several explanations have been offered . Regarding the
egg libraries, 15,774 and 20,800 unique tags from libraries SjE30
and SjE45, respectively, mapped to the S. japonicum genome (Table
S4). These datasets contain roughly an order of magnitude more
sequence than previous similar studies.
The short ncRNA transcripts were categorized according to
features related to primary and secondary structure (Figure 1 and
Table S5). The majority of the ncRNA transcripts were miRNAs
and TE-derived endo-siRNAs, accounting for 26.75% and
44.77%, respectively, of the total sncRNA pool (Figure 1A). Only
2.21% of the miRNAs identified in our libraries were novel,
indicating that most miRNAs have been recovered from the
genome. Long terminal retrotransposons (LTR) and un-annotated
transposons were predominant in the set of endo-siRNAs.
Interestingly, the sets of miRNAs and endo-siRNAs displayed
stage- or sex-related variation in expression (Figure 1B and C).
The percentage of miRNA was approximately double than that of
the TE-derived endo-siRNA set in the cercariae library; the
amount of miRNAs and endo-siRNAs was almost equal in
lungstage schistosomula, while endo-siRNAs were dominant in the
adult worms and eggs, especially in female worms and early
deposited eggs (6 times more than that of miRNAs). A class of
endo-siRNAs derived from unclassified transposons was
dominantly expressed in the male and female parasite compared to
other stages (Figure 1B). The clear pattern of preferential
expression of the genes encoding the two classes of small RNAs
suggests that they play stage-specific regulatory functions. Before
invasion into a mammalian host, the parasite is likely to mainly
utilize miRNA pathways to regulate gene expression, while
endosiRNA mediated regulation is suppressed. The high percentage of
TE-derived endo-siRNAs in females and early deposited eggs
suggests that siRNAs are more functional at these developmental
stages. Earlier studies in D. melanogaster and mouse oocytes
demonstrated that endo-siRNAs were critical elements for
maintaining genomic stability through suppression of TE activity
. S. japonicum possesses a faster reproductive rate than flies
or mice, and thousands of eggs are released by one female adult
worm each day. Efficient suppression of TE activity is likely a
prerequisite for continuity of parasite development and
transmission, a possible explanation for why TE-derived endo-siRNAs
were dominantly found in late-stage parasites.
miRNAs identified in different stages of S. japonicum
When the sequences of the small RNAs containing classical
miRNA structure were aligned to the Sanger miRBase (Release
15), 77 sequences homologous to known or well-characterized
miRNAs were identified. We found 74, 71, 69, 70, 18, and 25 such
sequences in libraries SjC, SjL, SjM, SjF, SjE30, and SjE45,
respectively. Only one miRNA, the previously reported
sja-miR-85p , was not detected in this study (Table S6). Among the set
of 77 known miRNAs, approximately 20 miRNAs were conserved
homologues of sequences from the planarian Schmidtea mediterranea,
the genus most closely related to Schistosoma, in previous
investigations [17,19,20,4143], indicated that phylum
Platyhelminthes contains common miRNAs that carry out similar
biological function. The maximum read number of a single
miRNA was 1,044,358 (library SjC, sja-miR-1; Table S7),
illustrating the sequencing depth of our investigation. The range
of read numbers was from the single-digits to millions, highlighting
the sequencing capacity of next-generation sequencing technology
and suggesting that expression variation of these miRNAs does
indeed exist. This observation most likely reflects functional
differentiation among the miRNAs.
Apart from the known miRNAs, 193 hairpins containing 45
conserved mature miRNAs derived from 19 families were
predicted in our sequence libraries. These miRNAs along with
their expression level (reflected by transcripts per million, TPM)
during development were shown in Table S8. Additionally, we
identified 199 novel miRNAs with various expression levels and
stage specificities (Table S9). In contrast with the common or
evolutionarily conserved miRNAs, most novel miRNAs identified
in this study possessed low read numbers, with the exceptions of
sequences sja-novel-23-5p and sja-novel-48-3p, which was mainly
expressed in female adult worms and cercariae, respectively.
Previous investigations of miRNA biogenesis revealed that
miRNA genes are located either in intergenic regions  that are
controlled by their own miRNA promoters and regulatory units
, or in introns, non-protein coding genes, or exons, and thus
they are likely to be regulated in concert with host genes . In
an earlier study, we found that many S. japonicum miRNA genes
were clustered together, and that genes within the same cluster
may be regulated independently . In the present study, we
mapped all identified miRNA sequences to the S. japonicum genome
and found that miRNAs were generated from 59 or 39 UTRs,
intragenic, and intergenic regions in the genome; however, a
majority of sequences (87.2% of the total miRNAs identified) were
transcripts derived from genes located in intergenic loci (Figure 2).
Thus, compared to Caenorhabditis elegans, S. japonicum has evolved
more sophisticated control mechanisms for regulation of miRNA
expression, possibly explaining the complicated nature of the
transcription profiles of individual miRNAs in various
developmental stages of the parasite.
Differential expression of miRNAs during parasite
Although the relative expression level of a particular miRNA
has been proposed to be represented by the number of sequence
reads, other investigations have argued that neither read counts
nor northern blot signal accurately reflect actual abundance or
expression level [19,47,48]. Here, the expression levels of each
unique tag in cercariae, lung-stage schistosomula, separated adult
Figure 1. Classification and percentage of S. japonicum sncRNA. A. Classification and percentage of S. japonicum sncRNA using mixed small
RNA data from all stages sequenced. MiRNAs took up more than 25% of total small RNAs. TE-siRNAs were mainly derived from LTR, LINE, TIR, and
MITE, and accounted for approximately 45% of total small RNAs (including those from unknown TEs). B. Classification and percentage of S. japonicum
sncRNA from different developmental stages. C. The percentage of miRNAs and TE-siRNAs during different developmental stages. The ratio of siRNA
to miRNA was gradually increased and accumulated to the top in female worms and early deposited eggs. Note that the color scheme used in section
B was the same as section A.
worms and eggs libraries were normalized to TPM as previously
described [18,49,50]. Thus, the read abundance should basically
reflect the expression level of the tags in the parasites. The scale of
the relative miRNA abundance during the various developmental
stages appears in Figure 3. Of 77 known miRNAs, 28 miRNAs
exhibited high expression levels in one or more developmental
stages. The expression levels of the novel miRNAs identified in this
study were generally low (Table S9). However, four miRNAs were
with relatively higher expression level in one particular stage, as
sja-Novel-23-3p and sja-Novel-23-5p were dominantly expressed
in the female parasite, while sja-Novel-48-3p and sja-Novel-74-3p
were substantially expressed in cercarial stage.
Like C. elegans, schistosomal parasites need to complete a series
of biological and physiological activities, including protease
secretion, tail detachment, glycocalyx shedding, and tegument
transformation before developing to the schistosomula stage
[51,52]. A particular gene repertoire of S. mansoni parasites was
previously shown to be up-regulated during the transition from
schistosomula to adult worms . Here, we observed that the
expression of a set of miRNAs including sja-bantam, sja-miR-1,
sja-miR-124-3p, sja-miR-2a-3p, sja-miR-3492, and sja-miR-36-3p
was substantially down-regulated in lung-stage schistosomula
compared to cercariae (Table S6), suggesting that the target
mRNAs of these miRNAs may encode proteins fulfilling important
functions at this stage.
Sex-biased miRNA expression
We further explored the differential expression of miRNA genes
between male and female adult worms. The expression of a set of
miRNAs, sja-miR-7-5p, sja-miR-61, sja-miR-219-5p,
sja-miR125a, sja-miR-125b, sja-miR-124-3p and sja-miR-1 were
dominant in male worms, while sja-bantam, sja-miR-71b-5p,
sja-miRFigure 2. Percentage of miRNAs generated from three different genomic loci in S. japonicum. MiRNA transcripts identified in S. japonicum
genome were derived from up- and down-stream UTR, intragenic and intergenic regions. Proportion of the miRNAs from the three regions was 2.4%,
14.4%, and 87.2%, respectively.
3479-5p, and sja-Novel-23-5p were predominantly found in the
female parasites (Table S6 and S9). The expression of these
sexbiased miRNAs was validated by stem-loop RT-PCR (Figure 4A).
The expression level of sja-miR-1 was relatively high in male adult
worms (1.09860.228) and female adult worms (0.35860.021)
when compared to other miRNAs, and was not shown in
Figure 4A. The correlation between the TPM values and qPCR
was investigated by a method described in a previous study
(R = 0.882, Spearmans Rho, p,0.0001, n = 11) . However,
among individual miRNAs, the qPCR results did not exactly
reflect the TPM values of the maximally expressed miRNAs,
probably due to the existence of extensive miRNA isomiRs, or
asymmetrical amplification during library construction. We
further validated the expression differences of ten sex-biased
miRNAs by northern blot analysis using the total RNA extracted
from adult male and female worms isolated from BALB/c mice
7weeks post infection (Figure 4B). The differential expression
pattern of these miRNAs except sja-miR-71b-5p between male
and female worms was quite consistent with the TPM values of
high-throughput sequencing and the qRT-PCR results. The
phenomenon was also observed in a recent study which noted
that several miRNAs were expressed at similar levels in
protoscoleces of G1 and G7 genotypes Echinococcus granulosus,
which parasitized in different hosts . Thus, these data
indicated that host factors may have little impact on the expression
profile or level of sncRNAs.
Figure 3. Hierarchical clustering of the known miRNAs during different developmental stages using Pearson correlation. Heatmap
was constructed based on the log10 of TPM value of miRNAs. Black indicated the expression value of the miRNA was 0 after normalization. Grey
indicated that the miRNA was not detected in that library. TPM value for each tag was calculated by the sum of total clean reads which were
within62 bp variations of the mature miRNAs on the precursor.
Although the function of these miRNAs remains to be
elucidated, the significant differential expression between male
and female adult worms indicated that they may participate in
regulation of sexual differentiation and maintenance, pairing
and reproduction of the parasite. Moreover, miRNAs may
cooperate with other small RNAs (such as endo-siRNAs) and
transcription factors to form a comprehensive network to
regulate growth, development, differentiation, and reproduction
for adaptation to a variety of environments . Further studies
on these miRNAs may contribute to better understanding of the
developmental mechanism of sexual dimorphism in this
Figure 4. Validation of sex-biased miRNAs by quantitative RT-PCR and Northern blot. A. The relative abundance of 9 known miRNAs and
one novel miRNA investigated by quantitative RT-PCR. Six miRNAs (from sja-miR-7-5p to sja-miR-124-3p) showed higher expression level in male
parasites, while the remaining four were dominantly expressed in the female parasites. B. Northern blot analysis of ten sex-biased miRNAs using RNAs
isolated from male and female adult worms perfused from BALB/c mice. M, ultra low range DNA ladder denatured in RNA loading Dye solution as the
total RNA done. W: male adult worms; X: female adult worms.
Recent observations of endo-siRNAs in D. melanogaster, mice,
and schistosome have added more complexity to our knowledge of
small RNA-mediated regulatory pathways [14,17,3840,5558].
So far, endo-siRNAs have been found to be mainly derived from
TEs, complementary annealed NATs, and the long fold-back
transcripts known as hairpin RNAs . We previously found that
the TE-derived siRNAs in S. japonicum were more predominant
than other endo-siRNAs, including NAT-derived siRNAs .
Here, we systematically analyzed the expression levels of sense and
antisense endo-siRNAs that derived from various TEs in cercariae,
lung-stages schistosomula, male and female adult worms (Table
S1014). The read numbers of endo-siRNAs in egg libraries were
much lower than the other libraries, leading us to exclude the egg
libraries from further analysis.
We observed that LINE, TIR, and LTR transposon classes
were the main sources of endo-siRNAs, while the endo-siRNAs
derived from other TEs were much less abundant (Figure 5).
Further, endo-siRNAs mapped to the top (sense siRNA) and
bottom (antisense siRNA) strands of LTR and non-LTR TEs. The
expression patterns of LTR-derived sense and antisense siRNAs
were relatively symmetrical, though there were obvious stage and
sex specificities in expression loci (Figure 5A, B, and C). Reads
mapped to the S. japonicum LTR retrotransposon SjCHGCS11 
were annotated as SACI-7_2p in our analysis (Figure 5A). Both
sense and antisense siRNAs were concentrated in the coding
region of reverse transcriptase in a manner similar to that observed
in D. melanogaster somatic cells .
Sjpido, SjR1, and SjR2 are three classes of non-LTR
retrotransposons that make up 5% of the S. japonicum genome; siRNAs
generated from these elements mainly mapped to certain sequence
regions (Figure 5B), contrary to our observations of LTR
retrotransposons. The expression levels of siRNAs derived from
SjR1 were much higher in cercariae than in male and female adult
worms, indicating that these siRNAs are more functional in the
earlier developmental stage (Figure 5B). Sj-alpha-1 derived siRNAs
were predominantly generated from the antisense strand, while
Sjalpha-2 derived siRNAs were generated from the sense strand;
however, both types of siRNAs had low expression levels
(Figure 5C). In cercariae and lung-stage schistosomula, the TIR
(Sj_Blaster_Recon_7337_MAP_14 annotated as SmTRC1_1p in
the genome) derived siRNAs were highly expressed, while the
MITE (Sj_Blaster_Grouper_1934_MAP_4) derived siRNAs were
mainly expressed in the adult worms, and predominantly
corresponded to the antisense strand (Figure 5D). Thus, the
TEderived endo-siRNAs of S. japonicum were more diverse than those
found in D. melanogaster. Although the origin of the antisense
siRNAs is not known (cis- or trans-transcription), their abundance
suggests that they are stable and likely participate in regulatory
Previous studies of mouse oocytes revealed that antisense
transcripts from pseudogenes formed double-strand RNAs with
their functional counterparts, the sources of the endo-siRNAs, and
the sense siRNAs were predominant in the endo-siRNA . It
has been proposed that the passenger strand of an siRNA is
unstable due to the thermodynamic asymmetry of the two strands
Figure 5. The abundance of sense and antisense siRNAs that mapped to the S. japonicum TEs. The TPM of the siRNA sequences generated
from TEs during different developmental stages was presented with bars in different colors. A. Endo-siRNAs mapped to the LTR retrotransposon
SjCHGCS11. The amount of siRNAs generated from the sense and antisense strands were similar but with different stage preferences. B. Endo-siRNAs
mapped to TE of LINE type, Sjpido (upper), SjR1 (middle), SjR2 (lower). Compared to those from LTR, siRNAs from these TEs were transcribed from
more concentrated region in the genes. C. Endo-siRNAs mapped to TE of SINE Type, Sj-alpha-1 (upper), Sj-alpha-2 (lower). D. Endo-siRNAs mapped to
TE of TIR type (Sj_Blaster_Recon_7337_MAP_14) and MITE type (Sj_Blaster_Grouper_1934_MAP_4). The sum and percentage of TPM of sense and
antisense siRNAs from each stage were displayed in Pie Chart.
. However, this hypothesis cannot explain our identification of
many reads corresponding to the antisense siRNAs; in some cases,
only the antisense strands were identified. Further dissection of the
function of the two endo-siRNA classes would be an essential step
toward understanding the network of gene regulation during the
Trans-NATs were the predominant sources of
NAT-derived siRNAs are a second source of endo-siRNAs;
these endo-siRNAs are further classified as cis-NAT- or
trans-NATderived endo-RNAs [56,61,62]. Cis-NAT-derived endo-siRNAs
are generated from transcripts from the same gene locu, while
trans-NAT-derived endo-siRNAs come from NAT transcripts of
distinct loci. We detected potential NAT pairs by aligning the
predicted mRNA sequences to each other. Only one cis-NAT pair
and 1772 trans-NAT pairs were identified in silico using data from
SGST. Our sequencing results were remarkably similar to the in
silico prediction; one cis-NAT pair and 225 trans-NAT pairs were
detected (Table S15), indicating that bi-directional transcription
was much less prevalent in schistosomal parasites and transcripts
from duplicated genes are more common. Thus,
trans-NATderived endo-siRNAs are likely the main sources of NAT-derived
siRNA in S. japonicum, a scenario that differs from other organisms
. However, we cannot rule out the possibility that most of the
cis-NAT pairs may be undetectable given the lack of information
about the non-protein-coding regions of the S. japonicum genome.
The identification of long non-coding RNAs in S. japonicum is still
underway, and may provide an important source of NAT-derived
A previous study of D. melanogaster somatic cells demonstrated
that endo-siRNAs mapped to protein-coding mRNAs rather than
to transcripts of transposons that regulate mRNA expression .
Here, we also mapped the endo-siRNAs to the predicted mRNA
sequences of S. japonicum, and found that nearly half of the
siRNArelated transcripts clustered within predicted mRNAs. These
mRNAs mainly encoded proteins from four categories: 1) proteins
similar to pol polyprotein and endonuclease-reverse transcriptase;
2) cytoskeletal proteins such as myosin, actin, and tropomyosin; 3)
enzymes or transporters such as COX1, COX2B, superoxide
dismutase 1, glyceraldehyde 3-phosphate dehydrogenase, lactate
dehydrogenase A, ATP-dependent RNA helicase,
cation-transporting ATPase, H+-transporting ATPase, and cathepsin B and L;
4) stress proteins including heat shock protein, cold shock protein,
and stress-induced phosphoprotein 1 (Table S16). We were unable
to distinguish whether siRNAs clustered in pol polyprotein and
endonuclease-reverse transcriptase transcripts were derived from
retrotransposons or NATs. We speculated that some of the
siRNAs in the other three categories were derived from
transNATs formed by transcripts of pseudogenes and their parental
genes, as suggested recently ; for example, the pseudogenes of
hsp70 and cathepsin B exist in schistosome genomes [66,67].
Furthermore, the pseudogenes of actin, COX, GAPDH, FABP,
and histone are common in mammalian genomes.
Pseudogenederived endo-siRNAs were previously detected in mouse oocytes,
with two transcripts, Hsp90ab1 (heat shock protein 90 kDa alpha,
class B member 1) and Dynll1 (dynein, light chain), possessing
features similar to our findings . Thus, unlike the silencing of
selfish genetic elements by TE-related siRNAs, trans-NAT-derived
endo-siRNAs mainly regulate the expression of mRNAs coding for
a diverse set of proteins.
Our current study generated comprehensive profiles of
endogenous small RNAs (miRNAs and endo-siRNAs) during the
four crucial developmental stages of S. japonicum. Reverse
expression patterns of miRNAs and endo-siRNAs during the
parasite development and differentiation process were observed.
Two classes of endo-siRNAs derived from TEs and trans-NATs
were identified, and the LTR retrotransposon derived siRNAs
were more abundant than siRNAs from non-LTR TEs. There are
likely two layers of regulatory function employed by the parasite;
the antisense siRNAs directly affect the stability of mRNA
transcripts, while the sense siRNAs may function indirectly by
affecting the amount of antisense transcripts. Thus, the small
RNA-mediated network in schistosomal parasites is more complex
than networks reported in other organisms.
Sequences of the primers used for stem-loop
Total data summary of the six libraries.
Small RNA classification.
Reads number of the known miRNAs.
We appreciate very much the kind assistance of Dr. Haibo Sun in the
bioinformatic analysis and the technicians at Shenzhen BGI for sequencing
Conceived and designed the experiments: PC HW QC. Performed the
experiments: PC NH XP SL. Analyzed the data: PC QC. Contributed
reagents/materials/analysis tools: HL FY JW QJ. Wrote the paper: PC
1. Fenwick A , Webster JP ( 2006 ) Schistosomiasis: challenges for control, treatment and drug resistance . Curr Opin Infect Dis 19 : 577 - 582 .
2. Gobert GN , Moertel L , Brindley PJ , McManus DP ( 2009 ) Developmental gene expression profiles of the human pathogen Schistosoma japonicum . BMC Genomics 10 : 128 .
3. Hirai H , Taguchi T , Saitoh Y , Kawanaka M , Sugiyama H , et al. ( 2000 ) Chromosomal differentiation of the Schistosoma japonicum complex . Int J Parasitol 30 : 441 - 452 .
4. Mone H , Boissier J ( 2004 ) Sexual biology of schistosomes . Adv Parasitol 57 : 89 - 189 .
5. Liu F , Lu J , Hu W , Wang SY , Cui SJ , et al. ( 2006 ) New perspectives on hostparasite interplay by comparative transcriptomic and proteomic analyses of Schistosoma japonicum . PLoS Pathog 2 : e29 .
6. Zhou Y , Zheng HJ , Chen YY , Zhang L , Wang K , et al. ( 2009 ) The Schistosoma japonicum genome reveals features of host-parasite interplay . Nature 460 : 345 - 351 .
7. Bartel DP ( 2009 ) MicroRNAs: target recognition and regulatory functions . Cell 136 : 215 - 233 .
8. Molnar A , Melnyk CW , Bassett A , Hardcastle TJ , Dunn R , et al. ( 2010 ) Small Silencing RNAs in Plants Are Mobile and Direct Epigenetic Modification in Recipient Cells . Science 328 : 872 - 875 .
9. Brennecke J , Aravin AA , Stark A , Dus M , Kellis M , et al. ( 2007 ) Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila . Cell 128 : 1089 - 1103 .
10. Kim VN ( 2005 ) Small RNAs: classification, biogenesis, and function . Mol Cells 19 : 1 - 15 .
11. Malone CD , Hannon GJ ( 2009 ) Small RNAs as guardians of the genome . Cell 136 : 656 - 668 .
12. Halic M , Moazed D ( 2010 ) Dicer-independent primal RNAs trigger RNAi and heterochromatin formation . Cell 140 : 504 - 516 .
13. Khraiwesh B , Arif MA , Seumel GI , Ossowski S , Weigel D , et al. ( 2010 ) Transcriptional control of gene expression by microRNAs . Cell 140 : 111 - 122 .
14. Kawamura Y , Saito K , Kin T , Ono Y , Asai K , et al. ( 2008 ) Drosophila endogenous small RNAs bind to Argonaute 2 in somatic cells . Nature 453 : 793 - 797 .
15. Golden DE , Gerbasi VR , Sontheimer EJ ( 2008 ) An inside job for siRNAs . Mol Cell 31 : 309 - 312 .
16. Copeland CS , Marz M , Rose D , Hertel J , Brindley PJ , et al. ( 2009 ) Homologybased annotation of non-coding RNAs in the genomes of Schistosoma mansoni and Schistosoma japonicum . BMC Genomics 10 : 464 .
17. Hao L , Cai P , Jiang N , Wang H , Chen Q ( 2010 ) Identification and characterization of microRNAs and endogenous siRNAs in Schistosoma japonicum . BMC Genomics 11 : 55 .
18. Huang J , Hao P , Chen H , Hu W , Yan Q , et al. ( 2009 ) Genome-wide identification of Schistosoma japonicum microRNAs using a deep-sequencing approach . PLoS One 4 : e8206 .
19. Wang Z , Xue X , Sun J , Luo R , Xu X , et al. ( 2010 ) An ''in-depth'' description of the small non-coding RNA population of Schistosoma japonicum schistosomulum . PLoS Negl Trop Dis 4 : e596 .
20. Xue X , Sun J , Zhang Q , Wang Z , Huang Y , et al. ( 2008 ) Identification and characterization of novel microRNAs from Schistosoma japonicum . PLoS One 3 : e4034 .
21. Simoes MC , Lee J , Djikeng A , Cerqueira GC , Zerlotini A , et al. ( 2011 ) Identification of Schistosoma mansoni microRNAs . BMC Genomics 12 : 47 .
22. Gobert GN , Tran MH , Moertel L , Mulvenna J , Jones MK , et al. ( 2010 ) Transcriptional changes in Schistosoma mansoni during early schistosomula development and in the presence of erythrocytes . PLoS Negl Trop Dis 4 : e600 .
23. McManus D , Loukas A ( 2008 ) Current status of vaccines for Schistosomaisis . Clinical Review of Microbiology . pp 225 - 242 .
24. Cai P , Bu L , Wang J , Wang Z , Zhong X , et al. ( 2008 ) Molecular characterization of Schistosoma japonicum tegument protein tetraspanin-2: sequence variation and possible implications for immune evasion . Biochem Biophys Res Commun 372 : 197 - 202 .
25. Li R , Li Y , Kristiansen K , Wang J ( 2008 ) SOAP: short oligonucleotide alignment program . Bioinformatics 24 : 713 - 714 .
26. Griffiths-Jones S , Grocock RJ , van Dongen S , Bateman A , Enright AJ ( 2006 ) miRBase: microRNA sequences, targets and gene nomenclature . Nucleic Acids Res 34 : D140 - 144 .
27. Griffiths-Jones S , Saini HK , van Dongen S , Enright AJ ( 2008 ) miRBase: tools for microRNA genomics . Nucleic Acids Res 36 : D154 - 158 .
28. Dsouza M , Larsen N , Overbeek R ( 1997 ) Searching for patterns in genomic data . Trends Genet 13 : 497 - 498 .
29. Griffiths-Jones S , Moxon S , Marshall M , Khanna A , Eddy SR , et al. ( 2005 ) Rfam: annotating non-coding RNAs in complete genomes . Nucleic Acids Res 33 : D121 - 124 .
30. Rice P , Longden I , Bleasby A ( 2000 ) EMBOSS: the European Molecular Biology Open Software Suite . Trends Genet 16 : 276 - 277 .
31. Jones-Rhoades MW , Bartel DP ( 2004 ) Computational identification of plant microRNAs and their targets, including a stress-induced miRNA . Mol Cell 14 : 787 - 799 .
32. Hofacker IL , Fontana W , Stadler PF , Bonhoeffer LS , Tacker M , et al. ( 1994 ) Fast folding and comparison of RNA secondary structures . Monatshefte fu r Chemie 125 : 167 - 188 .
33. Romualdi C , Bortoluzzi S , D'Alessi F , Danieli GA ( 2003 ) IDEG6: a web tool for detection of differentially expressed genes in multiple tag sampling experiments . Physiol Genomics 12 : 159 - 162 .
34. Chen C , Ridzon DA , Broomer AJ , Zhou Z , Lee DH , et al. ( 2005 ) Real-time quantification of microRNAs by stem-loop RT-PCR . Nucleic Acids Res 33 : e179 .
35. Livak KJ , Schmittgen TD ( 2001 ) Analysis of relative gene expression data using real-time quantitative PCR and the 2(- Delta Delta C(T)) Method. Methods 25 : 402 - 408 .
36. Kim SW , Li Z , Moore PS , Monaghan AP , Chang Y , et al. ( 2010 ) A sensitive non-radioactive northern blot method to detect small RNAs . Nucleic Acids Res 38 : e98 .
37. Pall GS , Hamilton AJ ( 2008 ) Improved northern blot method for enhanced detection of small RNA . Nat Protoc 3 : 1077 - 1084 .
38. Ghildiyal M , Seitz H , Horwich MD , Li C , Du T , et al. ( 2008 ) Endogenous siRNAs derived from transposons and mRNAs in Drosophila somatic cells . Science 320 : 1077 - 1081 .
39. Watanabe T , Totoki Y , Toyoda A , Kaneda M , Kuramochi-Miyagawa S , et al. ( 2008 ) Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes . Nature 453 : 539 - 543 .
40. Chung WJ , Okamura K , Martin R , Lai EC ( 2008 ) Endogenous RNA interference provides a somatic defense against Drosophila transposons . Curr Biol 18 : 795 - 802 .
41. Lu YC , Smielewska M , Palakodeti D , Lovci MT , Aigner S , et al. ( 2009 ) Deep sequencing identifies new and regulated microRNAs in Schmidtea mediterranea . Rna 15 : 1483 - 1491 .
42. Friedlander MR , Adamidi C , Han T , Lebedeva S , Isenbarger TA , et al. ( 2009 ) High-resolution profiling and discovery of planarian small RNAs . Proc Natl Acad Sci U S A 106 : 11546 - 11551 .
43. Palakodeti D , Smielewska M , Graveley BR ( 2006 ) MicroRNAs from the Planarian Schmidtea mediterranea: a model system for stem cell biology . Rna 12 : 1640 - 1649 .
44. Lau NC , Lim LP , Weinstein EG , Bartel DP ( 2001 ) An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans . Science 294 : 858 - 862 .
45. Lagos-Quintana M , Rauhut R , Lendeckel W , Tuschl T ( 2001 ) Identification of novel genes coding for small expressed RNAs . Science 294 : 853 - 858 .
46. Rodriguez A , Griffiths-Jones S , Ashurst JL , Bradley A ( 2004 ) Identification of mammalian microRNA host genes and transcription units . Genome Res 14 : 1902 - 1910 .
47. Reddy AM , Zheng Y , Jagadeeswaran G , Macmil SL , Graham WB , et al. ( 2009 ) Cloning, characterization and expression analysis of porcine microRNAs . BMC Genomics 10 : 65 .
48. Rajagopalan R , Vaucheret H , Trejo J , Bartel DP ( 2006 ) A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana . Genes Dev 20 : 3407 - 3425 .
49. Meyers BC , Tej SS , Vu TH , Haudenschild CD , Agrawal V , et al. ( 2004 ) The use of MPSS for whole-genome transcriptional analysis in Arabidopsis . Genome Res 14 : 1641 - 1653 .
50. Huang J , Hao P , Zhang YL , Deng FX , Deng Q , et al. ( 2007 ) Discovering multiple transcripts of human hepatocytes using massively parallel signature sequencing (MPSS) . BMC Genomics 8 : 207 .
51. Curwen RS , Ashton PD , Sundaralingam S , Wilson RA ( 2006 ) Identification of novel proteases and immunomodulators in the secretions of schistosome cercariae that facilitate host entry . Mol Cell Proteomics 5 : 835 - 844 .
52. Marikovsky M , Arnon R , Fishelson Z ( 1988 ) Proteases secreted by transforming schistosomula of Schistosoma mansoni promote resistance to killing by complement . J Immunol 141 : 273 - 278 .
53. Morey JS , Ryan JC , FM VD ( 2006 ) Microarray validation: factors influencing correlation between oligonucleotide microarrays and real-time PCR . Biol Proced Online 8 : 175 - 193 .
54. Cucher M , Prada L , Mourglia-Ettlin G , Dematteis S , Camicia F , et al. ( 2011 ) Identification of Echinococcus granulosus microRNAs and their expression in different life cycle stages and parasite genotypes . Int J Parasitol 41 : 439 - 448 .
55. Tam OH , Aravin AA , Stein P , Girard A , Murchison EP , et al. ( 2008 ) Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes . Nature 453 : 534 - 538 .
56. Okamura K , Balla S , Martin R , Liu N , Lai EC ( 2008 ) Two distinct mechanisms generate endogenous siRNAs from bidirectional transcription in Drosophila melanogaster . Nat Struct Mol Biol 15 : 581 - 590 .
57. Okamura K , Chung WJ , Ruby JG , Guo H , Bartel DP , et al. ( 2008 ) The Drosophila hairpin RNA pathway generates endogenous short interfering RNAs . Nature 453 : 803 - 806 .
58. Czech B , Malone CD , Zhou R , Stark A , Schlingeheyde C , et al. ( 2008 ) An endogenous small interfering RNA pathway in Drosophila . Nature 453 : 798 - 802 .
59. Okamura K , Lai EC ( 2008 ) Endogenous small interfering RNAs in animals . Nat Rev Mol Cell Biol 9 : 673 - 678 .
60. Carthew RW , Sontheimer EJ ( 2009 ) Origins and Mechanisms of miRNAs and siRNAs . Cell 136 : 642 - 655 .
61. Lapidot M , Pilpel Y ( 2006 ) Genome-wide natural antisense transcription: coupling its regulation to its different regulatory mechanisms . EMBO Rep 7 : 1216 - 1222 .
62. Borsani O , Zhu J , Verslues PE , Sunkar R , Zhu JK ( 2005 ) Endogenous siRNAs derived from a pair of natural cis-antisense transcripts regulate salt tolerance in Arabidopsis . Cell 123 : 1279 - 1291 .
63. Andre G , Even S , Putzer H , Burguiere P , Croux C , et al. ( 2008 ) S-box and T-box riboswitches and antisense RNA control a sulfur metabolic operon of Clostridium acetobutylicum . Nucleic Acids Res 36 : 5955 - 5969 .
64. Solda G , Suyama M , Pelucchi P , Boi S , Guffanti A , et al. ( 2008 ) Non-random retention of protein-coding overlapping genes in Metazoa . BMC Genomics 9 : 174 .
65. Ghildiyal M , Zamore PD ( 2009 ) Small silencing RNAs: an expanding universe . Nat Rev Genet 10 : 94 - 108 .
66. Koziol U , Iriarte A , Castillo E , Soto J , Bello G , et al. ( 2009 ) Characterization of a putative hsp70 pseudogene transcribed in protoscoleces and adult worms of Echinococcus granulosus . Gene 443 : 1 - 11 .
67. Smooker PM , Jayaraj R , Pike RN , Spithill TW ( 2010 ) Cathepsin B proteases of flukes: the key to facilitating parasite control? Trends Parasitol 26 : 506 - 514 .