A Deep Analysis of the Small Non-Coding RNA Population in Schistosoma japonicum Eggs
Citation: Cai P, Piao X, Hao L, Liu S, Hou N, et al. (
A Deep Analysis of the Small Non-Coding RNA Population in Schistosoma japonicum Eggs
Pengfei Cai 0
Xianyu Piao 0
Lili Hao 0
Shuai Liu 0
Nan Hou 0
Heng Wang 0
Qijun Chen 0
Emmanuel Dias-Neto, AC Camargo Cancer Hospital, Brazil
0 1 MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College , Beijing , People's Republic of China, 2 Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, Jilin University , Changchun , People's Republic of China, 3 Department of Microbiology and Parasitology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & School of Basic Medicine, Peking Union Medical College , Beijing , People's Republic of China, 4 College of Life Science and Technology, Southwest University for Nationalities , Chengdu , People's Republic of China
Background: Schistosoma japonicum is a parasitic flatworm that causes zoonotic schistosomiasis. The typical outcome of schistosomiasis is hepatic granuloma and fibrosis, which is primarily induced by soluble egg-derived antigens. Although schistosomal eggs represent an important pathogenic stage to the host, the biology of this critical stage is largely unknown. We previously investigated the expression profiles of sncRNAs during different developmental stages of this parasite. However, using small RNA extracted from egg-deposited liver tissues generated limited information about sncRNAs in eggs. Here, we characterized the complete small RNAome in this stage of the parasite after optimization of RNA purification. Methodology and Principal Findings: A library, SjE, was constructed with the small RNA extracted from S. japonicum eggs and subjected to high-throughput sequencing. The data were depicted by comprehensive bioinformatic analysis to explore the expression features of sncRNAs in the egg stage. MicroRNAs accounted for about one quarter of the total small RNA population in this stage, with a strongly biased expression pattern of certain miRNA family members. Sja-miR-71, sja-miR-715p, and sja-miR-36-3p were suggested to play important roles in embryo development. A panel of transfer RNA fragments (tRFs) precisely processed from the 59 end of mature tRNAs was identified for the first time, which represented a strong egg stage-biased expression. The tRNA-Ala derived small RNAs were the most highly expressed Sj-tRFs in eggs. Further, the expression of siRNAs from 29 types of well-defined transposable elements (TEs) was observed to be relatively stable among different developmental stages. Conclusions and Significance: In this study, we characterized the sncRNA profile in the egg stage of S. japonicum. Featured expression of sncRNAs, especially the tRNA-derived small RNAs, was identified, which was further compared with that of other developmental stages. These novel findings would facilitate a deeper understanding of the biology of schistosomal parasites.
Funding: This study was supported by the National Natural Science Foundation of China (NSFC 81270026 and 30901254), the National S & T Major Program
(Grant No. 2012ZX10004-220 and 2008ZX10004-011), and the intramural grant from Institute of Pathogen Biology, CAMS (2012IPB207). 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 the following interests. They very much appreciate the bioinformatic support of Dr. Haibo Sun at MininGene
Biotechnology and the efforts of the technicians at Shenzhen BGI for Solexa sequencing. This does not alter their adherence to all the PLOS ONE policies on
sharing data and materials.
Schistosomiasis, a debilitating disease, caused by blood flukes of
the genus Schistosoma afflicts more than 230 million people
en/index.html). The three major species infecting humans are
Schistosoma haematobium, S. mansoni, and S. japonicum. The pathology
of chronic infection with S. japonicum or S. mansoni is well known as
hepatosplenic schistosomiasis, with clinical symptoms of
granulomatous inflammation, periportal fibrosis, portal hypertension,
hepatosplenomegaly, ascites, and the formation of vascular shunts
[1,2]. The granulomatous responses induced by schistosome
soluble egg antigens (SEA) released from the eggshell-enclosed
miracidium are regarded as an evolutionary compromise, that is
critical for the survival of the infected host, and but also beneficial
for the transmission of eggs . As a classical immune regulatory
model, the host immune responses induced by SEA were
intensively investigated ; however, the gene expression
regulatory mechanism during schistosomal embryonic
development is still poorly understood.
Small non-coding RNAs (sncRNAs) with a size of 18,30 nt
have been found in most eukaryotes, and are increasingly
recognized as powerful regulators of gene expression and genome
stability [8,9]. Among them, microRNAs (miRNAs), small
interfering RNAs (siRNAs), and piwi-interacting RNAs (piRNAs)
are the three major categories. So far, numerous miRNAs have
been extensively identified in animals , plants , fungi ,
and viruses . In mammals, mutation or deletion of enzymes
involved in miRNA biogenesis has been observed to lead the
defects in germ-line division and differentiation, and embryonic
morphogenesis [14,15]. In the nematode of Meloidogyne incognita,
knockdown of drosha and pasha in undifferentiated eggs led to
irregular growth and embryonic lethality . Recent advances
have also proved that non-coding RNAs (ncRNAs) are key
regulators of embryogenesis, including miRNA-induced
degradation of mRNAs and long ncRNA-mediated modification of
chromatin . Two other classes of sncRNAs, siRNAs and
piRNAs, have known involvement in the defense against parasitic
DNA elements to maintain genome stability. PiRNAs have been
proposed to ensure germline stability in germ-line cells, whereas
siRNAs were observed to play roles in both somatic and germ-line
cells. These sncRNAs are loaded into the RNA-induced silencing
complex (RISC)  or RNA-induced transcriptional silencing
complex (RITS)  to function in chromatin architecture
modelling, post-transcriptional repression and mRNA
destabilization, mobile genetic elements suppression, and virus defence
[8,2022]. In schistosome, several Argonaute orthologues were
identified in both S. japonicum [23,24] and S. mansoni , and one
of the three Argonaute proteins in S. japonicum, SjAgo2 was
suggested to maintain genome stability via suppression of
In recent years, the knowledge regarding sncRNA biology has
rapidly expanded within the phylum Platyhelminthes, using
homology-based computational approach , molecular
cloning methodology [27,3032], or deep sequencing techniques
. SncRNA profiles in other important parasitic nematodes
have also been fractionally characterized . We previously
characterized the sncRNA profiles of S. japonicum at different
developmental stages, including cercariae, lung-stage
schistosomula, hepatic schistosomula, mixed and separated adult worms, and
liver tissue-trapped eggs [36,38]. However, using the RNA
extracted from egg-deposited liver tissue for sequencing generates
only limited information about sncRNAs in the egg stage of the
Since the tissue-trapped eggs are the major agents causing the
severe pathology of schistosomiasis and those released from the
host are relevant for the prevalence of the disease, it is
indispensable to explore a complete repertoire of sncRNAs in
schistosomal eggs, which will assist the discovery of novel
intervention targets. In this study, small RNA extracted from
purified S. japonicum eggs was subjected to high-throughput
sequencing and deep analysis. The data provide a unique
expression feature of egg sncRNAs, at a comparable level to those
from other developmental stages of S. japonicum, which will shed
light on the gene regulatory mechanisms during embryonic
morphogenesis of the schistosomal parasite.
Results and Discussion
Isolation and purification of S. japonicum eggs
Rapid isolation of viable S. japonicum eggs from host hepatic
tissue was a critical step for extracting intact total RNA. In our
previous study , we analyzed the sncRNAs by directly
sequencing the total small RNA from infected liver tissues.
However, the host small RNA population, which overwhelmed
that originating from the eggs, significantly reduced the resolution
of the egg-derived small RNAome. In this study, an improved
sieving and enzymatic methodology was applied. The purified egg
samples were examined under a light microscope, and we found
that most of the eggs contained a developing embryonic larva
(miracidium) (Figure S1), whereas small-sized ones with immature
embryos had either passed through the nylon mesh screens or
were removed from the suspension in the Percoll column after
centrifugation. Therefore, the data obtained here reflect mainly
the sncRNA of mature S. japonicum eggs, which were able to release
SEA to elicit the host hepatic granulomatous responses.
General features of the two small RNA libraries of
S. japonicum eggs
To investigate the small RNA profiles in the S. japonicum eggs,
three libraries were constructed with small RNAs extracted from
purified eggs and sequenced separately. Preliminary analysis
indicated that one tRNA-derived small RNA fragment was
preferentially amplified (<32% of total reads) in one library, but
not in the other two libraries. There were no significant differences
in term of read numbers and sequence length distribution between
the second and the third libraries (data not shown). Thus only the
data of the second library were further analyzed. In total,
34,244,779 reads were generated by Solexa sequencing of the
egg small RNA library (Table S1). In the library, <30.4 million
reads were high-quality clean reads, which could be merged into
3,053,121 unique tags (Table S1 and S2). The redundancy of the
library was 89.9 (Redundancy = 1002[Total Unique Clean
Reads/Total high-quality Clean Reads6100]), which was higher
than that of our previous small RNA libraries [36,38]. The match
rate of the library was more than 70% (Table S2), which is
dramatically higher than that of our previous libraries SjE30 and
SjE45 (both at <1%), which were constructed with total small
RNA isolated from egg-trapped liver tissues . Therefore, after
eliminating contaminated reads derived from host tissue, the data
presented here should reflect the authentic repertoire of sncRNA
in S. japonicum eggs. Since the egg small RNA library has the same
order of magnitude of reads number as other available S. japonicum
small RNA libraries, it is possible to compare the profile and
expression level of sncRNAs in the egg stage with that of other
Length distribution of small RNA reads in different
S. japonicum libraries
Here, we investigated the length distribution of small RNA
reads from the egg small RNA library along with libraries of other
developmental stages that perfectly matched the draft genome
sequences of S. japonicum. As shown in Figure 1, the dominant
species of small RNAs within S. japonicum are between 18 and
23 nt, which ruled out the piRNA (normally 2931 nt) pathway in
this species. The length distribution of the reads in SjE presented a
pattern similar to that of SjC and SjH, at both total and unique
levels, which featured as that the percentage of 22-nt and 23-nt
reads, particularly the latter, was significantly higher at total level
compare to that of unique level (Figure 1A, B, and D).
Classification of sncRNAs in different small RNA libraries
of S. japonicum
The sncRNA transcripts in different small RNA libraries of S.
japonicum were systematically analyzed using a more rigorous
bioinformatic pipeline than previously described . As shown in
Figure 2, the percentage of miRNAs in the egg libraries accounted
for <24%, which was slightly higher than that in the SjF library
(9.65%), but lower than that in other three libraries (SjC, SjL, and
SjH). The percentage of reads mapping to the 28S, 18S, and 5.8S
rRNA genes, as well as the intergenic spacer sequences among
these genes in the egg libraries was also modest (<25%) when
compared with that in the SjL, SjH, SjM, and SjF libraries.
Unexpectedly, the reads derived from tRNAs were dramatically
expanded in the egg libraries, compared with that of the other
libraries. The tRNA-derived small RNA reads accounted for
23.0% of the RNA population in SjE, suggesting that there may be
a specific processing mechanism of tRNA transcripts in the egg
stage of the parasite. In addition, the TE-derived siRNAs in the
egg libraries primarily originated from two types of retroelements,
long terminal retrotransposons (LTRs) and long interspersed
nucleotide elements (LINEs). There was no significant variation in
the transcription of TE-derived siRNAs among different
developmental stages of the parasite.
MiRNAs expressed in S. japonicum eggs
The clean reads from the six libraries were aligned to the 55 S.
japonicum miRNA precursors in the Sanger miRBase [43,44]
(Release 18). In the egg libraries, 75 out of 78 known S. japonicum
mature miRNAs were detected, significantly more than that of
previous egg libraries (18 and 25 known mature miRNAs were
found in the SjE30 and SjE45 libraries, respectively), suggesting
that those miRNAs with low expression in eggs were detected in
this study (Table S3). Of the miRNAs identified, sja-miR-71b-5p,
sja-miR-71, sja-miR-1, sja-miR-36-3p, and sja-124-3p were the
most abundant members at the egg stage (Figure 3A). These five
miRNAs accounted for approximately 86% of all known miRNAs
in the SjE library, which further supports our earlier finding that
there is a strongly biased expression of particular miRNA families
in each particular developmental stage of the parasite 
(Figure 3). A similar phenomenon was also observed in other
species, such as Clonorchis sinesis, in which members from the
miR71 family accounted for one third of the reads in the adult stage
. In the parasitic nematode Trichinella spiralis, members derived
from the miR-1 and let-7 families were predominantly expressed
in larvae . Combining the TPM value of miRNAs (Table S3)
and Northern blot analysis (Figure 4), we found that members of
the sja-miR-71 family were the most highly expressed ones in the
egg stage, implying that these miRNAs may play important
regulatory functions during this stage.
The miR-36 family has so far been observed only in helminthes
. A conserved ortholog of sja-miR-36-3p was identified in S.
mansoni by computational prediction . Recently, Liu et al. also
detected a putative miR-36 family member (tsp-Novel-21) in T.
spiralis, which was mainly expressed in the adult worms . The
alignment of the Sj-miR-36-3 sequence with orthologs from other
organisms is shown in Figure S2. All the orthologs shared a
conserved seed sequence CACCGGG except bma-miR-36a
and bma-miR-36b. In C. elegans, miR-36 was one of the eight
functionally redundant members of the cel-miR-35 family
(celmiR-35,42). Previously, Alvarez-Saavedra and colleagues
comprehensively analyzed the function of the miR-35 family members
in C. elegans, and found that mutation of seven members of this
family led to developmental suppression, including embryonic and
larval lethality . More recently, this family was found to
regulate the G1/S transition of intestinal cells and promote
proliferation of germ cells in C. elegans . Further, in the
parasitic nematode of Ascaris suum, each asu-miR-36 family
member was expressed in a narrow window from early to middle
embryogenesis, implying that each member from this family may
finely regulate the development of the parasite . Considering
the conserved seed sequence and its high expression in eggs
compared to adult worms, it is postulated that sja-miR-36-3p may
plays a similar role during embryonic development of S. japonicum.
However, sja-miR-36-3p was also abundantly expressed in
cercariae and lung-stage schistosomula (Table S3), suggesting that
subtle regulatory mechanisms may be exerted at different
developmental stages by one schistosomal miRNA.
tRNA-derived small RNA in S. japonicum
Previously, tRNA-derived RNA fragments (tRFs) precisely
processed from mature or precursor tRNAs were detected in
prostate cancer cell lines by ultra-high-throughput sequencing
. More recently, tRFs were identified in the halophilic
archaeon Haloferax volcanii  and in the plant pathogenic fungus,
Magnaporthe oryzae , indicating the existence of tRFs in various
organisms. However, because the tRFs were found to be processed
from different mature tRNAs by specific endonucleases (e.g.,
ELAC2 and Dicer) under stress responses and possibly on other
occasions , tRFs may be suppressed at other developmental
stages of S. japonicum, which may have prevented the identification
of this group of small RNAs in our previous studies [36,38]. Here,
after mapping to the predicted S. japonicum tRNA sequences
(sja.trna.bed), we unexpectedly found that small RNAs derived
from tRNAs were abundantly present in the SjE library. The
percentage of tRNA-derived small RNAs was significantly
upregulated in the egg stage compared with that of other stages
(Figure 2). For the first time, we defined a panel of highly
expressed Sj-tRFs processed from mature tRNAs of S. japonicum
(Table 1). The secondary structures of the tRNAs for generating
these Sj-tRFs are shown in Figure 5AK.
Previous studies indicated that tRFs can be derived from
different positions within tRNAs . Lee et al. categorized tRFs
into three series, of which tRF-5 and tRF-3 were aligned to the 59
and 39 ends of mature tRNA, respectively, whereas the tRF-1
series are located within pre-tRNA, and their 59 ends start
precisely after the 39 ends of the mature tRNA sequence .
Here, we found that the tRFs in S. japonicum were preferentially
processed from the 59 end of mature tRNAs (Table 1 and
Figure 5AK), which represent an extensive terminal and
asymmetric processing of tRNA, as recently reported .
Furthermore, like that observed in miRNAs, isoforms from
particular tRNA(s), such as Sj-tRF-0011,Sj-tRF-0014, were
also commonly identified (Table 1).
The tRF, Sj-tRF-0011, which could be processed from mature
tRNA-Ala (AGC) and tRNA-Ala (UGC) (Figure 5A), represented
the most abundant read deposited in the SjE library. However, the
hybridization signal corresponding to 24-nt small RNAs was
stronger than that of 19-nt (Figure 5J, left panel), which could be
due to the cross-hybridization to some other tRFs homologous to
Sj-tRF-0012. In addition, Sj-tRF-002 and Sj-tRF-006, which are
respectively derived from tRNA-Ala (CGC) and tRNA-Cys
(GCA), are highly homologous to Sj-tRF-0011, with only one
base mismatch. Therefore, the signal at the position of 19-nt may
GCCCGGUUAGCUCAGUCGGU GCCGGAGUAGCUCAGUUGGGAGAGC doi:10.1371/journal.pone.0064003.t001
reflect the expression of all three tRFs (Figure 5J, left panel).
SjtRF-0031 and Sj-tRF-0032 exhibited a biased expression in eggs
as well as their pre- and mature tRNAs (Figure 5J, middle panel).
Moreover, a relatively low expressed tRF, Sj-tRF-010, was also
detected mainly in the egg stage (Figure 5J, right panel). Although
the biogenesis and function of tRFs remains to be further clarified,
one exciting finding reported by Lee et al, was that tRF-1001 can
regulate cell proliferation in prostate cancer cell lines . As tRFs
are predominantly expressed in the egg stage of S. japonicum, we
postulate that some of these tRFs may play a role in regulating the
development of schistosomal embryos.
Small RNAs mapping to S. japonicum rDNA repeats
Deep analysis of the small RNA identified a large proportion of
sncRNAs that were derived from rDNA, especially in the hepatic
schistosomula and adult worms (Figure 2). A similar result was also
observed by Xue et al., who observed an over-representation of
fragmented rRNAs in the pool of short RNAs in their S. japonicum
library . They suggested that processing of the 28S large
rRNA subunit (the phenomenon known as nick in vivo in
flatworms) may be the source of the large proportion of rRNA
fragments. After precisely mapping the small RNAs to the
ribosomal DNA repeat sequences
(5.8S-ITS2-28S-IGS-18SITS1) of S. japonicum, we confirm that the majority of these small
RNAs were derived from the 28S rRNA (Figure 6). Notably, the
amount of small RNA reads,
59-CUGACCUCGGAUCAGACGUGAU(U)-39, derived from the 59-terminus of 28S rRNA was
predominant in the SjE library. Since the highly integrity of the
total RNA extracted from the eggs (Figure S3), it is suggested that
the high-abundant rRNA fragments derived from specific regions
of the 28S rRNA were not due to the random degradation of
rRNA transcripts. Whether these small RNAs are functionally
processed products of rRNA, as in the case of tRNA-derived
sncRNAs, remains to be determined.
Expression of 29 well-defined TE-derived endo-siRNAs in
TE sequences constitute 21.84% of the S. japonicum genome
, and the mobile genetic elements (MGEs) in the S. japonicum
genome, including short interspersed nucleotide elements
(SINEs)like retrotransposons, LTR retrotransposons, non-LTR
retrotransposons, and penelope-like retrotransposons, have been
characterized in several studies . Some of these elements are still
actively mobile within the genome , implying that they must
be under tight regulation to maintain the stability of the genome
. We recently demonstrated that TE-derived siRNAs in S.
japonicum were at least partially associated with the Argonaute
protein, Ago2 . In this study, the reads from the six libraries
were mapped to the 29 well-defined TEs, including 18 LTR
retrotransposons (SjCHGCS1-18), 7 non-LTR retrotransposons
(SjCHGCS19-22, SjR1, SjR2, and Sjpido), 3 Penelope-like
retrotransposons (Sj-penelope1-3), and Gulliver. The expression levels of
siRNAs derived from these retrotransposons in the related libraries
were presented based on their TPM value (Table 2). We found
that siRNAs derived from these 29 well-defined TEs were stably
expressed at different developmental stages, though slightly
suppressed in the male adult worms (Figure 7). However, the
expression of siRNAs derived from different TEs was relatively
diverse; for instance, the siRNAs originating from Gulliver,
SjCHGCS18, SjCHGCS21, SjCHGCS22, and Sj-penelope3 were
present nearly at undetectable levels, whereas those from
SjCHGCS3 were highly expressed. In addition, the expression of
both sense and antisense siRNAs from one specific TE showed a
symmetrical pattern, with the exception of SjCHGCS17, from
which the antisense siRNAs are predominant (Table 2). No major
variation in expression level was observed in all libraries,
supporting the earlier speculation that the main function of
TEderived siRNAs was to maintain the genomic stability of S.
In summary, we have further dissected the expression
characteristics of the small RNAome in the egg stage of S. japonicum.
Strong biased expression patterns of certain miRNA family
members were observed, of which, the expression of sja-miR-71,
sja-miR-71-5p, and sja-miR-36-3p were prominent in this
pathologically-related stage. Transfer RNA (tRNA)-derived small
RNA fragments, precisely processed mainly from the 59 side of
tRNA transcripts, were identified for the first time as a novel class
of small RNA in S. japonicum, which exhibited a significant
stagebiased expression pattern, indicating their potential regulatory
function in this stage. The most highly expressed tRF,
Sj-tRF-0011, has the potential to serve as an egg stage-specific bio-marker.
The TE-derived siRNAs, which showed less variation in
expression among different stages, also appeared to be an
important constituent of the small RNA population, and is likely
to protect the integrity of the genome against retroelements. The
data in this study provide novel insights into the small RNAome of
S. japonicum, which will facilitate a deeper understanding of the
biology of this important parasitic pathogen.
Materials and Methods
Animals and Parasites
S. japonicum-infected Oncomelania hupensis snails were purchased
from Jiangxi Provincial Institute of Parasitic Diseases, Nanchang,
China. The cercariae were shed by exposing the snails to light
conditions. A total of six New Zealand white rabbits were
randomly assigned to two groups. Each rabbit was percutaneously
infected with,1,200 cercariae. Hepatic schistosomula and mixed
sex adult worms were recovered at 2 and 6 weeks post-infection,
respectively, by hepatic-portal perfusion from the infected rabbits.
Male and female worms were manually separated with the aid of a
light microscope, and washed three times with phosphate buffered
saline (PBS). Liver tissues were also obtained from the infected
rabbits at 6 weeks post-infection. All procedures carried out on
animals within this study were conducted following the animal
husbandry guidelines of the Chinese Academy of Medical Sciences
and with permission from the Experimental Animal Committee of
the Chinese Academy of Medical Sciences with the Ethical Clearance Number IPB-2011-6.
The schistosomal eggs were isolated by an improved sieving and
enzymatic method . The egg-trapped liver tissues were
chopped with a scalpel blade and homogenated to a smooth
consistency in 500 ml ice-cold PBS. The suspension was
successively passed through 80, 120, 160, 200, and 260 mesh metal
sieves, and finally a 320 mesh nylon screen. After repeated washes
with PBS, the eggs on the nylon screen were collected in a 50 ml
tube. The eggs were washed three times by discarding the tissue
debris-containing suspension after natural sedimentation on ice.
The pellet was resuspended in 50 ml PBS containing 10 mg
collagenase B, 125 mg trypsin, 10 mg penicillin, and 20 mg
streptomycin, then incubated at 37uC for 3 h with gentle shaking.
The sample was then centrifuged at 1,500 rpm at 4uC for 5 min,
and the supernatant was removed. This washing procedure was
repeated twice more. The egg pellet was resuspended in 2 ml PBS
and layered on the top of a Percoll column (containing a mixture
of 2.4 ml of Percoll and 9.6 ml of 0.25 M sucrose) in a 15 ml tube.
The tube was centrifuged at 2,000 rpm at 4uC for 5 min. Liver
debris remaining in the supernatant was removed. The eggs were
resuspended in 2 ml PBS for two more Percoll separations. The
egg pellet was washed for 3 times with PBS then transferred to
1.5 ml tubes. The purity and integrity of the eggs was examined
with the aid of a light microscope.
Total RNA preparation
After centrifuging at 12,000 rpm for 1 min, the egg pellet was
ground in liquid nitrogen. Total RNA from the eggs was extracted
using Trizol reagent (Invitrogen, CA, USA) according to the
manufacturers protocol. Total RNA from hepatic schistosomula,
male adult worms, female adult worms, and normal rabbit livers were
also isolated using Trizol reagent. RNA quantification and quality
were examined with a Nanodrop ND-1000 spectrophotometer
(Nanodrop Technologies, Wilmington, DE) and standard agarose gel
electrophoresis. All RNA samples were stored at 280uC until use.
Small RNA library construction and deep sequencing
The egg total RNA sample was evaluated with an Agilent 2100
Bioanalyzer before library construction (Figure S3). Three small
RNA libraries were constructed as described previously . Small
RNAs between 1530 nucleotides (nt) were recovered from a 15%
TBE-Urea polyacrylamide gel electrophoresis (PAGE), and ligated
into Illuminas proprietary 59 and 39 adaptors. The product was
converted into single-stranded cDNA using Superscript III reverse
transcriptase (Invitrogen, CA, USA). The cDNA was then amplified
with Illuminas small RNA primer pair using Phusion high-fidelity
DNA polymerase (NEB) in 18 PCR cycles. The purified PCR
products were sequenced using Illuminas Genome Analyzer
platform at the BGI (Beijing Genomics Institute, Shenzhen, China).
Bioinformatics analysis of reads from different small RNAs
libraries of S. japonicum
Raw datasets were produced by deep sequencing of three
libraries. After primary analysis, only the data generated from one
library, designated as SjE, were further analyzed. The data were
simultaneously analyzed with the previous small RNA datasets
from cercariae, lung-stage schistosomula, hepatic schistosomula,
male adult worms and female adult worms. First, the low quality
reads, adaptor null reads, insert null reads, 59 adaptor
contaminants, and reads with poly(A) tail were filtered. Adapter sequences
were then trimmed from both ends of clean reads. Clean reads
were obtained after all identical sequences were counted and
merged as unique sequences These unique sequences were
mapped onto the S. japonicum genome of SGST (http://
lifecenter.sgst.cn) using the program SOAP version 2.20 .
We investigated the length distribution of the perfectly matched
small RNA reads in the six libraries . Further, these small
RNA reads were categorized using an optimal bioinformatic
pipeline. In our previous study , analysis was focused mainly
on miRNAs, whereas here, the unique sequences originating from
snoRNAs (small nucleolar RNA) were in the first step filtered out
, and sequences of rRNAs and tRNAs were investigated
separately. In detail, 28S, 18S, and 5.8S rRNA sequences and
rRNA intergenic spacer sequences (GenBank Accession Number:
Z46504.4 , AY157226.1 , FJ852569.1 , and
EU835685.1 ) of S. japonicum were retrieved from the NCBI
GenBank database . The putative tRNA gene sequences of S.
japonicum were downloaded from http://www.bioinf.uni-leipzig.
de/publications/supplements/08-014 . Reads from different
small RNA libraries mapped to those rRNA and tRNA sequences,
other than to the data of Rfam as in the previous study , were
respectively defined as rRNA-derived and tRNA-derived small
RNAs. The secondary structures of mature tRNAs were predicted
using an on line algorithm, ARAGORN . The remaining
perfectly matched reads were then BLAST-searched against the 77
known mature miRNAs of S. japonicum deposited in the Sanger
miRBase [43,44] (Release 18) using the program Patscan , and
were further BLAST-searched against the conserved and novel S.
japonicum miRNAs reported in our previous study . Next, the
reads were matched to the transposable elements in the S. japonicum
genome predicted by using REPET software (http://urgi.
versailles.inra.fr/index.php/urgi/Tools/REPET), in the order of
LINE (Long Interspersed Elements), SINE (Short Interspersed
Elements), LTR (Transposable elements with Long Terminal
Repeats), TIR (Terminal inverted repeat), MITE (Miniature
inverted-repeat transposable elements), and unknown TEs. The
remaining small RNAs were aligned to S. japonicum predicted
mRNA sequences (sjr_mRNA.fasta) downloaded from SDSPB
using SOAP 2.20 aligner, and perfectly matched reads were
retained as mRNA-related siRNA. Finally, the remaining reads
were labeled as unknown small RNAs. We employed IDEG6 
to identify miRNAs or tRFs showing statistically significant
difference in relative abundance (as reflected by TPM, transcripts
per million) between any two small RNA libraries. The general
Chi-square test was applied to determine whether one particular
miRNA or tRF was significantly differentially expressed between
any two samples (P value, = 0.01) (Table S3 and S4).
To further characterize the small RNAome, full length
sequences of 29 classes of retrotransposons [54,56,68,69] were
retrieved from the NCBI GenBank database . The small RNA
reads from the libraries SjC, SL, SjH, SjF, SjM, and SjE were
mapped to the sequences of rDNA repeat and the above
mentioned retrotransposons. The abundance of rDNA-derived
small RNAs or retrotransposon-derived siRNAs was reflected
based on their expression values (TPM). A set of graphs depicting
the distribution and abundance of these small RNAs was
constructed as previously described . All sequence data of
the six small RNA libraries have been submitted to NIH Short
Read Archive with the Accession numbers of SRR786675 (for
SjE), SRR786666 (for SjC), SRR786671 (for SjL), SRR786672
(for SjH), SRR786673 (for SjM), and SRR786674 (for SjF).
Confirmation of sncRNA expression by Northern blot
The 59-DIG-labeled miRCURY LNA probes were synthesized
by Exiqon (Vedbaek, Denmark) (Http://www.exiqon.com) (Table
S5). Northern blot analysis was performed as described previously
. Total RNAs (10 mg each) from different S. japonicum stages
were resolved by 15% denaturing PAGE (7 M urea). The samples
were then transferred to neutral nylon membranes (Hybond-NX,
GE) by capillary with 206SSC, and cross-linked to the membrane
using an EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide)
method . The blots were rinsed thoroughly with double
distilled water and pre-hybridized at 37uC for 3 h in DIG Easy
Granule (Roche). Hybridization was carried out in fresh DIG Easy
Granule containing 1 nM DIG-labeled LNA probe at the
recommended temperature (RNA Tm230uC) overnight. Blots
were then washed sequentially in a low stringency buffer (26SSC,
0.1% w/v SDS) and a high stringency buffer (0.16SSC, 0.1% w/v
SDS) at the hybridization temperature. After briefly rinsing in
washing buffer, the blots were incubated in blocking buffer at
room temperature for at least 2 h (DIG washing and blocking
buffer Set, Roche). Subsequently, the blots were incubated with a
10,000-fold dilution of anti-DIG-AP Fab fragment (Roche) in
blocking buffer at room temperature for 30 min then washed 5
times for 15 min each in washing buffer. Blots were then rinsed in
detection buffer for 5 min. Anti-DIG-AP was detected using
CDPstar chemiluminescent substrate for alkaline phosphatase (Roche).
Blots were stripped by boiling for 1 min at 100uC in 10 mM
TrisHCl, 5 mM EDTA, and 0.1% SDS and probed up to three times.
Figure S1 Viable S. japonicum eggs purified from the
hepatic tissues of infected rabbits. A majority of the eggs
contains a developing miracidium.
Figure S2 Sequence alignment of sja-miR-36-3p with its
orthologs from other species. Alignment of sja-miR-36-3p
with homologous sequences from S. mansoni (sma), S. mediterranea
(sme), A. suum (asu), Capitella teleta (cte), T. spiralis (tsp), Caenorhabditis
briggsae (cbr), C. elegans (cel), Brugia malayi (bma), D. melanogaster (dme),
Drosophila mojavensis (dmo), Apis mellifera (ame), Bombyx mori (bmo),
Tribolium castaneum (tca) and Anopheles gambiae (aga), was performed by
DNAMAN version 6.0 and further refined with GeneDoc software.
Figure S3 Agilent 2100 Bioanalyzer analysis of total
RNA sample extracted from the purified eggs.
Table S4 Statistical analysis for determining whether
one specific tRF was significantly differentially
expressed between any two small RNA libraries.
probes used in
We very much appreciate the bioinformatic support of Dr. Haibo Sun at
MininGene Biotechnology and the efforts of the 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 publicly available on line.
Conceived and designed the experiments: PC QC. Performed the
experiments: PC XP LH SL NH. Analyzed the data: PC QC. Contributed
reagents/materials/analysis tools: HW. Wrote the paper: PC QC.
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