Transcriptome of Small Regulatory RNAs in the Development of the Zoonotic Parasite Trichinella spiralis
et al. (2011) Transcriptome of Small Regulatory RNAs in the Development of the Zoonotic Parasite Trichinella
spiralis. PLoS ONE 6(11): e26448. doi:10.1371/journal.pone.0026448
Transcriptome of Small Regulatory RNAs in the Development of the Zoonotic Parasite Trichinella spiralis
Xiaolei Liu 0
Yanxia Song 0
Huijun Lu 0
Bin Tang 0
Xianyu Piao 0
Nan Hou 0
Shuai Peng 0
Ning Jiang 0
Jigang Yin 0
Mingyuan Liu 0
Qijun Chen 0
Michael Freitag, Oregon State University, United States of America
0 1 Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, Jilin University , Changchun , China , 2 Laboratory of Parasitology, Institute of Pathogen Biology, Chinese Academy of Medical Sciences/Peking Union Medical College , Beijing , China , 3 Biological Therapy Center, The First Affiliated Hospital, Jilin University , Changchun , China
Background: Trichinella spiralis is a parasite with unique features. It is a multicellular organism but with an intracellular parasitization and development stage. T. spiralis is the helminthic pathogen that causes zoonotic trichinellosis and afflicts more than 10 million people worldwide, whereas the parasite's biology, especially the developmental regulation is largely unknown. In other organisms, small non-coding RNAs, such as microRNAs (miRNA) and small interfering RNAs (siRNA) execute post-transcriptional regulation by translational repression or mRNA degradation, and a large number of miRNAs have been identified in diverse species. In T. spiralis, the profile of small non-coding RNAs and their function remains poorly understood. Methodology and Principal Findings: Here, the transcriptional profiles of miRNA and siRNA in three developmental stages of T. spiralis in the rat host were investigated, and compared by high-throughput cDNA sequencing technique (''RNA-seq''). 5,443,641 unique sequence tags were obtained. Of these, 21 represented conserved miRNAs related to 13 previously identified metazoan miRNA families and 213 were novel miRNAs so far unique to T. spiralis. Some of these miRNAs exhibited stage-specific expression. Expression of miRNAs was confirmed in three stages of the life cycle by qRT-PCR and northern blot analysis. In addition, endogenous siRNAs (endo-siRNAs) were found mainly derived from natural antisense transcripts (NAT) and transposable elements (TE) in the parasite. Conclusions and Significance: We provide evidence for the presence of miRNAs and endo-siRNAs in T. spiralis. The miRNAs accounted for the major proportion of the small regulatory RNA population of T. spiralis, while fewer endogenous siRNAs were found. The finding of stage-specific expression patterns of the miRNAs in different developmental stages of T. spiralis suggests that miRNAs may play important roles in parasite development. Our data provide a basis for further understanding of the molecular regulation and functional evolution of miRNAs in parasitic nematodes.
Funding: This study was supported by the National Natural Science Foundation of China (NSFC 30625029, 30825033, 31030064, 30950110328, 81070311), the
intramural grant from Institute of Pathogen Biology, CAMS (2008IPB204), and the national science and technology specific projects (2008ZX-10004-011,
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.
. These authors contributed equally to this work.
Parasites of the genus Trichinella are a group of pathogens with
diverse biological and pathological features. Phylogenetic analysis of
the mitochondrial DNA of the species identified so far indicated that
there are two genetic clades that form unique monophyletic lineages
. One clade is represented by T. pseudospiralis, which does not
capsulate in the muscle cells, while the other clade is represented by T.
spiralis while does encapsulate in muscle cells, named nurse cells, with
a parasite inside the cell, surrounded by a sick layer of collagen.
Parasites of Trichinella genus are unique intracellular pathogens.
Interestingly, their entire life cycle can be completed within an animal,
and trichinellosis in human and other mammals was caused through
the ingestion of parasite-contaminated meat. This is a typical zoonotic
disease that affects more than 10 million people worldwide .
After being ingested with the infected muscle tissue, L1 larvae
are released and activated in the small intestine, enter the
epithelial layer and undergo four times of moultings before
maturation into adult worms. Mating is initiated on day 2 after
infection and newborn larvae are released by the females into the
mucosa as early as 4 days post infection (dpi) . The larvae
migrate through the lymphatic and blood vessels, invade striated
muscle cells and develop into the infective Ll stage over a period of
23 weeks to the next host which complete the life cycle . Thus,
unlike other nematodes, T. spiralis does not have an embryonic
developmental stage in the egg, which differs markedly in
biological and molecular characteristics from other nematodes,
especially the well-characterized free-living worm Caenorhabditis
elegans. Intriguingly, compared to other nematodes, T. spiralis has a
much smaller genome with 64 Mb in nuclear DNA, which
contains <15,808 genes . The availability of genome sequence
information has made it possible to dissect parasite biology.
Small non-coding RNAs (sncRNAs) are a large group of small
endogenous RNAs that have been widely identified in animals ,
plants , fungi [8,9] and some viruses . They are generally 21
23 nucleotides in length, which guide various processes involving
sequence-specific silencing through chromatin modification,
mRNA degradation, and translational repression . Based
on their origins, structures, associated proteins and biological roles,
sncRNAs are divided into three general categories: microRNAs
(miRNAs), endogenous small interfering RNAs (endo-siRNAs), and
piwi-interacting RNAs (piRNAs) . MiRNA and endo-siRNAs
have been discovered in diverse animals and plants and fungi 
, while piRNAs are found only in animals . miRNAs are
generated from precursor transcripts by two RNase III-type
enzymes, Drosha and Dicer. In animals, single-stranded miRNA
is incorporated into the argonaute (Ago) protein complexes (Ago)
known as RNA induced silencing complexes (RISC) and binds by
partially or completely complementary to the 39 untranslated region
(39UTR) of a target mRNA , this results negative control of gene
expression by cleavage or inhibition of translation or other
regulatory functions . The biological function of miRNAs was
first demonstrated in C. elegans, where two miRNAs (Let-7 and Lin-4)
were shown to be regulators for stage-specific differentiation of the
worm [17,18]. Endogenous siRNAs are mainly derived from three
sources: transposable elements (TEs), complementary annealed
transcripts, also called natural antisense transcripts (NAT) and long
fold-back transcripts called hairpin RNAs (hpRNAs) . Unlike
miRNAs, endo-siRNAs function requires perfect match with the
target mRNA [6,20]. MiRNAs and endogenous siRNAs play
important roles in the regulation of fundamental cellular processes,
including cell differentiation, stress response, apoptosis, proliferation
 and metabolism [21,22]. Recent studies further differentiated
the functions of endo-siRNAs based on the sources they were
generated from . The TE-derived siRNAs are likely functional
in the germline cells by repression of transposon activity and
consequently keep genome stability, while NAT-derived siRNAs are
more functional in somatic cells .
In this study, the transcriptional profiles of both miRNA and
endo-siRNA in three developmental stages, namely adult (Ad),
new born larvae (NBL) and muscle larvae (ML) of T. spiralis were
systematically investigated and compared by high throughput
cDNA sequencing technology (RNA-seq). We found that
miRNAs were mainly expressed in the adult worm stage and
endo-siRNAs were predominantly derived from transposable
elements in the genome.
Results and Discussion
Summary of small RNA sequencing
To identify miRNA and endo-siRNA involved in the
development of T. spiralis, small RNA libraries were generated from three
life cycle stages, the new-born larvae, musclar larvae and adult
worms. The libraries were directly sequenced using Solexa
sequencing technology. A total of about 40 million unfiltered
sequence reads (12,368,833 reads from adult worms, 12,867,246
reads from newborn larvae and 14,495,649 reads from muscular
larvae) with sizes between 18 and 30 nucleotides were obtained,
respectively. After removal of low quality reads and adaptor
sequences, the clean reads obtained in Ad, NBL and ML were
11,878,917 (96.0% of total reads), 12,357,960 (96.0% of total
reads) and 14,078,375 (97.1% of total reads) respectively.
Sequence characterization suggested that the small RNA pools
contained miRNAs (20.63%), other non-coding RNAs (rRNA,
tRNA, snoRNA) (0.55%), mRNA-related small RNAs (32.13%),
TE-related small RNAs (0.02%) and unknown small RNA
transcripts (46.66%) (Fig. 1A, Table S1). The GC content of the
small RNAs was around 38.7% and the distribution of the small
RNA populations in the libraries generally followed the patterns
identified in other organisms, such as schistosomal parasites [23
29]. The libraries generated in this study have significantly high
coverage than those in a recent report, which only showed a
proportion of miRNAs in one single stage of the parasite .
As shown in Fig. 1B, the unique clean reads obtained in Ad,
NBL and ML were 1,816,014, 1,691,015 and 1,936,612,
respectively (Table S2). The number of small RNAs existed in
all three life cycle stages was 393,026 reads, which accounted for
21.6%, 20.3% and 23.2% of the unique clean reads of Ad, NBL
and ML stage, respectively (Fig. 1B). The small RNAs commonly
expressed in Ad stage and ML stage were 3 times more than that
between Ad stage and NBL stage (Fig. 1B), indicating that many
genes of Ad stage were activated already in ML and NBL had
more stage-specific small RNAs.
Length variation of small RNAs in T. spiralis
The length of predicted small RNAs varied from 18 to 30 nt in
the three life stages, with most of them between from 18 to 27 nt.
Here the size and number of sequences in Ad and ML almost
followed the same pattern, whereas the number of sequences
between 18 and 22 nt was enriched in NBL (Fig. 2 and Table S3).
This implied that, apart from stage-specific activation in NBL,
small RNAs may undergo size differentiation during early
development of the parasite. A previous study indicated that the
length of the non-conserved connector helix in Dicer was the main
determinant of product size of the mature small RNAs ,
providing a possible explanation for the stage-specific variation we
observed. The mechanism behind the stage-specific small RNA
processing remains unknown. Since both piRNA and endo-siRNA
were more common in the germline, it is likely that the small
RNAs found in NBL were predominantly miRNAs, which may
play fundamental roles in stage-specific differentiation. Longer
RNAs may be more important for parasite reproduction .
Identification of miRNAs in T. spriralis
To identify candidate miRNAs of T. spiralis, the clean unique reads
obtained were mapped to the draft T. spiralis genome sequences
spiralis/assembly/Trichinella_spiralis-1.0/)  sing SOAP. A total of
72,110 (out of 2,099,966) unique small RNA reads that perfectly
matched are referred to as miRNA candidates. After screening for
secondary structure of the inverted repeats (found with Einverted of
Emboss) with RNAfold and evaluation by MirCheck, a total of 240 T.
spiralis predicted miRNAs were identified.
To further characterize miRNAs in T. spiralis, all predicted
miRNAs obtained above were compared against a miRNA
database, miRBase (Release 15.0). In total, 21 miRNAs were
found that had been identified in other species, and belong to 13
different miRNA families (Table 1, Table S4 and S5). Most
miRNAs contain a 7 nt region (typically positioned at the 59 side
28 nt) known as the miRNA seed sequence (Fig. 3). It has been
suggested that the seed regions serv to anchor miRNAs to their
mRNA targets .
The sequencing data showed that, of the 21 conserved miRNAs,
tsp-let-7 and tsp-miR-87 were found to locate only in the 39 arm of
their pre-miRNAs, and tsp-miR-31 was located only in the 59 arm of
the hairpin structures. The remaining 18 miRNAs were derived
from both arms of the pre-miRNAs (Table S4 and S5). According to
the current model of miRNA maturation, Dicer recognizes the
Figure 1. Small non-coding RNA identified in three developmental stages of T. spiralis. A The composition in percentages of clean reads of
small RNAs. B Numbers and proportions (bracketed) of unique small RNAs identified in the three stages.
double-strand of pre-miRNAs, cleaves away the loop structure and
de-associates the duplex to generate antisense and sense strands, i.e.
the mature miRNA and miRNA* . Although the antisense and
sense strands may have different thermostability, which also
determined there relative abundance in different tissues, both
strands could be functional in miRNA-mediated regulatory
pathways . For instance, tsp-miR-1-3p was detected at 1,515
TPM (transcripts per million) in the Ad stage, 9,759 in the NBL
stage and 3,351 in the ML stage, respectively, whereas its
counterpart tsp-miR-1-5p was much less abundant (Fig. 4, Table
S5). The TPM of tsp-miR-100-5p and tsp-miR-100-3p,
tsp-miR-1255p and tsp-miR-125-3p, tsp-miR-9-1-5p and tsp-miR-9-1-3p,
tspmiR-9-2-3p and tsp-miR-9-2-5p behaved in similar fashion (Fig. 4,
Fig. 5A and Table S5). Thus the anti-sense strands complementary
to mRNA targets may play a main regulatory role. However, it
cannot be ruled out that both strands generated from the same
premiRNA execute similar functions. This is because, in some
miRNAs, both strands were either similarly expressed or showed
stage-specific expression patterns (Fig. 5 A and B; Table S5 and S6).
We found 213 novel miRNAs (Table 2, Table S7). Comparison
of novel miRNAs expressed in the three developmental stages of
T. spiralis revealed stage-specific expression patterns (Fig. 5). A
majority of the novel miRNAs in NBL stage has a lower expression
level than those in Ad stage and ML stage (Fig. 5B, Table S6 and
S7). For instance, tsp-novel-21, tsp-novel-50a-3p, tsp-novel-50b-3p
were relatively enriched in adult worm, tsp-novel-46 were
abundant in muscular larvae, whereas tsp-novel-108 and
tspnovel-83 exhibited a high abundance in all the three stages (Table
S7). Of the novel miRNAs, the expression patterns of
tsp-novel-935p and tsp-novel-93-3p, tsp-novel-50a-3p and tsp-novel-50b-3p,
tspnovel-101-3p and tsp-novel-101a-5p and tsp-novel-101b-5p were
interesting. Tsp-novel-93-5p and tsp-novel-93-3p were derived
from the same pre-miRNA, but tsp-novel-93-5p was mainly
expressed in the NBL stage, less in ML and least in Ad. While
tsp-novel-93-3p was much less expressed than tsp-novel-93-5p in all
three stages. (Fig. 5B, Table S7). Tsp-novel-50a-3p and
tsp-novel50b-3p were derived from the 39 arm of two gene copies with a
similar sequence. Tsp-novel-50a-3p was dominantly expressed in
Figure 2. Length distribution of small RNAs in different developmental stages. Length of small RNAs is given on the x-axis in base pairs.
And he left Y-axes indicate the percentage of small RNAs afer removal of low quality sequences but prior to selection for complete matches to the
Ad, while tsp-novel-50b-3p was mainly expressed in NBL and Ad
but less in ML. The genes coding for tsp-novel-101-3p and
tspnovel-101a-5p and tsp-novel-101b-5p and their expression patterns
were even more complicated. They were encoded by three genes
in the genome of T. spiralis. The sequences of tsp-novel-101-3p
derived from the 3 genes were the same, while tsp-novel-101b-5p
was derived from 1 gene which had a single nucleotide change
from tsp-novel-101a-5p (Fig. 5B and Table S6 and S7). The later
was derived from two gene copies. All these data suggested that
there may be multiple layers of control for the stage-specific
expression of miRNA genes, as well as their potential regulatory
function in the development of the parasite . Although the
mRNA targets of these regulatory miRNAs have not been
identified, with the availability of the genome sequence of the
parasite, genome-wide association study can be pursued. For
instance, miR-1 has been found in many species, from Drosophila to
human , suggesting that they are evolutionary conserved,
which has been characterized to play essential functions in
regulating proliferation and differentiation of muscle cell [41,44].
One homologous to mir-1, tsp-miR-1, was observed to express in
Most abundant sequence
aY indicates that sequences from both arms of a pre-miRNA species were found, while N means that only a sequence from one arm was identified.
bThe abundance value of each miRNA was normalized to transcripts per million (TPM). If the value after normalization was less than 1, the normalized value was set as
three developmental stages of T. spiralis and may have similar
functions. Further, MiR-100 and let-7, the two conserved miRNA
in metazoa, play a role in regulation of developmental timing
[18,42,45]. Their homologs, tsp-miRNA-100 and tsp-let-7, were
found throughout the life cycle of T. spiralis. Tsp-let-7 showed very
low expression in NBL stage, whereas tsp-miRNA-100 was
detected in rather high abundance at the same development
stage. The abundance of tsp-miRNA-100 was almost identical with
that of tsp-let-7 in both Ad and ML stage, indicating that
miRNA100 may be more functional in NBL stage.
Characterization of stage-associated miRNAs in T. spiralis
Quantitative real-time PCR and northern blots were performed
to validate miRNAs identified in T. spiralis and their relative
expression levels at different developmental stages. Five conserved
miRNAs (tsp-miR-228, tsp-miR-100, tsp-let-7, tsp-miR-1 and
tspmiR-31) and five novel miRNAs (tsp-novel-108, tsp-novel-83,
tspnovel-46, tsp-novel-86 and tsp-novel-21) with relatively higher TPM
values identified by sequencing were validated by qRT-PCR and
Northern blot. First, the above miRNAs identified in sequencing
were all amplified by qRT-PCR (Fig. 6A) and except the tsp-let-7
which was found less expressed in NBL, the qRT-PCR results were
all consistent with the TPM values of sequencing results. The reason
for tsp-let-7 being found less common in NBL is not known. This
may be due to preferential amplification in the tag-generation step
before RNA-seq or other unidentified factors. Further, all qPCR
amplicons were cloned into the pMD-18T vector (Takara, Dalian,
China) and sequenced. The sequencing results demonstrated that
the amplicons were identical to the miRNAs sequences (data not
shown). Four miRNAs were selected for characterization by
northern blots. The results confirmed the qPCR data. However,
the probe for tsp-mir-100 did not show any signal with total RNA
purified from the Ad stage; this was likely due to probe modification
or other unidentified factors that prevented annealing of the probes
with the targets (Fig. 6B). Since this miRNA was found by both
Figure 5. Clustering of expression level of conserved (A) and novel (B) miRNAs. Log 10 (expression) is used instead of raw expression. The
abundance of each miRNA was normalized to transcripts per million (TPM). If the value after normalization was less than 1, the normalized value
was set as 1 to avoid the negative values.
sequencing and qPCR in the Ad stage, we have no doubt that
tspmir-100 was indeed expressed at this stage.
Identification of Endogenous siRNA
Endogenous siRNA are extremely diverse and normally not
conserved across species. Several types of endogenous siRNAs
have been found in Drosopila melanogaster, C. elegans, Schistosoma
japonicum, fungi, Arabidopsis thaliana and mice [8,9,27,33,36,4650].
Most of these endogenous siRNAs derive from transposable
elements (TE), complementary annealed transcripts (also called
natural antisense transcripts, NAT) and long fold-back transcripts
called hairpin RNAs (hpRNAs) . TE and NAT are the main
sources of endogenous siRNAs. Thus, the small RNA transcript
reads perfectly matched to TE and NAT were regarded as
endogenous siRNAs (endo-siRNAs) [47,48].
TEs are major components of the intergenic regions in the
genomes of eukaryotic organisms [47,48]. Based on their structures
and modes of integration, TEs are comprised of two main classes
. One class includes retrotransposons and retrotransposon-like
elements such as Long Interspersed Nuclear Elements (LINE), Long
Terminal Repeat Elements (LTR), and Short Interspersed Elements
(SINE). The other group includes DNA transposons. We predicted
TE structures in the T. spiralis genome with RepeatMasker (http://
www.repeatmasker.org/), and endo-siRNAs matched to TEs were
further analyzed. As reported recently , the repetitive sequences
in the T. spiralis genome estimated to be around 18%, which is much
lower than in schistosomes and Drosophila melanogaster [52,53]. Thus,
TE-derived siRNAs accounted for a minor portion of the sncRNAs
identified here (Fig. 1A).
The numbers for TE-derived siRNAs identified in the Ad, NBL
and ML stages were 2,055, 2,234 and 2908 respectively. No
obvious stage-related variations in endo-siRNA expression were
found, suggesting that TE components in the genome might not be
very active during embryogenesis or during other development
stages. Further, most of these siRNAs were derived from
transposons of Long Interspersed Nucleotide Elements (LINE)
family and the transposon Charlie 24 accounted for a major
portion (Table S8). Sequencing analysis showed that more siRNA
are derived from the antisense strand than from the sense strands
(data not shown). This suggested that antisense-derived siRNA
may execute regulatory functions through hybridization with the
mRNA template generated from the sense strand.
Since NAT-derived siRNAs were generated from
doublestranded RNAs formed by complementary annealed transcripts
(NAT) and long fold-back transcripts (called hairpin RNAs,
hpRNAs) , T. spiralis NAT-siRNAs were identified from
predicted overlapping genes. The number of NAT-derived
siRNAs identified in Ad, NBL and ML was 21,157, 16,243 and
21,251, respectively (Table S9). Thus the number of NAT-derived
endo-siRNAs was much higher than that from TE, which suggests
that these siRNAs played more regulatory roles in the
development and parasitization of T. spiralis. Further, all NAT-derived
siRNAs were trans-NAT siRNAs and no cis-NAT siRNAs were
found (Fig. 7 and data not shown), which differs from observations
with D. melanogaster and mice [33,4749]. This also suggests that
there are fewer coding sequences with internal inverted repeat
sequences in the genome of T. spiralis.
In summary, we identified and analyzed the expression of
miRNAs and endo-siRNAs in three development stages of T. spiralis
through high through-put RNA sequencing techniques. We found
vastly more transcripts of miRNAs than that of endo-siRNAs. A total
of 21 conserved miRNAs in 13 metazoan miRNA familis and 213
Most abundant sequence
aY indicates that the sequences from both strands of a pre-miRNA species were found, while N means that only the sequence from one arm was identified.
bThe abundance value of each miRNA was normalized to transcripts per million (TPM). If the value after normalization was less than 1, the normalized value was set as 1.
novel miRNAs were identified in the parasite. Some showed clear
stage-specific expression patterns, suggesting a potential regulatory
function in the corresponding developmental stages. Endo-siRNAs
were mainly derived from natural antisense transcripts with
TEderived siRNAs accounting for only minor proportion of the small
RNA population. Thus, the function of endo-siRNAs in T. spiralis is
likely to regulate gene expression instead of maintaining genome
stability. The data of this study provide insight information for
further dissection of the parasites biology.
Materials and Methods
Muscle larvae (ML) of T. spiralis (strain ISS534) were obtained
from rats 35 days post infection (dpi) by digestion of minced
skeletal muscle in 1% pepsin, 1% HCl for 3 h at 37uC with
agitation as previously described . To purify adult worms and
newborn larvae, Wistar rats at 6 weeks of age were orally
inoculated with T. spiralis (strain ISS534) with a dose of 8000
larvae per rat. At 30 h or 6 dpi, all rats were killed and the entire
intestines were removed, opened longitudinally and cut into small
pieces (about 0.51 cm). The fragmented intestine was put on a
layer of gauze which was immersed into 0.9% sodium chloride
solution at 37uC and incubated for 3 h. Adult T. spiralis worms
migrate into the liquid phase, which was harvested by
centrifugation. To obtain new-born larvae, adult worms collected at 6 dpi
were incubated in Iscoves Modified Dulbeccos Medium (IMDM)
in 75-cm2 cell culture plates at 37uC, and the newborn larvae were
harvested every 12 h. Our study was reviewed and approved by
the Ethics Committee of Jilin University (Ethical clearance
application number IZ-2009-III). All animal work was conducted
according to Chinese and international guidelines.
Total RNA of T. spiralis (Ad, NBL and ML) was extracted using
Trizol reagent (Invitrogen, CA, USA) according to the
manufacturers instructions. RNAs were dissolved in diethylpyrocarbonate
(DEPC)-treated water, aliquoted and stored at 280uC. RNA was
quantified by measuring the absorbance at 260 nm with a
Nanodrop 1000 machine (Thermo Scientific CA, USA).
Construction of small RNA libraries and sequencing
For small RNA library construction and deep sequencing, the
1530 nt size range base-pair fraction of each RNA sample from
the three life cycle stages was first enriched by 15% TBE urea
polyacrylamide gel electrophoresis and the Illuminas proprietary
adaptors (UCAGAGUUCUACAGUCCGACGAUC and
UCGUAUGCCGUCUUCUGCUUGUidT) were ligated to the 59
and 39 termini of the purified small RNAs which were converted
into single-stranded cDNA with Superscript II reverse
transcriptase (Invitrogen, CA, USA) and Illuminas small RNA RT-Primer.
The cDNAs were PCR-amplified with high fidelity Phusion
DNApolymerase (Finnzymes Oy, Finland) in 18 PCR cycles using
Illuminas small RNA primer sets. After purification using 6%
TBE urea PAGE gels, the PCR products were sequenced by
Solexas sequencing-by-synthesis method (Fig. S1).
Mapping the sequence reads onto the reference genome
The bioinformatic analysis and work flow is shown in Figure S1.
After removing the low quality sequence reads and the adapter
sequences according to the criteria of Illumina, all identical sequences
were retained with associated count numbers as their expression
abundances To determine whether these clean small RNA sequences
were candidate miRNAs, the unique reads were mapped onto the T.
spiralis genome of the Genome Sequencing Center (GSC) at
Washington University St. Louis, (http://genome.wustl.edu/pub/
spiralis-1.0/) with SOAP  (http://soap.genomics.org.cn).
The perfectly matched reads were searched against the Metazoa
mature miRNA of Sanger miRBase with Patscan . Sequence
Figure 7. NAT-derived siRNA that matched to the sense and antisense strands. The origin of the genomic sequence (genomic loci) is
named on top. siRNAs matched to the sense strand are in blue and those matched to antisense strand are in red. The read numbers and reading
direction of the siRNA are listed on the right side.
tags of more than 5 reads that matched perfectly or near-perfectly
(no more than 1 mismatch and the mismatch not positioned in the
seed region) were regarded to be conserved miRNA candidates.
For reads that did not match to the miRNA database, we used the
software Einverted of Emboss  to find the inverted repeats
(stem loops or hairpin structure). Each inverted repeat was
extended 10 nt on each side, and the secondary structure of the
inverted repeat was predicted by RNAfold . Unique reads
with a folding free energy of at least 25 kcal/mole( gGufolding
#225 kcal/mol) were evaluated by MirCheck  with modified
parameters. Finally, precursors (hairpins) of miRNA that passed
MirCheck were inspected manually in order to remove false
predictions. The reads passing the inspections were regarded as
Similar to credibility interval approaches reported for the
analysis of SAGE data , we employed IDEG6  to identify
miRNAs showing statistically significant differences in relative
abundance (as reflected by the total count of individual sequence
reads) between the three libraries (corresponding to the three
developmental stages of the parasite). We used the general
Chisquare method for comparison analysis, which has been
commonly applied by others [27,36]. Finally, miRNA with a P
value#0.01 were deemed to be significantly different between the
samples of the three developmental stages of the parasite.
The repeated sequences, i.e. potential transposable elements, in
the T. spiralis genome were predicted by using RepeatMasker
(http://www.repeatmasker.org/) and the sequences of typical
transposons were annotated (data not shown). The sequencing
reads that perfectly matched the T. spiralis genome were aligned to
repeats (TE) using SOAP. The reads that perfectly matched TEs
were considered TE-derived siRNAs.
Natural antisense transcripts (NATs) were detected by aligning
the T. spiralis predicted genes to each other. If a pair of overlapping
genes were matched on opposite strands with an E value of #1e-9
, this pair of overlapping genes was defined as a NAT pair.
The reads that perfectly matched the T. spiralis genome were
aligned to overlapped sequences of NAT pairs with SOAP. The
reads that perfectly matched the overlapped regions were
considered NAT-derived siRNAs.
miRNAs quantification by real-time PCR
Total RNA purified from the three stages were polyadenylated with
E. coli poly(A) polymerase (E-PAP) following the manufacturers
protocol of the poly(A)-tailing kit (Ambion, CA, USA). The RNA
samples were purified separately from the parasites of the same
developmental stages for sequencing purpose. After
phenol-chloroform extraction and ethanol precipitation, the polyadenylated
products were dissolved in DEPC-treated water and
reversetranscribed with 200 U of SuperScriptTM III Reverse Transcriptase
(Invitrogen, CA) and 39 RACE Adapter
(59-GCGAGCACAGAATTAATACGACTCACTATAGGT12VN-39) in the FirstChoice
RLM-RACE Kit (Ambion) according to the manufacturers protocol.
The 20 ml RT reaction contained 1 mg total RNA, 2 ml 39 RACE
Adapter, 0.5 mM dNTP mix (Takara, Dalian, China), 10 U RNase
inhibitor and 200 U SuperScriptTM III Reverse Transcriptase.
In quantitative RT-PCR reactions, GAPDH
(glyceraldehyde-3phosphate dehydrogenase) was chosen as an endogenous
reference. The forward and reverse primers for GAPDH were
59-CTAAGCCATTGGTAGTGC-39. PCR was done on an Applied Biosystems 7500
system. The following forward primers were designed to confirm
the sequencing results of miRNAs that showed differential
expression patterns: tsp-miR-100 59-AAC CCG TAG ATC
CGA ACT TGT GT-39; tsp-let-7 59-TGA GGT AGT AGG
TTG TAT AGT T-39; tsp-miR-228 59-AAT GGC ACT GGA
TGA ATT CAC GG-39; tsp-miR-1 59-TGG AAT GTA AAG
AAG TAT GTA G-39; tsp-miR-31 59-AGG CAA GAT GTT
GGC ATA GCT GA-39; tsp-novel-108 59-CTT GGC ACT GTA
AGA ATT CAC AGA-39; tsp-novel-83 59-TTG AGC AAT TTT
GAT CGT AGC-39; tsp-novel-46 59-TGG ACG GCG AAT TAG
TGG AAG-39; tsp-novel-86 59-TGA GAT CAC CGT GAA AGC
CTT T-39; tsp-novel-21 59-TCA CCG GGT AAT AAT TCA
CAG C-39. The sequence 59-GCG AGC ACA GAA TTA ATA
CGA CT-39 (complementary to the adaptor) was used as a
common reverse primer. Relative expression was calculated by the
comparative Ct method . ANOVA and Tukeys HSD
posthoc test were used to analyze significant differences among three
stage; P,0.05 was considered significant.
Northern blot analysis of miRNA expression
Total RNA of T. spiralis (muscle larvae, adult worms and
newborn larvae) were separated by electrophoresis on a 12.5%
polyacrylamide gel under denaturing (8 M urea) conditions and
transferred to Hybond-N+ nylon membranes (GE Healthsystems,
Uppsala, Sweden). The membranes were crossslinked in a UV
crosslinker and baked for 1 h at 80uC. Probes complementary to
small RNA sequences were end-labeled with DIG at 59 termini
(Takara, Dalian, China). Prehybridization and hybridization were
both performed overnight at 53uC in Northernmax Hybridization
buffer (Ambion, CA, USA). The blots were washed four times for
30 min in 26SSC, 0.05% SDS and twice for 15 min in 0.16SSC,
0.1% SDS at room temperature. The hybridization signal was
detected using a DIG Detection Kit (Roche) following
manufacturers instructions. The oligonucleotide probes used for
hybridization are as follows:
tsp-let-7 probes: 59AACTAT*ACA*ACCT*ACT*ACCTCA39
(LNA (Locked nucleic acid) substitutions are indicated by a *).
General information of the small RNA libraries.
Length distribution of small RNAs in T.spiralis.
Novel miRNAs identified in different developmental
siRNAs derived from DNA transposons.
siRNAs derived from NAT.
We very much appreciate the effort of scientists at BGI Genomics for their
kind assistance with the sequencing of small RNA libraries and Dr. Haipo
Sun for assistance with bioinformatics.
Conceived and designed the experiments: XL ML QC. Performed the
experiments: XL YS HL BT XP NH SP NJ. Analyzed the data: XL QC.
Contributed reagents/materials/analysis tools: JY. Wrote the paper: XL
1. Zarlenga DS , Rosenthal BM , La Rosa G , Pozio E , Hoberg EP ( 2006 ) PostMiocene expansion, colonization, and host switching drove speciation among extant nematodes of the archaic genus Trichinella . Proc Natl Acad Sci U S A 103 : 7354 - 7359 .
2. Dupouy-Camet J ( 2000 ) Trichinellosis: a worldwide zoonosis . Vet Parasitol 93 : 191 - 200 .
3. Liu MY , Zhu XP , Xu KC , Lu Q , Boireau P ( 2001 ) Biological and genetic characteristics of two Trichinella isolates in China; comparison with European species . Parasite 8 : S34 - 38 .
4. Wakelin D , Goyal PK ( 1996 ) Trichinella isolates: parasite variability and host responses . Int J Parasitol 26 : 471 - 481 .
5. Mitreva M , Jasmer DP , Zarlenga DS , Wang Z , Abubucker S , et al. ( 2011 ) The draft genome of the parasitic nematode Trichinella spiralis . Nat Genet 43 : 228 - 235 .
6. Ambros V ( 2004 ) The functions of animal microRNAs . Nature 431 : 350 - 355 .
7. Zhang B , Pan X , Cobb GP , Anderson TA ( 2006 ) Plant microRNA: a small regulatory molecule with big impact . Dev Biol 289 : 3 - 16 .
8. Lee HC , Li L , Gu W , Xue Z , Crosthwaite SK , et al. ( 2010 ) Diverse pathways generate microRNA-like RNAs and Dicer-independent small interfering RNAs in fungi . Mol Cell 38 : 803 - 814 .
9. Lagos-Quintana M , Rauhut R , Lendeckel W , Tuschl T ( 2001 ) Identification of novel genes coding for small expressed RNAs . Science 294 : 853 - 858 .
10. Cullen BR ( 2006 ) Viruses and microRNAs . Nat Genet 38 Suppl: S25 - 30 .
11. Carrington JC , Ambros V ( 2003 ) Role of microRNAs in plant and animal development . Science 301 : 336 - 338 .
12. Bartel DP ( 2004 ) MicroRNAs: genomics , biogenesis, mechanism, and function. Cell 116 : 281 - 297 .
13. Zhang B , Pan X , Anderson TA ( 2006 ) Identification of 188 conserved maize microRNAs and their targets . FEBS Lett 580 : 3753 - 3762 .
14. Carthew RW , Sontheimer EJ ( 2009 ) Origins and Mechanisms of miRNAs and siRNAs . Cell 136 : 642 - 655 .
15. Kim VN ( 2006 ) Small RNAs just got bigger: Piwi-interacting RNAs (piRNAs) in mammalian testes . Genes Dev 20 : 1993 - 1997 .
16. Vasudevan S , Tong Y , Steitz JA ( 2007 ) Switching from repression to activation: microRNAs can up-regulate translation . Science 318 : 1931 - 1934 .
17. Lee RC , Feinbaum RL , Ambros V ( 1993 ) The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin -14. Cell 75 : 843 - 854 .
18. Reinhart BJ , Slack FJ , Basson M , Pasquinelli AE , Bettinger JC , et al. ( 2000 ) The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans . Nature 403 : 901 - 906 .
19. 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 .
20. Filipowicz W , Jaskiewicz L , Kolb FA , Pillai RS ( 2005 ) Post-transcriptional gene silencing by siRNAs and miRNAs . Curr Opin Struct Biol 15 : 331 - 341 .
21. Poy MN , Eliasson L , Krutzfeldt J , Kuwajima S , Ma X , et al. ( 2004 ) A pancreatic islet-specific microRNA regulates insulin secretion . Nature 432 : 226 - 230 .
22. Gauthier BR , Wollheim CB ( 2006 ) MicroRNAs: 'ribo-regulators' of glucose homeostasis . Nat Med 12 : 36 - 38 .
23. Xu MJ , Liu Q , Nisbet AJ , Cai XQ , Yan C , et al. ( 2010 ) Identification and characterization of microRNAs in Clonorchis sinensis of human health significance . BMC Genomics 11 : 521 .
24. 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 .
25. Poole CB , Davis PJ , Jin J , McReynolds LA ( 2010 ) Cloning and bioinformatic identification of small RNAs in the filarial nematode, Brugia malayi . Mol Biochem Parasitol 169 : 87 - 94 .
26. 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 .
27. 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 .
28. Simoes MC , Lee J , Djikeng A , Cerqueira GC , Zerlotini A , et al. ( 2011 ) Identification of Schistosoma mansoni microRNAs . BMC Genomics 12 : 47 .
29. Chen MX , Ai L , Xu MJ , Zhang RL , Chen SH , et al. ( 2011 ) Angiostrongylus cantonensis: Identification and characterization of microRNAs in male and female adults . Exp Parasitol 128 : 116 - 120 .
30. Chen MX , Ai L , Xu MJ , Chen SH , Zhang YN , et al. ( 2011 ) Identification and characterization of microRNAs in Trichinella spiralis by comparison with Brugia malayi and Caenorhabditis elegans . Parasitol Res.
31. MacRae IJ , Zhou K , Doudna JA ( 2007 ) Structural determinants of RNA recognition and cleavage by Dicer . Nat Struct Mol Biol 14 : 934 - 940 .
32. Saito K , Siomi MC ( 2010 ) Small RNA-Mediated Quiescence of Transposable Elements in Animals . Dev Cell 19 : 687 - 697 .
33. 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 .
34. Wang G , Reinke V ( 2008 ) A C. elegans Piwi, PRG-1, regulates 21U-RNAs during spermatogenesis . Curr Biol 18 : 861 - 867 .
35. Das PP , Bagijn MP , Goldstein LD , Woolford JR , Lehrbach NJ , et al. ( 2008 ) Piwi and piRNAs act upstream of an endogenous siRNA pathway to suppress Tc3 transposon mobility in the Caenorhabditis elegans germline . Mol Cell 31 : 79 - 90 .
36. Ruby JG , Jan C , Player C , Axtell MJ , Lee W , et al. ( 2006 ) Large-scale sequencing reveals 21U-RNAs and additional microRNAs and endogenous siRNAs in C. elegans. Cell 127 : 1193 - 1207 .
37. Batista PJ , Ruby JG , Claycomb JM , Chiang R , Fahlgren N , et al. ( 2008 ) PRG-1 and 21U-RNAs interact to form the piRNA complex required for fertility in C. elegans. Mol Cell 31 : 67 - 78 .
38. 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 .
39. Guo L , Lu Z ( 2010 ) The fate of miRNA* strand through evolutionary analysis: implication for degradation as merely carrier strand or potential regulatory molecule? PLoS One 5: e11387 .
40. Lim LP , Lau NC , Weinstein EG , Abdelhakim A , Yekta S , et al. ( 2003 ) The microRNAs of Caenorhabditis elegans . Genes Dev 17 : 991 - 1008 .
41. Chen JF , Mandel EM , Thomson JM , Wu Q , Callis TE , et al. ( 2006 ) The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation . Nat Genet 38 : 228 - 233 .
42. Sokol NS , Xu P , Jan YN , Ambros V ( 2008 ) Drosophila let-7 microRNA is required for remodeling of the neuromusculature during metamorphosis . Genes Dev 22 : 1591 - 1596 .
43. Bentwich I , Avniel A , Karov Y , Aharonov R , Gilad S , et al. ( 2005 ) Identification of hundreds of conserved and nonconserved human microRNAs . Nat Genet 37 : 766 - 770 .
44. Christodoulou F , Raible F , Tomer R , Simakov O , Trachana K , et al. ( 2010 ) Ancient animal microRNAs and the evolution of tissue identity . Nature 463 : 1084 - 1088 .
45. Caygill EE , Johnston LA ( 2008 ) Temporal regulation of metamorphic processes in Drosophila by the let-7 and miR-125 heterochronic microRNAs . Curr Biol 18 : 943 - 950 .
46. 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 : 998 .
47. 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 .
48. 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 .
49. 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 .
50. Allen E , Xie Z , Gustafson AM , Carrington JC ( 2005 ) microRNA-directed phasing during trans-acting siRNA biogenesis in plants . Cell 121 : 207 - 221 .
51. Gogvadze E , Buzdin A ( 2009 ) Retroelements and their impact on genome evolution and functioning . Cell Mol Life Sci 66 : 3727 - 3742 .
52. Berriman M , Haas BJ , LoVerde PT , Wilson RA , Dillon GP , et al. ( 2009 ) The genome of the blood fluke Schistosoma mansoni . Nature 460 : 352 - 358 .
53. Biemont C , Vieira C ( 2006 ) Genetics: junk DNA as an evolutionary force . Nature 443 : 521 - 524 .
54. Despommier DD , Campbell WC , Blair LS ( 1977 ) The in vivo and in vitro analysis of immunity to Trichinella spiralis in mice and rats . Parasitology 74 : 109 - 119 .
55. Li R , Li Y , Kristiansen K , Wang J ( 2008 ) SOAP: short oligonucleotide alignment program . Bioinformatics 24 : 713 - 714 .
56. Dsouza M , Larsen N , Overbeek R ( 1997 ) Searching for patterns in genomic data . Trends Genet 13 : 497 - 498 .
57. Rice P , Longden I , Bleasby A ( 2000 ) EMBOSS: the European Molecular Biology Open Software Suite . Trends Genet 16 : 276 - 277 .
58. Hofacker IL , Fontana W , Stadler PF , Bonhoeffer LS , Tacker M , Schuster P ( 1994 ) Fast Folding and Comparison of RNA Secondary Structures . Monatshefte fur Chem Chem Month 125 : 167 - 188 .
59. Jones-Rhoades MW , Bartel DP ( 2004 ) Computational identification of plant microRNAs and their targets, including a stress-induced miRNA . Mol Cell 14 : 787 - 799 .
60. Matsubara H , Takeuchi T , Nishikawa E , Yanagisawa K , Hayashita Y , et al. ( 2007 ) Apoptosis induction by antisense oligonucleotides against miR-17-5p and miR-20a in lung cancers overexpressing miR -17-92. Oncogene 26 : 6099 - 6105 .
61. 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 .
62. Li YY , Qin L , Guo ZM , Liu L , Xu H , et al. ( 2006 ) In silico discovery of human natural antisense transcripts . BMC Bioinformatics 7 : 18 .
63. Livak KJ , Schmittgen TD ( 2001 ) Analysis of relative gene expression data using real-time quantitative PCR and the 2(2Delta Delta C (T)) Method . Methods 25 : 402 - 408 .