Identification and Characterization of Argonaute Protein, Ago2 and Its Associated Small RNAs in Schistosoma japonicum
Ago2 and Its Associated Small RNAs in
Schistosoma japonicum. PLoS Negl Trop Dis 6(7): e1745. doi:10.1371/journal.pntd.0001745
Identification and Characterization of Argonaute Protein, Ago2 and Its Associated Small RNAs in Schistosoma japonicum
Pengfei Cai 0
Xianyu Piao 0
Nan Hou 0
Shuai Liu 0
Heng Wang 0
Qijun Chen 0
Aaron G. Maule, Queen's University Belfast, United Kingdom
0 1 MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College , Beijing , People's Republic of China, 2 Department of Microbiology and Parasitology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College , Beijing , People's Republic of China, 3 Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, Jilin University , Changchun , People's Republic of China
Background: The complex life cycle of the genus Schistosoma drives the parasites to employ subtle developmentally dependent gene regulatory machineries. Small non-coding RNAs (sncRNAs) are essential gene regulatory factors that, through their impact on mRNA and genome stability, control stage-specific gene expression. Abundant sncRNAs have been identified in this genus. However, their functionally associated partners, Argonaute family proteins, which are the key components of the RNA-induced silencing complex (RISC), have not yet been fully explored. Methodology/Principal Findings: Two monoclonal antibodies (mAbs) specific to Schistosoma japonicum Argonaute protein Ago2 (SjAgo2), but not SjAgo1 and SjAgo3, were generated. Soluble adult worm antigen preparation (SWAP) was subjected to immunoprecipitation with the mAbs and the captured SjAgo2 protein was subsequently confirmed by Western blot and mass spectrometry (MS) analysis. The small RNA population associated with native SjAgo2 in adult parasites was extracted from the immunoprecipitated complex and subjected to library construction. High-through-put sequencing of these libraries yielded a total of <50 million high-quality reads. Classification of these small RNAs showed that endogenous siRNAs (endo-siRNAs) generated from transposable elements (TEs), especially from the subclasses of LINE and LTR, were prominent. Further bioinformatics analysis revealed that siRNAs derived from ten types of well-defined retrotransposons were dramatically enriched in the SjAgo2-specific libraries compared to small RNA libraries constructed with total small RNAs from separated adult worms. These results suggest that a key function of SjAgo2 is to maintain genome stability through suppressing the activities of retrotransposons. Conclusions/Significance: In this study, we identified and characterized one of the three S. japonicum Argonautes, SjAgo2, and its associated small RNAs were found to be predominantly derived from particular classes of retrotransposons. Thus, a major function of SjAgo2 appears to associate with the maintenance of genome stability via suppression of retroelements. The data advance our understanding of the gene regulatory mechanisms in the blood fluke.
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 (2008ZX10401). 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 caused by the
parasitic blood flukes of the genus Schistosoma, which afflicts more
than 230 million individuals in 77 endemic countries (http://www.
schistosomes have a complex developmental life cycle characterized by
an asexual multiplication phase (mother sporocysts and daughter
sporocysts) in the molluscan hosts and a sexual development and
reproduction phase (lung-stage schistosomula, juvenile, adult male
and female worms, and eggs) in mammalian hosts, as well as the
aquatic free-swimming phase including miracidia and cercariae
. It is well known that the schistosome parasites undergo
dramatic morphological transformation and rapid physiological
adaptation to its life niche during development , which is
essentially controlled by subtle gene regulatory mechanisms
[1,36]. The decoding of the genomes of the three major
pathogenic blood flukes, Schistosoma japonicum, Schistosoma mansoni,
and Schistosoma haematobium, has provided a valuable entity for a
systematic dissection of the parasite biology [4,7,8].
In the past decade, small non-coding RNAs (sncRNAs) have
emerged as critical regulators of gene expression both at
transcriptional and post-transcriptional levels in metazoans, plants,
fungi, and viruses . In schistosomes, sncRNA repertoires at
different developmental stages of the parasites have been revealed
. Both microRNAs (miRNAs) and small endogenous
interfering RNAs (endo-siRNAs) are expressed in a stage- and
Schistosomiasis, a chronic disease caused by agents of the
genus Schistosoma, still afflicts more than 230 million
people worldwide. The genomes of the three major
pathogenic blood flukes, Schistosoma japonicum,
Schistosoma mansoni, and Schistosoma haematobium, have been
decoded as valuable entities for a systematic dissection of
the biological characteristics of the parasites. Transposable
elements constitute a major component in the genome of
schistosomes and have been recognized as remnants of
evolutionary events, but some of them are still active
today. Thereby, the activity of these active mobile genetic
elements should be restricted by elaborate mechanisms to
protect genome stability. Our study showed that one of
the three S. japonicum Argonaute proteins, SjAgo2, is
involved in such mechanisms. By using specific mAb,
native SjAgo2 protein was immunoisolated from a soluble
adult worm antigen preparation, and its associated small
RNAs were extracted for deep sequencing. We found that
SjAgo2 is mainly associated with particular types of
retrotransposon-derived siRNAs. For instance, siRNAs
generated from 10 classes of well-defined retrotransposons
were significantly enriched in the SjAgo2-specific libraries.
Thus, a major function of Ago2 in S. japonicum is proposed
to be the maintenance of genome stability via
retrotransposon suppression. Our findings advance
understanding of the putative gene regulatory mechanisms in
a flatworm parasite.
gender-biased manner. MiRNA transcripts are generated
primarily from the intergenic regions of the genome, whereas
endosiRNAs are principally originated from the transposable elements,
including transposons and retrotransposons. The preferential
expression of these sncRNAs in different developmental stages
and sexes suggests that they play distinct roles in modulating
development, maturation, and reproduction of the parasite [14
To exert their activities, sncRNAs must be selectively loaded
onto their relevant machinery, the RNA-induced silencing
complex (RISC), and guide the RISC to their complementary
templates. Argonaute family proteins are at the heart of RISCs,
which can be divided into Ago and PIWI subfamilies , and a
third clade, termed group III Argonautes is worm-specific for
binding secondary siRNAs . Although small RNA pathways
are evolutionally conserved, the number of Argonaute genes varies
dramatically in different organisms, ranging from one in the fission
yeast Schizosaccharomyces pombe to twenty-seven in the nematode
Caenorhabditis elegans . Different small RNA regulatory
pathways (SRRPs) may be mediated by one Argonaute protein, such as
metazoan-like Argonaute in the single-cell parasite Toxoplasma
gondii , or entangled with multiple Argonaute proteins, which
compete and collaborate with each other to form regulatory
networks . In S. mansoni, four Argonaute proteins were
identified by two groups mainly based on bioinformatic analysis,
but SmAgo3 and SmAgo4 seemed to be generated from an
alternatively spliced mRNA . Argonaute orthologs in S.
japonicum (SjAgos) have been also reported by two groups [27,28].
Both of them tried to determine the full-length sequences of the
three Argonaute proteins and described the molecular
characteristics of SjAgos. Chen et al. also reported the differential expression
of SjAgos during the parasite development and suggested that
SjAgos coordinated in different SRRPs may be involved in
regulating schistosome development . In addition, no PIWI
homologue was identified in S. japonicum, though it was found in its
closely related genus Schmidtea mediterranea [29,30].
Although abundant small non-coding RNAs have been
identified in schistosomes, the authentic function of Argonaute proteins
in different SRRPs is still largely unknown. SjAgo1 has been
previously speculated to participate in the miRNA pathway due to
its high homology with miRNA-associated Argonautes in flies,
humans, and worms, although experimental support for this idea is
still lacking . In this study, by using SjAgo2-specific mAb
(27A9), native SjAgo2 complex and the associated small RNAs in
the parasite were identified and deeply analyzed. Classification of
the small RNAs led us to propose that suppression of parasitic
retrotransposons within the genome may be the primordial
biological function of SjAgo2.
Materials and Methods
Parasites and animals
The parasite-infected Oncomelania hupensis were provided by
Jiangxi Institute of Parasitic Diseases, Nanchang, China. The
freshly released cercariae of S. japonicum were harvested for Total
RNA isolation. To obtain hepatic schistosomula and adult worms,
New Zealand White rabbits were percutaneously infected with S.
japonicum cercariae (1000 to 1500 per rabbit). Hepatic
schistosomula were isolated from the rabbits at 2 weeks post-infection, while
mixed adult worms were obtained after 6-weeks post infection by
hepatic-portal perfusion. Male and female adult worms were
manually separated with the aid of a light microscope. Eggs were
isolated from liver tissues of infected rabbits by enzyme digestion
method . 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 of Chinese Academy of
Medical Sciences with the Ethical Clearance Number IPB-2011-6.
Total RNA isolation and quality control
Total RNAs of S. japonicum at different developmental stages
(cercariae, hepatic schistosomula, separated adult male and female
worms, and eggs) were extracted using RNeasy Mini kit
(QIAGEN) and the contaminating genomic DNA was removed
from RNA samples with TURBO DNA-freeTM kit (Ambion, CA,
USA). RNA quantification and quality control was conducted by
denaturing agarose gel electrophoresis and Nanodrop ND-1000
spectrophotometer (Nanodrop Technologies, Wilmington, DE).
One mg total RNA from S. japonicum adult worms was used to
synthesize the first strand cDNA using SuperScriptTM III Reverse
Transcriptase Kit (Invitrogen, CA, USA), with oligo (dT) 15
primer. The 59 UTR of SjAgo2 gene was amplified with a SMART
RACE cDNA Amplification Kit according to the manufacturers
instructions (Clontech, CA, USA). The amplicons were cloned into
T-Vector and sequenced. The primers used for 59 RACE were
listed in Table S1.
xQRT-PCR was performed to quantitate the expression level
of SjAgo1, SjAgo2, and SjAog3 transcripts at different
developmental stages of the parasite and between separated adult worms. For
each sample, 1 mg total RNA was reverse transcribed into
strand cDNA using SuperScriptTM III Reverse Transcriptase Kit
(Invitrogen) with Oligo dT (15) primer by incubation for 5 min
at 25uC, 60 min at 50uC, and 15 min at 70uC. The resulting
cDNA products were diluted 20-fold with nuclease-free water
before qPCR. Each 25 ml PCR reaction contained 12.5 ml of
26Brilliant II SYBR Green QPCR Master Mix (Agilent, USA),
1 ml cDNA, 1 ml of the forward and reverse primer pair (Table
S1), and 10.5 ml of sterile water. The PCR conditions included 40
cycles with denaturation at 95uC for 30 s, followed by annealing
and extension at 60uC for 1 min. A dissociation step (95uC for
15 s, 60uC for 1 min, 95uC for 15 s, and 60uC for 15 s) was
added to confirm the amplification specificity for each gene. The
PCR products were separated on a 2.5% agarose gel to confirm
the presence of a single band with the expected size.
Quantification of the expression for each SjAgo gene during the parasite
development was performed by normalizing against a novel
house-keeping gene, PSMD4 (26S proteasome non-ATPase
regulatory subunit 4, GenBank accession number: FN320595)
 and applying the comparative 22DDCt method using the
software SDS 1.4.
Preparation of SWAP for immunoprecipitation
The SWAP (soluble adult worm antigen preparation) was
prepared mainly as previously described with minor modification.
Briefly, the S. japonicum adult worms were washed in PBS for five
times to reduce contamination of host components, homogenized
on ice in lysis buffer containing 20 mM Tris-HCl (pH 7.4),
200 mM NaCl, 2.5 mM MgCl2, 0.05% NP-40, EDTA-free
protease inhibitor cocktail (Roche) and RNasin (Promega) at a
final concentration of 0.1 U/ml. The homogenate was then
centrifuged at 14,000 g for 10 min at 4uC and the supernatant
was collected carefully to avoid the top lipid layer. This procedure
was repeated until the supernatant was clear. The supernatant was
stored at 280uC for further use.
Construction of the plasmids for generation of
The DNA fragments encoding tSjAgo1 (aa198-1009), SjAgo2
(aa1-935), and SjAgo3 (aa1-923) were amplified from S.
japonicum adult worm cDNA using high fidelity Phusion DNA
polymerase (Finnzymes Oy, Finland) with KpnI and NotI
endonucleases site added at their 59 and 39 terminus,
respectively (Primer sets were listed in Table S1). The PCR was
performed with an initial denaturation for 1 min at 98uC. Ten
PCR cycles were performed as follows: 98uC for 8 s, 50uC for
30 s and 72uC for 1 min, followed by another twenty PCR
cycles: 98uC for 8 s, 55uC for 30 s and 72uC for 1 min, with a
final extension at 72uC for 5 min. The amplicons were digested
with KpnI and NotI restriction endonucleases, and cloned into
pcDNA3-FLAG3C vector. The recombinant plasmids were
transformed into DH5a (DE3) Escherichia coli and positive clones
were selected for sequencing. The correct recombinant plasmids
were designated as FLAG-tSjAgo1, FLAG-SjAgo2, and
Cell culture and transfection
To generate Flag-tagged recombinant SjAgos, human 293T
cells were grown in Dulbeccos modified Eagles medium
supplemented with 2 mM L-glutamine and 10% fetal bovine
serum. 293T cells were transfected at 90% confluency in
60-mm dishes with 8 mg of FLAG-tSjAgo1, FLAG-SjAgo2,
FLAG-SjAgo3, or the empty vector, respectively, using
LipofectamineTM 2000 (Invitrogen). The cells were further
cultured for 36 h at 37uC in a 5% CO2 incubator. Cell extracts
were prepared in 200 ml lysis buffer containing 20 mM
TrisHCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, and protease
Production of mouse monoclonal antibodies to SjAgo2
Monoclonal antibodies 27A9 and 11E8 to SjAgo2 protein were
produced by Abmart Inc (Shanghai, China). Briefly, the optimal
peptide immunogens were selected from SjAgo2 by an in-house
peptide selection database called Antibody Designer. Two cDNA
fragments encoding aa1-232 and aa34305 of SjAgo2 were
subcloned into pET30a vector (Novagen) between BamHI and
HindIII endonuclease sites. The recombinant plasmids were
transformed into BL21 (DE3) E. coli. Two His-tag recombinant
proteins were then expressed in E. coli and purified with Ni-NTA
agarose beads. Six BALB/c mice were immunized subcutaneously
with the peptides. Spleen cells obtained from immunized mice
were fused with SP2/0 myeloma cells according to the standard
procedure. Positive hybridomas were cloned, and immunoglobulin
G (IgG) was purified by protein G affinity chromatography from
Immunoprecipitation and Western blot analysis
Immunoprecipitations were carried out essentially as described
by Kiriakidou et al. . For immunoprecipitation of endogenous
SjAgo2 protein, a procedure of sequential depletion by absorption
was adapted. One ml SWAP was first mixed with 100 ml of
Protein-A/G agarose slurry (50%) (Abmart, Shanghai, China) and
incubated at 4uC for 2 h with gentle rotation (Mock). After
centrifugation at 2,500 rpm for 5 min, the supernatant was
recovered and subsequently mixed with 4 mg normal mouse IgG
(Santa Cruz Biotechnology) and incubated with gentle rotation at
4uC for 2 h. Then, 40 ml of Protein-A/G agarose was added and
continually incubated at 4uC for another 2 h. The agarose beads
were collected by centrifugation for 5 min at 2,500 rpm (moIgG
IP). The supernatant was divided into equal parts and respectively
mixed with 4 mg of 11E8 or 27A9 mAb, and gently incubated at
4uC for 4 h. The immunocomplex were captured by addition of
20 ml of Protein-A/G agarose beads and gently rotating for 2 h at
4uC. The beads were collected by centrifugation for 5 min at
2,500 rpm (mAb IP). The beads in the three sequential IP assays
(without antibodies, with moIgG, and with specific mAbs) were
further washed with 1 ml ice-cold lysis buffer for 5 times and
resuspended with 16SDS loading buffer. The protein samples
were boiled for 10 min. After centrifugation, the supernatant was
collected and used for further analysis.
Western blot analysis was performed as previously described
. Cell extracts with over-expressed tSjAgo1, SjAgo2, SjAgo3,
SWAP, as well as immunoprecipitates were separately mixed with
SDS-PAGE loading buffer and separated on SDS-PAGE gels, and
transferred to the PVDF membrane. The membrane was blocked
with 5% SMP in TBS for 90 min at room temperature. Anti-Flag
mAb M2 (1:2,000 dilution, Sigma) or anti-SjAgo2 mAbs (at a final
concentration of 10 mg/ml) was used for detection of the target
proteins. The HRP (horseradish peroxidase)-conjugated goat
antimouse IgG (Zhongshan, China) at a dilution of 1:10,000 was used
as a secondary antibody and signal was detected with a
luminolbased chemiluminescent substrate (CSN).
Orbitrap Mass Spectrometry Analysis
To confirm that SjAgo2 was truly precipitated by the mAbs, two
IP and MS assays were performed. In the first assay, the
immunocomplex directly precipitated by mAb 27A9 from SWAP
was resolved on a 10% SDS-PAGE gel and visualized by
Coomassie Brilliant Blue staining. Protein bands with different
molecular weights (.170 kDa, 130170 kDa, 90130 kDa, 70
80 kDa, 6070 kDa, and 4252 kDa) were excised and subjected
to Orbitrap MS analysis. In the second assay, SWAP was
sequentially incubated with Protein-A/G agarose beads (Mock),
normal mouse IgG, and eventually with mAb 27A9. The
immunoprecipitates were resolved on a 10% SDS-PAGE gel.
Protein bands with sizes of <7090 kDa and <90120 kDa
were excised from the gel (Figure S1) and digested with trypsin.
The resulting peptides were analyzed by Orbitrap MS
and identified by blasting against the protein datasets of S.
japonicum downloaded from SDSPB (http://lifecenter.sgst.cn/
schistosoma/en/schdownload.do) and Uniprot (http://www.
uniprot.org/uniprot/?query = taxonomy%3a6182&format = *).
Small RNA library construction and sequencing
The SjAgo2 associated small RNAs were extracted as previously
described . RNA quantification and quality were evaluated by
an Agilent 2100 Bioanalyzer (Figure S2). Small RNA libraries
were constructed mainly as previously described . Briefly,
RNAs between 1540 nucleotides (nt) were excised from a 15%
TBE urea polyacrylamide gel electrophoresis (PAGE). The RNA
sample was purified and their 59 and 39 termini were ligated with
Illuminas proprietary adapters, which was further used as
templates to synthesize first-strand cDNA. The cDNA was
amplified by PCR with a high fidelity Phusion DNA polymerase
and the Illuminas small RNA primer set. The libraries were
sequenced on the Illumina Genome Analyzer II platform at the
BGI (Beijing Genomics Institute, Shenzhen, China). IP assays
were performed from two independent biological repeats with
mAb 27A9, and the RNAs were separately applied for library
construction and sequencing. The two libraries were designated as
SP1 and SP2, respectively.
Mapping sequence reads to the reference genome
Raw datasets produced by Solexa sequencing from the two
libraries were tagged and 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 trimmed from 59 and 39 ends of clean
reads. All identical sequences were counted and merged as unique
sequences. These unique reads affiliated with read counts were
mapped to the S. japonicum genome draft (sjr2_contig.fasta) (http://
lifecenter.sgst.cn/schistosoma/en/schdownload.do) using the
program SOAP version 2.20 .
Bioinformatic analysis of small RNA libraries
First, we investigated the length distribution of small RNA
reads in the two libraries that perfectly matched the genome draft
of S. japonicum, and the small RNAs were categorized by the
bioinformatic pipeline as described . Afterwards, an
alternative bioinformatic pipeline was designed to classify the small RNA
reads that perfectly matched the reference genome. Briefly, 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 an
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
TE. 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. Next, the endo-siRNAs
depleted reads were then BLAST-searched against the 78 known
mature miRNAs of S. japonicum deposited in Sanger miRBase
[37,38] (Release 17) using the program Patscan , and further
BLAST-searched against the conserved and novel S. japonicum
miRNAs reported in our previous study . Finally, homologs
to rRNA, tRNA, snoRNA, and other small RNAs  were
filtered and the remaining reads were labeled as unknown small
To further characterize the small RNAs identified, full length
sequences of 29 classes of retrotransposons [4,4143] were
retrieved from the NCBI GenBank database . The small
RNA reads from the SP1, SP2, SjM, and SjF libraries were
mapped to these retrotransposons. The abundance of these
retrotransposon-derived siRNAs was reflected based on their
expression values (TPM, transcripts per million). A set of graphs
depicting the distribution and abundance of
retrotransposonderived siRNAs were further constructed as previously described
. Briefly, the expression of each base on these TEs was the sum
of the TPM value of siRNAs that mapped to the position. A
proper bin (10 or 50 bases) was then selected based on the length
of TE sequences, and the average expression value was calculated
for each bin.
Results and Discussion
Genes encoding the three Argonaute paralogues are
differentially expressed in S. japonicum
To investigate the possibility of functional or stage specificity
of the three Argonaute paralogues in S. japonicum, we determined
the transcription levels of the three Argonaute genes in the
parasite before and after host invasion using qRT-PCR with that
of 26S proteasome non-ATPase regulatory subunit 4 (PSMD4) as
an endogenous control . The overall expression level of the
three genes was much lower in cercariae than in other stages
within the host (Figure 1). This observation suggests that the
SjAgos were mainly functional in the late developmental stages
of the parasite. The expression of SjAgo1 in eggs, miracidium,
cercariae, schistosomula, and adult worms has been reported
earlier by Lou et al. . Even though the trend of the expression
of SjAgo1 was found similar between the two studies, our results
were more profound than those reported previously. The
difference was most likely caused by the different endogenous
controls used in the two studies. The SjGAPDH gene was recently
found to be unstably transcribed during the parasite
development, which means that it is not suitable as an endogenous
transcriptional control . SjAgo2 and SjAgo3 presented a
reversed expression pattern between male and female adult
worms (Figure 1B and C). The expression of SjAgo2 was
upregulated in schistosomula and female parasites, whereas SjAgo3
was highly expressed in schistosomula and male worms.
Previously, Chen et al. reported that the expression of SjAgo1
was significantly higher in eggs than that in adult worms and the
expression of SjAgo2 and SjAgo3 was not significantly different
between male and female parasites . This inconsistency is
likely due to different experimental conditions, especially the
endogenous controls applied in the two studies. However, it
cannot be ruled out that the parasite strains in the two studies
might be different.
Determination of the full-length of SjAgo2
The two groups mentioned above also reported their analysis on
the Argonaute family members in S. japonicum but with different
results [27,28]. In light of the uncertainty of the size of SjAgo2
protein, we performed 59 RACE to determine the N-terminal
region of the protein, for the C-terminus of the protein has been
definitively defined. We confirmed that the full-length SjAgo2
protein contains 935 amino acids as reported by Chen et al. ,
but not 945 amino acids as reported by Luo et al. .
Generation of specific mAbs to SjAgo2
In order to obtain specific mAbs against SjAgo2, two optimal
peptide immunogens, aa1-232 and aa34-305 of SjAgo2, that
avoided the major homologous regions with SjAgo3, were selected
for immunizing BALB/c mice and two mAbs, 11E8 and 27A9,
were generated. To determine the specificity of the mAbs against
SjAgo2, we cloned the ORFs of the three Argonaute genes in the
eukaryotic vector pcDNA3-FLAG3C, and the SjAgos were
expressed in human 293T cells. Western blot analysis confirmed
that SjAgo2 and SjAgo3, but not SjAgo1, were expressed in the
293T cells. Next, we tried to express a truncated form of SjAgo1
(tSjAgo1, aa198-1009), since the N-terminus of SjAgo1 displayed
very low similarity with SjAgo2 and SjAgo3 , it is unlikely that
the mAb to the N-terminus of SjAgo1 would cross react with
SjAgo2 and SjAgo3. The tSjAgo1 was successfully expressed in
293T cells, though the expression level was relatively lower than
that of SjAgo2 and SjAgo3 (Figure 2A). The recognition of the
recombinant SjAgo2 by mAb 11E8 or 27A9 was confirmed by
Western blot analysis (Figure 2B). To determine whether both
mAbs would cross-react with SjAgo1 and SjAgo3, equal amounts
of the recombinant tSjAgo1, SjAgo2, and SjAgo3 were loaded in
each lane (Figure S3). The blot was further detected by mAb 11E8
or 27A9, and both mAbs only specifically recognized SjAgo2, but
not tSjAgo1 and SjAgo3 (Figure 2C).
Identification of native SjAgo2 in SWAP
Immunoprecipitates from all experimental groups were
separated by 10% SDS-PAGE (Figure S4) and followed by Western
blot analysis (Figure 3A). Two prominent bands at a molecular
weight of approximately 100 kDa were observed. However, the
lower band (asterisked) also appeared in the immunoprecipitates
captured by normal mouse IgG, indicating that it may have been
caused by non-specific binding to mouse IgG. As indicated by the
molecular weight, we speculated that the lower band might be the
IgG-binding protein paramyosin (PMY) . In contrast, the
upper band (arrowed) is more close to the theoretical molecular
weight of SjAgo2 (105.9 kDa). Western blot analysis was
performed to determine the reactivity and specificity of the mAb
27A9 directly against SWAP, and two bands (arrowed) with the
size of ,100 kDa were detected (Figure 3B).
By using Orbitrap MS analysis, 38 peptides derived from
SjAgo2 were identified from bands between ,90130 kDa in
27A9 immunoprecipitates, whereas no peptides derived from
SjAgo1 and SjAgo3 were detected in the immunoprecipitates
(Table 1), which further confirmed the specificity of the mAb
27A9 to SjAgo2. The RISC forming proteins like TRBP and
DDX6 were not identified in the immunoprecipitates. This could
be due to the experimental condition which may not be suitable
for the coprecipitation of these proteins; or due to the missing
sequence information of the two proteins in the S. japonicum
database which prevented the identification of these two proteins
in the MS analysis. The appearance of the 13 peptides derived
from PMY in the Orbitrap MS analysis supported our
speculation that this was due to its IgG-binding property of the
molecule (Table 1). In addition to PMY, several other
cytoskeleton and motor proteins, including actin, myosin, dynein,
spectrin, and kinesin, were also detected in the
immunoprecipitates (Table S2), which were presumably co-purified through
interaction with PMY . Strikingly, several members of the
heat shock protein (HSP) family (90, 97, and 110 kDa
respectively), and three isoforms of the HSP70 protein were identified
(Table S2). However, these proteins also appeared in the mock
group in the second MS analysis (Table S3), indicating that they
were non-specifically captured by the protein-G/A agarose
beads. This finding was consistent with the previous observation
that the HSP70 homologue in S. mansoni (SCHMA-HSP70) can
readily bind to protein-G Sepharose . Recent studies in
human and flies revealed that HSP90 protein can chaperone
Argonautes and facilitate the loading of small RNA duplexes [49
51]. More recently, HSP90 was reported to participate in the
Piwi-interacting RNA (piRNA) pathway and function in
canalization . Our results here suggest that HSP members in S.
japonicum do not directly interact with SjAgos; thus, whether they
can participate in the assembly of RISC complex remains
unclear. Nevertheless, as no SjAgo1 and SjAgo3 were detected in
27A9 immunoprecipitates, these co-precipitated contaminating
proteins have no influence on analyses of the small RNA
population associated with SjAgo2.
Overview of S. japonicum small RNA libraries
32,876,012 and 21,822,050 high quality reads were obtained
respectively from the two small RNA libraries, SP1 and SP2 (both
were established from the SjAgo2 complex with mAb 27A9)
(Table S4). The redundancy level of both libraries was ,85%
(Redundancy = 1002(Total Unique Clean Reads/Total
Highquality Clean Reads 6100)) (Table S5), which presented a similar
sequencing depth as our previous study .
We investigated the length distribution of small RNA reads in
the SP1 and SP2 libraries that perfectly matched the draft genomic
sequence of S. japonicum (Figure 4). The length distribution of the
reads in both libraries presented a quite similar pattern, both at
total and unique level. The 20 nt reads were predominant in both
libraries, which accounted for 46.1% (SP1) and 55.7% (SP2) of the
reads, respectively, followed by the 21 nt reads. Thus, the reads
length of sncRNAs associated with SjAgo2 was closer to that of
endogenous siRNAs bound to Drosophila Ago2, which peaks at
21 nt [25,53], rather than miRNAs, whose sizes are typically
<22 nt [54,55].
Classification of sncRNAs associated with native SjAgo2
We systematically defined the sncRNAs in both libraries SP1
and SP2 (Figure 5A and B), using the bioinformatic pipeline as
Figure 5. Classification and percentage of small non-coding RNAs in different libraries. A. Classification of small RNAs in the SP1 and B.
SP2 libraries using the bioinformatic pipeline described in . C. Small RNA classification of the SjM and D. SjF libraries using the data from our
previous study . E. Small RNA classification of the SP1 and F. SP2 libraries using an alternative bioinformatic pipeline as described in the Materials
reported previously . We also compared the data to that
obtained from the adult worm libraries SjM and SjF, which were
constructed with total small RNA (Figure 5C and D) . The
proportions of LTR- and LINE-derived siRNAs were significantly
higher than that of miRNA, rRNA, TIR- and MITE-derived
siRNAs in the two libraries compared to that constructed with
total small RNAs. For the LINE-derived siRNAs, the proportion
increased from <3% in the adult small RNA libraries to an
average of 17% in the SjAgo2-specific libraries. For the
LTRderived siRNAs, the proportion in the SjAgo2-specific libraries
was at least 5-fold higher than that in the libraries SjF and SjM
(from <4% to an average of 22%). This difference strongly
suggests that SjAgo2 preferentially associated with siRNAs derived
from LINE and LTR retrotransposons.
Regarding the mRNA related small RNAs, the proportion of
this group in SjAgo2-specific libraries was twice as high as that in
the small RNA libraries of separated adult worms (Figure 5A, B,
C, and D). This is due to the reason that numerous TE-derived
transcripts were deposited in the predicted S. japonicum database as
mRNA sequences (sjr_mRNA.fasta). Thus, a mass of TE-derived
siRNAs may have been categorized as mRNA-related small
RNAs. Therefore, an optimized bioinformatic pipeline was
designed to sort the small RNAs from SjAgo2-specific libraries.
As a result, the proportion of mRNA-related small RNAs
substantially decreased in contrast to that of
retrotransposonderived siRNAs, in particular LTR-derived siRNAs, which
increased nearly one-third (Figure 5E and F). This observation
further implies that SjAgo2 predominantly interacts with
SiRNAs interacted with SjAgo2 were restricted to several
classes of retrotransposons
TE components have been recognized as one of the principal
forces driving genome diversity and evolution . However, too
many insertions of TEs into the genome may be deleterious,
imposing that they must be under appropriate control to keep the
integrity of the genome . In S. japonicum, the repetitive elements
account for more than 40% of the genome sequences . And the
Table 2. Transcriptional levels (TPM) of 29 types of well-defined retrotransposons and the corresponding siRNAs in different
libraries from adult worms of S. japonicum.
GenBank Accession number
Total distinct tag
1Whole-transcriptome RNA-seq library from male adult worm of S. japonicum.
2Whole-transcriptome RNA-seq library from female adult worm of S. japonicum.
mobile genetic elements (MGEs) in S. japonicum have been
categorized into several classes, including short interspersed
nucleotide elements (SINEs)-like retrotransposons , LTR
[4,41], non-LTR [4,42], and Penelope-like retrotransposons .
We therefore further investigated whether the small RNAs
interacted with SjAgo2 were restricted to any particular class of
retrotransposons. The expression levels of siRNAs derived from 29
well-defined retrotransposons in the SP1, SP2, SjM, and SjF
libraries were presented based on their TPM value (Table 2). We
found that siRNAs in the SjAgo2-specific libraries were mainly
derived from 11 classes of retrotransposons (Table 2, Top 11). For
example, siRNAs generated from retrotransposon SjCHGCS11,
SjCHGCS13, SjCHGCS14, and Sj-penelope1 were 46 fold more in
the SjAgo2-specific libraries than that in the libraries SjM and SjF
(Figure 6A and B). Sense siRNAs generated from LINE
SjCHGCS21 were also enriched in the SP1 and SP2 libraries
(Figure 6C). In contrast, the abundance of siRNAs derived
from SjCHGCS10, Sjpido, SjCHGCS1, SjCHGCS2, SjCHGCS19,
SjCHGCS20, SjR2, and SjCHGCS3 was decreased in the
SjAgo2specific libraries compared to that in the SjM and SjF libraries
(Table 2, and Figure 6C), suggesting that the function of siRNAs
from these classes of retrotransposons were correlated to SjAgo2.
The potential function of S. japonicum Argonaute
We further evaluated the correlation between the transcription
levels of the well-defined retrotransposons of S. japonicum and the
enrichment of the siRNAs in the SjAgo2 complex by analysis of
the whole-transcriptome data generated from separated adult
worms (Piao et al., unpublished data). Interestingly, for several
classes of retrotransposons, an obvious inverse relationship was
observed between the abundance of mRNA transcripts and
amount of relevant siRNAs in the SjAgo2-specific libraries. For
instance, siRNAs derived from retrotransposon SjCHGCS6,
Sjpenelope1, Sj-penelope2, SjCHGCS21, SjCHGCS9, and SjCHGCS4
were highly enriched in the SjAgo2 libraries, whereas the levels of
the corresponding transcripts of these mobile elements were much
lower (Table 2). On the contrary, siRNAs derived from
retrotransposon SjCHGCS20, SjR2, and SjCHGCS3 were much less in
the SjAgo2 libraries, the transcripts of these retroelements were
relatively more (Table 2). These findings suggest that siRNAs
enriched in the SjAgo2 libraries were not affected by the
transcription levels of the retrotransposons, and SjAgo2 may be
functionally specialized to suppress a group of transposable
elements in the parasite. However, this regulatory model cannot
be applied to all types of retrotransposons. It can be explained by
the facts that the transcriptome data reflect the transcriptional
levels of retroelements within the whole worms, while the
expression of SjAgo2 in the parasite may be tissue-specific as its
ortholog in S. mansoni .
Based on the property of its associated small RNA population,
we postulated that SjAgo2 is mainly involved in such a mechanism
by regulating retrotransposon at the transcriptional level. A
similar function of Argonaute protein has previously been
suggested in studies of Trypanosoma brucei, D. melanogaster, and mice
[25,53,6063]. In addition, the Ago2 transcripts in S. mansoni
exhibited a germline-specific expression in both adult female and
male worms . This observation indicates that, in schistosome
adult worms, Ago2 functions in the maintenance of genome
stability in germline cells by retrotransposons silencing. Previous
studies in Drosophila and vertebrates have shown that the
endosiRNA pathway is involved in transposons silencing in somatic
tissues [25,53,57,60,63]; whereas transposons are mainly
controlled by the piRNA pathway in germline cells, which functions
through Piwi subclade proteins [64,65]. However, the piRNA
pathway does not appear to be specialized in schistosome as no
Piwi homolog has been discovered in its genome [15,31]. The
siRNA pathway mediated by SjAgo2 in schistosome germline
could, to some extent, compensate for the absence of the piRNA
pathway as suggested previously . Given the fact that SjAgo2 is
ubiquitously expressed during various developmental stages of the
parasite, though at different levels, SjAgo2 may be bi-functional in
both somatic and germline cells. However, further studies are
needed to dissect it out.
Though the PAZ and Piwi domains were highly homologous
between SjAgo2 and SjAgo3, substantial differences exist in the
region corresponding to the typical Mid domain, which has been
definitively established to play role in 59 end recognition of the
guide strand [27,66]. The reverse expression pattern of SjAgo2 and
SjAgo3 genes in male and female adult worms was also observed
(Figure 1). Both of these observations indicate that SjAgo3 may
play an analogous, but non-redundant role to SjAgo2 in S.
japonicum, such as suppressing the activities of TEs in somatic cells.
One line of evidence supporting this is that a substantial portion of
small RNAs derived from DNA transposons TIR and MITE was
detected in adult worms, with an amount even more than that of
LTR- and LINE-derived siRNAs in both male and female worms
(Figure 5C and D). Another possibility is that SjAgo3 may also
restrict the activities of retrotransposons, such as SjR1, SjR2, and
Sjpido, via binding with siRNAs that not enriched in the
SjAgo2specific libraries (Figure 6C).
Only a small proportion of miRNAs was found to be associated
with SjAgo2 (Figure 5A and B), which is in line with the suggestion
that the miRNA pathway in schistosomes is mainly mediated by
Drosha, Dicer, and Ago1, as Ago1 is more closely related to
Argonaute orthologs involved in the miRNA pathway in flies,
humans, and worms . Our findings here as well as those found
in Drosophila suggest that some miRNAs were still bound to their
unconventional partner, Ago2, in addition to being strongly
associated with Ago1 [67,68]. Thus, the phenomenon that some
miRNAs sorted onto the SjAgo2 complex exhibits the complexity
of a small RNA regulatory network in schistosome parasite and
suggests that different silencing pathways may cross-link with each
other and share or compete the apparatus required in the
biogenesis of different small RNAs. In Drosophila, miRNA are
generated in a Dicer1-dependent manner, whereas siRNAs are
produced in a Dicer2-dependently manner . However, the
dsRNA-binding protein Loquacious (Loqs), a typical miRNA
factor associated with Dicer1, may actually be required for the
biogenesis of endo-siRNAs [25,69]. Since only one Dicer gene was
found in S. japonicum , the miRNA pathway and endo-siRNA
system in schistosomes may share one Dicer in the production of
miRNA and siRNA duplex, cross-linking both pathways at
In summary, using a mAb specific to SjAgo2, we have
systematically investigated the small RNAs bound to the protein.
SjAgo2 was determined to associate mainly with endo-siRNAs
derived from LINE and LTR types of transposable elements in
adult S. japonicum. The enrichment of siRNAs in the
SjAgo2specific libraries was found to be restricted to particular types of
retrotransposons. These results emphasize the potential role of
SjAgo2 in maintaining genomic stability in germ-line and/or
somatic cells by repressing retrotransposons.
Figure S1 SWAP immunoprecipitates were resolved
on 10% SDS-PAGE. Sequential IP assays were carried
out as described in the Materials and Methods. Protein
bands with different molecular weights located in the squares
were excised from SDS-PAGE gel and anylzed by MS.
Figure S2 Agilent 2100 Bioanalyzer analysis of small
RNA samples co-precipitated with SjAgo2 by two 27A9 IP
assays. The predominant species of the small RNAs was around
Figure S3 The expression of Flag-tagged SjAgos in 293T
cells was detected by Western blot with mAb M2 (anti-Flag)
after adjusting the loading volumes of protein samples.
Figure S4 SWAP was sequentially incubated with pure
Protein-A/G agarose beads only (Mock), and normal
mouse IgG, mAb 11E8, and 27A9. The precipitated protein
complexes were resolved on 10% SDS-PAGE, stained with
Coomasie brilliant blue.
We appreciate very much the bioinformatic support of Dr. Haibo Sun at
MininGene Biotechnology and the efforts of technicians at Shenzhen BGI
for sample sequencing. We also thank the Schistosoma japonicum Genome
Sequencing and Functional Analysis Consortium for making the S.
japonicum genome available on the public domain.
Conceived and designed the experiments: PC QC. Performed the
experiments: PC XP NH SL. Analyzed the data: PC QC. Contributed
reagents/materials/analysis tools: HW. Wrote the paper: PC QC.
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