BACKGROUND
small noncoding rnas  are distributed in a wide variety of species from plants to mammals.
to date, several kinds of small rnas, including microrna , small interfering rna , and piwi-associated rna , have been discovered and demonstrated to function in various biological and cellular processes through regulation of gene expression at the post-transcriptional level .
the most characterized class of small regulatory rnas is mirnas, which are 19– <dig> nucleotides  single strand rnas and highly phylogenetically conserved across almost all species.
mature mirnas are produced from longer mirna precursors by two successive rnase iii cleavages.
after transcribed by rna polymerase ii, the primary mirna transcripts  are bound by the rna-binding protein dgcr <dig>  and initially processed by drosha to form a ~70 nt hairpin-shaped pre-mirna in the nucleus, and then the pre-mirnas are transported to the cytoplasm where they are further processed by dicer into an mirna:mirna* duplex.
only one strand of the duplex, the mature mirna , preferentially combined with the argonaut protein to form a rna-induced silencing complex .
the mirna then guides the risc to their target mrna through perfectly or imperfectly base-paring with the seed sequence, a 7– <dig> nucleotide section at the 5′ end of the mirna.
this binding either degrades the target rna or blocks protein production, resulting in the suppression of the target gene expression .
unlike mirnas, pirnas are 23–30 nt single-stranded rnas, which are deprived from a dicer-independent pathway and their sequences are poorly conserved among species  <cit> .
pirnas are abundantly expressed in the germ lines and interact with the piwi subfamily of argonaute proteins and repress the expression of selfish genetic elements, such as transposons  <cit> .
a strict translational regulation is essential for oocyte maturation  <cit> .
during oocyte maturation, oocytes that escape g2-arrest undergo germinal vesicle breakdown .
the expression of different maternal rnas must be precisely spatio-temporally regulated in this process.
generally, the mrna transcription is terminated in the gvbd-oocytes  <cit> , since mature oocytes are arrested in the metaphase i  or ii  of meiotic division until reactivation at fertilization.
in xenopus and mouse, large amounts of selective maternal mrnas are deadenylated and translationally repressed during meiotic maturation .
the proteins being made during oocyte maturation are mostly cell cycle regulators needed to advance the oocyte through meiosis.
given that discovery of mirnas and their roles in the regulation of gene expression through translational suppression or degradation of their target mrna, there should be large numbers of such small rnas in the ovary, which may be important regulators of gene expression during oocyte maturation.
oocyte gvbd, meiotic maturation is governed by the maturation-promoting factor , cyclin b and cdc <dig> kinase complex, cyclin b synthesis and degrade during meiosis i, ii.
it has been demonstrated that gonadotropin-releasing hormone  can induce oocyte maturation through regulating mpf activity  <cit> .
cytoplasmic polyadenylation element  and translation control element  in the 3′-untranslated region  of cyclin b mrna play important roles in the translational regulation of gene expression in clam, drosophila, xenopus and mouse  <cit> .
the cpe can activate translation of cyclin b during oocyte maturation by helping consort with the polyadenylation signal  <cit> .
on the contrary, tce in 3′-utr is involved in translational repression of the transcripts in the pole cells of the early drosophila embryo  <cit> .
in our previous study, eight potential cpes but no tce are found in the unique long 3′-utr of the cyclin b in the chinese mitten crab eriocheir sinensis
<cit> .
interestingly, the mirna-binding sites, namely gy-box, brd-box, and k-box motifs for translational regulation of the enhancer of split complex  and the bearded-complex  family genes in drosophila  <cit> , are simultaneously present in the 3′-utr of the cyclin b in chinese mitten crab  <cit> , suggesting that the translation or post-transcription of the crab cyclin b might be regulated cooperatively via cpe and the mirna-binding sites.
to examine the crab ovarian mirna expression and test whether mirna can regulate the cyclin b expression, we generated an ovarian small rna library and characterized the mirna transcriptom by combining the solexa sequencing with bioinformatics analysis.
we identified a total of  <dig> mirnas, including  <dig> known mirnas and  <dig> novel mirna candidates.
mir- <dig> and mir- <dig> were revealed to have a role in the regulation of 3′-utr of the crab cyclin b gene.
this study represents the first survey of the crab mirna in the ovary and provides the first evidence for the involvement of mirna in the crab oocyte maturation.
RESULTS
global analysis of small rnas in the crab ovary
to retrieve as many small rnas as possible with less cost, a cdna library of the crab small rnas was constructed from a mixture of total rnas from various ovarian stages and was high-throughput sequenced using the solexa system.
a total of  <dig> ,  <dig> reads were obtained from the sequencing machine.
after discarding low quality reads, adaptor sequence, contaminating mrna, rrna, trna and snorna sequences by illumina pipeline filter ,  <dig> , <dig> clean reads  were obtained.
among the clean reads,  <dig> , <dig> reads  of sequences were found to be 15–30 nt in length and more than two copies, representing  <dig>  unique sequences.
as shown in figure  <dig>  the size  of the small rna from the crab ovary exhibited a distinct double peak distribution.
one of peak for the  <dig> to 23 nt size class represents the typical mirna of dicer-processed product.
another peak for the  <dig> to 27 nt size class mostly represents longer pirna-like small rnas in the crab ovary .
obviously, the peak represented by the long size class  was much higher than that for the shorter size class , suggesting more abundant for the longer-sized small rnas in the ovary.figure  <dig>
length distribution and abundance of small rnas in the ovaries of the mitten crab.
abundance of each size class of small rnas based on nucleotide  length plotted using total small rna reads  and unique reads .
identification of mirnas in the crab ovaries
to identify potential mirnas in the mitten crab ovary, the screened dataset were blasted against the known mirna precursors  and mature mirnas  of arthropoda species in mirbase  <dig>   by miralign.
because the mitten crab genomic information is unavailable, a small crustacean daphnia genome was selected as a reference one.
among the screened  <dig> , <dig> reads,  <dig>  reads that represent  <dig>  unique sequences were mapped to the known mirs/mirs in mirbase  <dig>  and/or daphnia genome.
the mappable sequences are divided into five groups:   <dig>  reads representing  <dig> unique sequences which were mapped to both daphnia mirs/mirs in mirbase and daphnia genome;   <dig>  reads representing  <dig> unique sequences which were mapped not only to the locations of known mir/mirs but also to other locations in the genome.
the extended sequences at the mapped positions of the genome formed new hairpins;   <dig>  reads representing  <dig> unique sequences which mapped to both arthropoda mirs and other genome, but unmapped to daphnia genome;   <dig>  reads representing  <dig> unique sequences which were mapped to arthropoda mirs, but unmapped to genome;   <dig>  reads representing  <dig> unique sequences which were not mapped to arthropoda mirs, but mapped to the genome.
the extended sequences  at the mapped genome positions have the propensity of forming hairpins.
finally, we identified  <dig> known mirnas mapped to selected mirs/mirs  and  <dig> novel mirnas un-mapped to selected mirs/mirs in mirbase .
among the known mirnas,  <dig> belongs to  <dig> families, while the other two does not belonging to any family.
these mirnas in a family share the same seed sequence in the mitten crab as in other metazoan.
there are some nucleotide substitutions in the flanking regions of the seed sequences in mir- <dig>  mir- <dig>  mir- <dig>  mir- <dig> and mir- <dig>  in addition, we identified seven mirna* that aligned to 3′ end region of known mirna precursors.
the relative abundance of mirnas varies greatly among the known mirnas .
the top  <dig> most abundant mirnas include mir- <dig>  mir- <dig>  mir-9b and let- <dig>  each of these mirnas has more than  <dig>  reads.
identification of the mirnas targeting the crab cyclin b gene
to identify mirnas targeting the crab cyclin b gene, the full sequences of 3′-utr of cyclin b and mirna candidates were submitted to rnahybrid online searching  based on the degree of mirna:target sequence complementarity and the free energy level of rna-rna duplexes  <cit> .
as shown in figure  <dig>  the 5′ seed sequences of mir- <dig>  mir- <dig> mir- <dig> and mir- <dig> were revealed to be complementary to their corresponding target sites such as gy-box, brd-box, and k-box in 3′-utr of the crab cyclin b gene.
the mir- <dig> was identified with the smallest free energy value .
by taking advantage of a pair of primers designed according to the sequence of mir-2* and mir- <dig> , the precursor sequence for mir- <dig> was successfully amplified and cloned from the crab genomic dna  and its hairpin structure was predicted .
the crab mir- <dig> precursor shared high sequence homology with the mir- <dig> precursor from other invertebrates such as daphnia pulex and drosophila melanogaster.figure  <dig>
the potential mirna target sites of mir- <dig>  mir- <dig>  mir- <dig> and mir- <dig> in the 3′-utr of the crab cyclin b as detected by rnahybrid  <cit> .
shadow indicated complementary region of seed sequence of the mirna and their putative binding sites in the 3′-utr.
mfe: match free energy.
the sequence and hairpin structure of the crab mir- <dig> precursor.
alignments of mir- <dig> precursor sequences from the mitten crab e. sinensis and other invertebrates including daphnia pulex, drosophila melanogaster, apis mellifera, tribolium castaneum, lottia gigantean, ascaris suum, nasonia vitripennis, ixodes scapularis, caenorhabditis remanei.
the identical nucleotides were shaded with color table.
the mature mirna sequence is underlined.
the hairpin structure of the crab mir- <dig> precursor predicted using the rna-fold server on the website http://mfold.rna.albany.edu/?q=mfold.
the mature mirna sequence is shown in red.
quantification of the expression of the mirnas in ovaries
to identify mirnas differentially expressed during the crab oocyte maturation, the relative abundance of mir- <dig>  mir- <dig>  mir- <dig>  mir- <dig> and other six selected mirnas in the ovaries at gv and mi stages was assessed by quantitative real-time pcr.
the selected mirnas include top four most abundance mirnas  in the ovaries and two mirnas  related to oogenesis in fruit fly  <cit> .
because our previous studies have shown that gnrh can induce gvbd of the crab oocyte , we further extended our investigation to the potential effect of the gnrh on the mirna expression during the oocyte maturation.
the expression level of the  <dig> mirnas was measured over a period of 30 days after injection of gnrh.
as indicated in figure  <dig>  gvbd was first observed at 15 days post injection of gnrh.
no gvbd was found till 30 days in non-injected animals and control animals injected with pbs.
after normalized against u <dig> snrna, the relative expression level of mir- <dig> and mir- <dig> significantly increased at mi  whereas the other eight mirnas exhibited stable expression from gv to mi among all the groups .figure  <dig>
quantitative real-time pcr analysis of mir- <dig>  and mir- <dig>  expression in the ovaries of the mitten crab.
the crabs were injected with pbs solution or gnrh, and the blank control received no injection as shown on the top.
the expression was normalized against u <dig> snrna levels.
bar represents the standard error  of the mean value for 3– <dig> individuals, asterisks indicate significant difference  between gv and mi stages.
to further test whether the differential expression of mir- <dig> and mir- <dig> at mi results from the induction of gnrh, we examined the detail expression profile of mir- <dig> and mir- <dig> in a period of 24-hour after injection of gnrh.
surprisingly, no significant difference of the expression level was found for the both mirnas tested in 24 hours , indicating gnrh cannot induce expression of mir- <dig> and mir- <dig>  therefore, we inferred that the increased expression of mir- <dig> and mir- <dig> observed at mi was independent of gnrh injection.
validation of regulation of the mirnas for the 3′-utr of the crab cyclin b gene
to determine whether there are direct interactions between mir- <dig>  mir- <dig>  mir- <dig>  mir- <dig> and their target sites of the crab cyclin b gene, we used a luciferase 3′-utr reporter assay to measure the inhibitory effects of these mirnas.
the full sequence of the cyclin b 3′-utr harboring gy-box, brd-box, and k-box motifs or mutated boxes was cloned into the downstream of luciferase reporter gene.
after cotransfected hek 293 t cells, mir- <dig> and mir- <dig> mimics significantly reduce the luciferase activity from the reporter construct containing the cyclin b 3′-utr, whereas mir- <dig>  mir- <dig> and negative control mimics have no effect on the luciferase activity .
no effect was also detected with a construct containing mutated seed sites in the cyclin b 3′-utr .
these results indicated that mir- <dig> and mir- <dig> can downregulate the target gene expression by mirna binding sites in the 3′-utr of cyclin b.figure  <dig>
luciferase 3′-utr reporter assays of the mirnas silence effects using pgl3/cyclin b 3′-utr  and pgl3/cyclin b 3′-utr mutant  reporter vectors.
firefly luciferase activity was evaluated 24 h after cotransfection.
values are mean ± se of three independent experiments.
asterisks indicate significant difference  compared with negative control  using pgl <dig> empty vector.
to verify whether the protein level of cyclin b drops during meiosis of oocytes, gv-, gvbd-oocytes and fertilized eggs were submitted to western blot analysis using an antibody against the crab cyclin b. the results showed that the crab cyclin b protein was present in gv- and gvbd-oocytes but disappeared in fertilized eggs at the time of transition from mi to anaphase of meiosis , suggesting the potential role for the mir- <dig> and mir- <dig> in regulating the destruction of the cyclin b protein.figure  <dig>
western blot analysis of the expression of the cyclin b protein  and mpf activity assay  in gv-, gvbd-oocytesand fertilized eggs .
asterisks indicate significant difference  compared with those of gv-, gvbd-oocytes.
furthermore, relative mpf activity was measured using a cdc2-cyclin b kinase assay kit.
the data showed that mpf activity reach the highest level at the time of gvbd and sharply drop to the basal level at fertilization , indicating that the destruction of the cyclin b protein in the fertilized egg results in a rapid and complete suppression of mpf activity.
discussion
over thirty thousands of mirna have now been identified in a multitude of organisms .
however, there is rare information available regarding mirna in decapod species.
in this study, we attempted to identify small rnas from the crab ovary as the first step toward understanding the regulatory roles of small rnas in oogenesis.
by combining the solexa sequencing with bioinformatics analysis, we identified a total of  <dig> mirnas, including  <dig> known mirnas and  <dig> novel mirna candidates.
to the best of our knowledge, this is the first time that such a high number of mirnas have been isolated from a decapod brachyuran.
furthermore, expression analysis revealed that mir- <dig> and mir- <dig> exhibited higher expression at mi arrest of meiosis and both of mir- <dig> and mir- <dig> can bind the 3′-utr of the crab cyclin b, indicating that mir- <dig> and mir- <dig> is involved in the regulation of cyclin b expression during meiotic maturation of oocyte in the mitten crab.
the survey of small rnas in the crab ovary
a global survey of small rnas in the crab ovary would contribute to understanding the function and evolution of small rnas in crustacean.
as in the insect species silkworm  <cit>  and locust  <cit> , our results indicate that there is a distinct bimodal distribution in the size of the small rnas in the crab ovary at different developmental stages .
one peak around 21–23 nt represents mirnas and another distinct peak around 24–27 nt mostly represents longer pirna-like small rnas.
the pirna-like small rnas make up a larger proportion in the crab ovary compared with mirnas, indicating their potential functions in ovarian development, although we could not annotate them for the time being.
given that the whole genome sequence data of the mitten crab are unavailable currently, it is essential for screening mirnas to use reference genomes from other closely related species.
thus the daphinia genome was employed as a reference in our analysis to identify the mirnas from the crab deep sequencing data.
the retrieved mirnas shared conserved regions of the seed sequences, although there are some species-specific nucleotide substitutions in the flanking regions of the seed sequences in some mirnas.
retrieval of mir-2*, together with predicted stem-loop structure for the precursor , strongly supported that the mature mirna is processed from stem-loop structure.
in the ovarian mirna transcriptom, the reads of mirnas sequences unevenly distributed within a wide range from  <dig> to  <dig>  .
quantitative real-time pcr assay showed that the expression of the top four most abundant mirnas  is stable during the crab oocyte maturation.
intriguingly, previous studies in the fruit fly drosophila showed that mir- <dig> plays a crucial role in the female germline development.
loss of mir- <dig> leads to multiple severe defects during oogenesis and early embryogenesis  <cit> .
mir- <dig> and let- <dig> are involved in developmental timing in the nematode and the fly  <cit> .
further analysis of the expression profile of these mirnas would ultimately lead to functional characterization of these abundant mirnas in regulation of the crab ovarian development and early embryogenesis.
mir- <dig> and mir- <dig> can regulate the 3′-utr of the crab cyclin b gene
accumulating evidence has showed that mirnas are involved in regulation of cell cycle by controlling the expression of cyclins.
for instance, mir-15b induced cell cycle arrest at g0/g <dig> phase by targeting cyclin e in glioma cells  <cit> ; mir- <dig> induced g <dig> arrest partially by targeting cyclin d <dig>  <cit> ; mir-122a can modulate cyclin g <dig> expression in human hepatocellular carcinoma-derived cell lines  <cit> .
the regulative effect of mirnas on mrnas during meiotic maturation of oocytes was initially investigated in mice lacking dicer.
dicer null oocytes showed abnormal gene expression and arrested with defects in spindle organization at mi stage, indicating that dicer is essential for meiotic maturation of mouse oocytes  <cit> .
however, subsequent studies in dgcr <dig> knockout mice revealed that limited mirna-associated mrna degradation and mirna activity is globally suppressed in mouse oocytes and early embryos  <cit> , suggesting that the abundance of mirnas in eggs may have no effect on the expression of their target genes.
this data does not support the notion that mirnas extensively modulate gene expression in mouse oocytes.
the exact role of the mirnas in the oocytes remains to be elucidated, since the fecundity of dgcr <dig> knockout mice becomes diminished.
surprisingly, a recent study showed that mir-27a, which represses the translation of map2k <dig> and functions in the cell cycle, has activity in porcine oocytes, indicating that mirna activity was not globally suppressed in porcine oocytes  <cit> .
furthermore,  <dig> human mirnas were revealed to be differential expression in gv and mii oocytes  <cit> , suggesting that mirnas may play specific, rather than global, regulatory functions in gene expression during oocyte maturation.
as in many oviparous invertebrates, the mature gvbd-oocyte in the mitten crab arrests in mi till fertilization  <cit> .
fertilization is required for progression through meiosis i and completion of meiotic division.
the present study showed that two mirnas  exhibited significantly increased expression from gv to mi stage .
interestingly, many putative binding sites for mirnas including mir- <dig> and mir- <dig> were found in 3′-utr of the crab cyclin b transcript .
thus we postulated that the high expression of mir- <dig> and mir- <dig> at mi arrest might be related to translation inhibition of the cyclin b. to test this hypothesis, the direct interactions between the mirnas and their target sites of the crab cyclin b gene were examined using a luciferase 3′-utr reporter assay.
the result showed that mir- <dig> and mir- <dig> downregulated the luciferase activity by targeting the 3′-utr of the cyclin b , indicating that mir- <dig> and mir- <dig> are involved in inhibiting the expression of the crab cyclin b gene.
the result was confirmed by western blot analysis and mpf activity assay, in which the crab cyclin b protein disappeared and cyclinb-cdc <dig> kinase activity sharply dropped to the basal level in fertilized eggs at the transition from mi to anaphase i of meiosis, supporting the notion that cyclin b could be a direct target for mir- <dig> and mir- <dig> and the degradation of cyclin b is required for the fertilized egg to exit from mi to anaphase i of meiosis.
gnrh can induce gvbd but has not effect on mir- <dig> and mir- <dig> expression
in vertebrates, reproductive success relies on the coordinated actions of the hypothalamic- pituitary-gonadal axis.
gnrh is the central regulator of the reproductive hormonal cascade that is released from hypothalami into the hypophyseal portal system to stimulate the biosynthesis and secretion of gonadotropic hormones lh and fsh from pituitary.
several studies indicated that mirna expression in reproductive tissues varied in response to pituitary and gonadal hormones  <cit> .
thirteen mirnas were differentially expressed in periovulatory granulosa cells post-hcg  induction.
two mirnas, mir- <dig> and mir- <dig> were found to be highly up-regulated following lh/hcg induction  <cit> .
our present studies showed that the gnrh injection in vivo can induce gvbd of the crab oocyte as in vertebrates.
although mir- <dig> and mir- <dig> displayed a higher expression at mi meiosis, no significant difference of the expression level was found for all the selected mirnas including mir- <dig> and mir- <dig> in 24 hours post gnrh injection, indicating gnrh cannot induce expression of the mirnas.
there must be other unknown mechanism for regulation of differential expression of mir- <dig> and mir- <dig> during oocyte maturation.
also, more mirnas need to be selected for assay in the future.
CONCLUSIONS
a high number of mirnas have been isolated from the crab ovary for the first time.
this data provides basis for better understanding of mirna roles in regulating the ovarian development.
mir- <dig> and mir- <dig>  which were predicted to target the crab cyclin b gene, displayed high expression in mi arrest stage relative to gv stage.
luciferase reporter genes assay demonstrated that mir- <dig> and mir- <dig> have activity and can downregulate the 3′-utrs of the cyclin b gene.
this result strongly suggest that mir- <dig> and mir- <dig> are involved in regulating the expression of the cycin b at the transition from mi to anaphase of meiosis i, and that mirnas might have not been globally suppressed during meiosis of the crab oocyte.

 small noncoding rnas are distributed in a wide variety of species from plants to mammals. to date, several kinds of small rnas, including microrna, small interfering rna, and piwi-associated rna, have been discovered and demonstrated to function in various biological and cellular processes through regulation of gene expression at the post-transcriptional level. the most characterized class of small regulatory rnas is mirnas, which are 19– nucleotides single strand rnas and highly phylogenetically conserved across almost all species. mature mirnas are produced from longer mirna precursors by two successive rnase iii cleavages. after transcribed by rna polymerase ii, the primary mirna transcripts are bound by the rna-binding protein dgcr and initially processed by drosha to form a ~70 nt hairpin-shaped pre-mirna in the nucleus, and then the pre-mirnas are transported to the cytoplasm where they are further processed by dicer into an mirna:mirna* duplex. only one strand of the duplex, the mature mirna, preferentially combined with the argonaut protein to form a rna-induced silencing complex. the mirna then guides the risc to their target mrna through perfectly or imperfectly base-paring with the seed sequence, a 7– nucleotide section at the 5′ end of the mirna. this binding either degrades the target rna or blocks protein production, resulting in the suppression of the target gene expression. unlike mirnas, pirnas are 23–30 nt single-stranded rnas, which are deprived from a dicer-independent pathway and their sequences are poorly conserved among species. pirnas are abundantly expressed in the germ lines and interact with the piwi subfamily of argonaute proteins and repress the expression of selfish genetic elements, such as transposons. a strict translational regulation is essential for oocyte maturation. during oocyte maturation, oocytes that escape g2-arrest undergo germinal vesicle breakdown. the expression of different maternal rnas must be precisely spatio-temporally regulated in this process. generally, the mrna transcription is terminated in the gvbd-oocytes, since mature oocytes are arrested in the metaphase i or ii of meiotic division until reactivation at fertilization. in xenopus and mouse, large amounts of selective maternal mrnas are deadenylated and translationally repressed during meiotic maturation. the proteins being made during oocyte maturation are mostly cell cycle regulators needed to advance the oocyte through meiosis. given that discovery of mirnas and their roles in the regulation of gene expression through translational suppression or degradation of their target mrna, there should be large numbers of such small rnas in the ovary, which may be important regulators of gene expression during oocyte maturation. oocyte gvbd, meiotic maturation is governed by the maturation-promoting factor, cyclin b and cdc kinase complex, cyclin b synthesis and degrade during meiosis i, ii. it has been demonstrated that gonadotropin-releasing hormone can induce oocyte maturation through regulating mpf activity. cytoplasmic polyadenylation element and translation control element in the 3′-untranslated region of cyclin b mrna play important roles in the translational regulation of gene expression in clam, drosophila, xenopus and mouse. the cpe can activate translation of cyclin b during oocyte maturation by helping consort with the polyadenylation signal. on the contrary, tce in 3′-utr is involved in translational repression of the transcripts in the pole cells of the early drosophila embryo. in our previous study, eight potential cpes but no tce are found in the unique long 3′-utr of the cyclin b in the chinese mitten crab eriocheir sinensis. interestingly, the mirna-binding sites, namely gy-box, brd-box, and k-box motifs for translational regulation of the enhancer of split complex and the bearded-complex family genes in drosophila, are simultaneously present in the 3′-utr of the cyclin b in chinese mitten crab, suggesting that the translation or post-transcription of the crab cyclin b might be regulated cooperatively via cpe and the mirna-binding sites. to examine the crab ovarian mirna expression and test whether mirna can regulate the cyclin b expression, we generated an ovarian small rna library and characterized the mirna transcriptom by combining the solexa sequencing with bioinformatics analysis. we identified a total of mirnas, including known mirnas and novel mirna candidates. mir- and mir- were revealed to have a role in the regulation of 3′-utr of the crab cyclin b gene. this study represents the first survey of the crab mirna in the ovary and provides the first evidence for the involvement of mirna in the crab oocyte maturation.  global analysis of small rnas in the crab ovary to retrieve as many small rnas as possible with less cost, a cdna library of the crab small rnas was constructed from a mixture of total rnas from various ovarian stages and was high-throughput sequenced using the solexa system. a total of, reads were obtained from the sequencing machine. after discarding low quality reads, adaptor sequence, contaminating mrna, rrna, trna and snorna sequences by illumina pipeline filter,, clean reads were obtained. among the clean reads,, reads of sequences were found to be 15–30 nt in length and more than two copies, representing unique sequences. as shown in figure  the size of the small rna from the crab ovary exhibited a distinct double peak distribution. one of peak for the to 23 nt size class represents the typical mirna of dicer-processed product. another peak for the to 27 nt size class mostly represents longer pirna-like small rnas in the crab ovary. obviously, the peak represented by the long size class was much higher than that for the shorter size class, suggesting more abundant for the longer-sized small rnas in the ovary.figure length distribution and abundance of small rnas in the ovaries of the mitten crab. abundance of each size class of small rnas based on nucleotide length plotted using total small rna reads and unique reads. identification of mirnas in the crab ovaries to identify potential mirnas in the mitten crab ovary, the screened dataset were blasted against the known mirna precursors and mature mirnas of arthropoda species in mirbase by miralign. because the mitten crab genomic information is unavailable, a small crustacean daphnia genome was selected as a reference one. among the screened, reads, reads that represent unique sequences were mapped to the known mirs/mirs in mirbase and/or daphnia genome. the mappable sequences are divided into five groups: reads representing unique sequences which were mapped to both daphnia mirs/mirs in mirbase and daphnia genome; reads representing unique sequences which were mapped not only to the locations of known mir/mirs but also to other locations in the genome. the extended sequences at the mapped positions of the genome formed new hairpins; reads representing unique sequences which mapped to both arthropoda mirs and other genome, but unmapped to daphnia genome; reads representing unique sequences which were mapped to arthropoda mirs, but unmapped to genome; reads representing unique sequences which were not mapped to arthropoda mirs, but mapped to the genome. the extended sequences at the mapped genome positions have the propensity of forming hairpins. finally, we identified known mirnas mapped to selected mirs/mirs and novel mirnas un-mapped to selected mirs/mirs in mirbase. among the known mirnas, belongs to families, while the other two does not belonging to any family. these mirnas in a family share the same seed sequence in the mitten crab as in other metazoan. there are some nucleotide substitutions in the flanking regions of the seed sequences in mir- mir- mir- mir- and mir- in addition, we identified seven mirna* that aligned to 3′ end region of known mirna precursors. the relative abundance of mirnas varies greatly among the known mirnas. the top most abundant mirnas include mir- mir- mir-9b and let- each of these mirnas has more than reads. identification of the mirnas targeting the crab cyclin b gene to identify mirnas targeting the crab cyclin b gene, the full sequences of 3′-utr of cyclin b and mirna candidates were submitted to rnahybrid online searching based on the degree of mirna:target sequence complementarity and the free energy level of rna-rna duplexes. as shown in figure  the 5′ seed sequences of mir- mir- mir- and mir- were revealed to be complementary to their corresponding target sites such as gy-box, brd-box, and k-box in 3′-utr of the crab cyclin b gene. the mir- was identified with the smallest free energy value. by taking advantage of a pair of primers designed according to the sequence of mir-2* and mir-, the precursor sequence for mir- was successfully amplified and cloned from the crab genomic dna and its hairpin structure was predicted. the crab mir- precursor shared high sequence homology with the mir- precursor from other invertebrates such as daphnia pulex and drosophila melanogaster.figure the potential mirna target sites of mir- mir- mir- and mir- in the 3′-utr of the crab cyclin b as detected by rnahybrid. shadow indicated complementary region of seed sequence of the mirna and their putative binding sites in the 3′-utr. mfe: match free energy. the sequence and hairpin structure of the crab mir- precursor. alignments of mir- precursor sequences from the mitten crab e. sinensis and other invertebrates including daphnia pulex, drosophila melanogaster, apis mellifera, tribolium castaneum, lottia gigantean, ascaris suum, nasonia vitripennis, ixodes scapularis, caenorhabditis remanei. the identical nucleotides were shaded with color table. the mature mirna sequence is underlined. the hairpin structure of the crab mir- precursor predicted using the rna-fold server on the website http://mfold.rna.albany.edu/?q=mfold. the mature mirna sequence is shown in red. quantification of the expression of the mirnas in ovaries to identify mirnas differentially expressed during the crab oocyte maturation, the relative abundance of mir- mir- mir- mir- and other six selected mirnas in the ovaries at gv and mi stages was assessed by quantitative real-time pcr. the selected mirnas include top four most abundance mirnas in the ovaries and two mirnas related to oogenesis in fruit fly. because our previous studies have shown that gnrh can induce gvbd of the crab oocyte, we further extended our investigation to the potential effect of the gnrh on the mirna expression during the oocyte maturation. the expression level of the mirnas was measured over a period of 30 days after injection of gnrh. as indicated in figure  gvbd was first observed at 15 days post injection of gnrh. no gvbd was found till 30 days in non-injected animals and control animals injected with pbs. after normalized against u snrna, the relative expression level of mir- and mir- significantly increased at mi whereas the other eight mirnas exhibited stable expression from gv to mi among all the groups.figure quantitative real-time pcr analysis of mir- and mir- expression in the ovaries of the mitten crab. the crabs were injected with pbs solution or gnrh, and the blank control received no injection as shown on the top. the expression was normalized against u snrna levels. bar represents the standard error of the mean value for 3– individuals, asterisks indicate significant difference between gv and mi stages. to further test whether the differential expression of mir- and mir- at mi results from the induction of gnrh, we examined the detail expression profile of mir- and mir- in a period of 24-hour after injection of gnrh. surprisingly, no significant difference of the expression level was found for the both mirnas tested in 24 hours, indicating gnrh cannot induce expression of mir- and mir- therefore, we inferred that the increased expression of mir- and mir- observed at mi was independent of gnrh injection. validation of regulation of the mirnas for the 3′-utr of the crab cyclin b gene to determine whether there are direct interactions between mir- mir- mir- mir- and their target sites of the crab cyclin b gene, we used a luciferase 3′-utr reporter assay to measure the inhibitory effects of these mirnas. the full sequence of the cyclin b 3′-utr harboring gy-box, brd-box, and k-box motifs or mutated boxes was cloned into the downstream of luciferase reporter gene. after cotransfected hek 293 t cells, mir- and mir- mimics significantly reduce the luciferase activity from the reporter construct containing the cyclin b 3′-utr, whereas mir- mir- and negative control mimics have no effect on the luciferase activity. no effect was also detected with a construct containing mutated seed sites in the cyclin b 3′-utr. these results indicated that mir- and mir- can downregulate the target gene expression by mirna binding sites in the 3′-utr of cyclin b.figure luciferase 3′-utr reporter assays of the mirnas silence effects using pgl3/cyclin b 3′-utr and pgl3/cyclin b 3′-utr mutant reporter vectors. firefly luciferase activity was evaluated 24 h after cotransfection. values are mean ± se of three independent experiments. asterisks indicate significant difference compared with negative control using pgl empty vector. to verify whether the protein level of cyclin b drops during meiosis of oocytes, gv-, gvbd-oocytes and fertilized eggs were submitted to western blot analysis using an antibody against the crab cyclin b. the results showed that the crab cyclin b protein was present in gv- and gvbd-oocytes but disappeared in fertilized eggs at the time of transition from mi to anaphase of meiosis, suggesting the potential role for the mir- and mir- in regulating the destruction of the cyclin b protein.figure western blot analysis of the expression of the cyclin b protein and mpf activity assay in gv-, gvbd-oocytesand fertilized eggs. asterisks indicate significant difference compared with those of gv-, gvbd-oocytes. furthermore, relative mpf activity was measured using a cdc2-cyclin b kinase assay kit. the data showed that mpf activity reach the highest level at the time of gvbd and sharply drop to the basal level at fertilization, indicating that the destruction of the cyclin b protein in the fertilized egg results in a rapid and complete suppression of mpf activity. discussion over thirty thousands of mirna have now been identified in a multitude of organisms. however, there is rare information available regarding mirna in decapod species. in this study, we attempted to identify small rnas from the crab ovary as the first step toward understanding the regulatory roles of small rnas in oogenesis. by combining the solexa sequencing with bioinformatics analysis, we identified a total of mirnas, including known mirnas and novel mirna candidates. to the best of our knowledge, this is the first time that such a high number of mirnas have been isolated from a decapod brachyuran. furthermore, expression analysis revealed that mir- and mir- exhibited higher expression at mi arrest of meiosis and both of mir- and mir- can bind the 3′-utr of the crab cyclin b, indicating that mir- and mir- is involved in the regulation of cyclin b expression during meiotic maturation of oocyte in the mitten crab. the survey of small rnas in the crab ovary a global survey of small rnas in the crab ovary would contribute to understanding the function and evolution of small rnas in crustacean. as in the insect species silkworm and locust, our results indicate that there is a distinct bimodal distribution in the size of the small rnas in the crab ovary at different developmental stages. one peak around 21–23 nt represents mirnas and another distinct peak around 24–27 nt mostly represents longer pirna-like small rnas. the pirna-like small rnas make up a larger proportion in the crab ovary compared with mirnas, indicating their potential functions in ovarian development, although we could not annotate them for the time being. given that the whole genome sequence data of the mitten crab are unavailable currently, it is essential for screening mirnas to use reference genomes from other closely related species. thus the daphinia genome was employed as a reference in our analysis to identify the mirnas from the crab deep sequencing data. the retrieved mirnas shared conserved regions of the seed sequences, although there are some species-specific nucleotide substitutions in the flanking regions of the seed sequences in some mirnas. retrieval of mir-2*, together with predicted stem-loop structure for the precursor, strongly supported that the mature mirna is processed from stem-loop structure. in the ovarian mirna transcriptom, the reads of mirnas sequences unevenly distributed within a wide range from to. quantitative real-time pcr assay showed that the expression of the top four most abundant mirnas is stable during the crab oocyte maturation. intriguingly, previous studies in the fruit fly drosophila showed that mir- plays a crucial role in the female germline development. loss of mir- leads to multiple severe defects during oogenesis and early embryogenesis. mir- and let- are involved in developmental timing in the nematode and the fly. further analysis of the expression profile of these mirnas would ultimately lead to functional characterization of these abundant mirnas in regulation of the crab ovarian development and early embryogenesis. mir- and mir- can regulate the 3′-utr of the crab cyclin b gene accumulating evidence has showed that mirnas are involved in regulation of cell cycle by controlling the expression of cyclins. for instance, mir-15b induced cell cycle arrest at g0/g phase by targeting cyclin e in glioma cells; mir- induced g arrest partially by targeting cyclin d; mir-122a can modulate cyclin g expression in human hepatocellular carcinoma-derived cell lines. the regulative effect of mirnas on mrnas during meiotic maturation of oocytes was initially investigated in mice lacking dicer. dicer null oocytes showed abnormal gene expression and arrested with defects in spindle organization at mi stage, indicating that dicer is essential for meiotic maturation of mouse oocytes. however, subsequent studies in dgcr knockout mice revealed that limited mirna-associated mrna degradation and mirna activity is globally suppressed in mouse oocytes and early embryos, suggesting that the abundance of mirnas in eggs may have no effect on the expression of their target genes. this data does not support the notion that mirnas extensively modulate gene expression in mouse oocytes. the exact role of the mirnas in the oocytes remains to be elucidated, since the fecundity of dgcr knockout mice becomes diminished. surprisingly, a recent study showed that mir-27a, which represses the translation of map2k and functions in the cell cycle, has activity in porcine oocytes, indicating that mirna activity was not globally suppressed in porcine oocytes. furthermore, human mirnas were revealed to be differential expression in gv and mii oocytes, suggesting that mirnas may play specific, rather than global, regulatory functions in gene expression during oocyte maturation. as in many oviparous invertebrates, the mature gvbd-oocyte in the mitten crab arrests in mi till fertilization. fertilization is required for progression through meiosis i and completion of meiotic division. the present study showed that two mirnas exhibited significantly increased expression from gv to mi stage. interestingly, many putative binding sites for mirnas including mir- and mir- were found in 3′-utr of the crab cyclin b transcript. thus we postulated that the high expression of mir- and mir- at mi arrest might be related to translation inhibition of the cyclin b. to test this hypothesis, the direct interactions between the mirnas and their target sites of the crab cyclin b gene were examined using a luciferase 3′-utr reporter assay. the result showed that mir- and mir- downregulated the luciferase activity by targeting the 3′-utr of the cyclin b, indicating that mir- and mir- are involved in inhibiting the expression of the crab cyclin b gene. the result was confirmed by western blot analysis and mpf activity assay, in which the crab cyclin b protein disappeared and cyclinb-cdc kinase activity sharply dropped to the basal level in fertilized eggs at the transition from mi to anaphase i of meiosis, supporting the notion that cyclin b could be a direct target for mir- and mir- and the degradation of cyclin b is required for the fertilized egg to exit from mi to anaphase i of meiosis. gnrh can induce gvbd but has not effect on mir- and mir- expression in vertebrates, reproductive success relies on the coordinated actions of the hypothalamic- pituitary-gonadal axis. gnrh is the central regulator of the reproductive hormonal cascade that is released from hypothalami into the hypophyseal portal system to stimulate the biosynthesis and secretion of gonadotropic hormones lh and fsh from pituitary. several studies indicated that mirna expression in reproductive tissues varied in response to pituitary and gonadal hormones. thirteen mirnas were differentially expressed in periovulatory granulosa cells post-hcg induction. two mirnas, mir- and mir- were found to be highly up-regulated following lh/hcg induction. our present studies showed that the gnrh injection in vivo can induce gvbd of the crab oocyte as in vertebrates. although mir- and mir- displayed a higher expression at mi meiosis, no significant difference of the expression level was found for all the selected mirnas including mir- and mir- in 24 hours post gnrh injection, indicating gnrh cannot induce expression of the mirnas. there must be other unknown mechanism for regulation of differential expression of mir- and mir- during oocyte maturation. also, more mirnas need to be selected for assay in the future.  a high number of mirnas have been isolated from the crab ovary for the first time. this data provides basis for better understanding of mirna roles in regulating the ovarian development. mir- and mir- which were predicted to target the crab cyclin b gene, displayed high expression in mi arrest stage relative to gv stage. luciferase reporter genes assay demonstrated that mir- and mir- have activity and can downregulate the 3′-utrs of the cyclin b gene. this result strongly suggest that mir- and mir- are involved in regulating the expression of the cycin b at the transition from mi to anaphase of meiosis i, and that mirnas might have not been globally suppressed during meiosis of the crab oocyte. 
a high number of mirnas have been identified from the crab ovarian small rna transcriptom for the first time. further expression analysis using double-luciferase reporter genes assay showed that mir- and mir- can downregulate the 3′-utrs of the crab cyclin b gene, indicating that they could inhibit the translation of the cyclin b. western blot analysis confirmed that cyclin b protein is completely disappeared in fertilized egg at the metaphase-anaphase transition of meiosis i, suggesting that mir- and mir- could function in destruction of cyclin b near the end of mi. our previous studies showed that many mirna-binding sites are present in the 3′-utr of the cyclin b in the chinese mitten crab eriocheir sinensis, suggesting that the translation or post-transcription of the crab cyclin b might be regulated by mirnas during meiosis of oocyte. this result is inconsistent with recent finding that mirna activity is globally suppressed in mouse oocytes. of, reads, known mirnas representing mirna families were identified and novel mirna candidates were predicted. mir- and mir- exhibit differential expression during the meiotic maturation of the oocytes and have activity in regulating the 3′-utr of the crab cyclin b gene. to identify ovarian mirnas in the mitten crab, ovarian small rnas were subjected to high-throughput sequencing using an illumina genome analyzer. up to now, rare information for the mirnas is available in decapod crustaceans. ovarymicrorna transcriptomeoocyte meiosischinese mitten crabissue-copyright-statement© the author 2014 the 5′ seed sequences of four mirnas, mir- mir- mir- and mir- were revealed to complementary to mirna binding sites in 3′-utr of the cyclin b. quantitative real time pcr analysis showed that mir- and mir- are much more abundant in the first metaphase of meiosis than in germinal vesicle stage.
 micrornas are small non-coding rna molecules that downregulate gene expression by base pairing to the 3′-untranslated region of target messenger rnas. up to now, rare information for the mirnas is available in decapod crustaceans. our previous studies showed that many mirna-binding sites are present in the 3′-utr of the cyclin b in the chinese mitten crab eriocheir sinensis, suggesting that the translation or post-transcription of the crab cyclin b might be regulated by mirnas during meiosis of oocyte.  to identify ovarian mirnas in the mitten crab, ovarian small rnas were subjected to high-throughput sequencing using an illumina genome analyzer. of, reads, known mirnas representing mirna families were identified and novel mirna candidates were predicted. the 5′ seed sequences of four mirnas, mir- mir- mir- and mir- were revealed to complementary to mirna binding sites in 3′-utr of the cyclin b. quantitative real time pcr analysis showed that mir- and mir- are much more abundant in the first metaphase of meiosis than in germinal vesicle stage. but their increasing expressions are independent of induction of gonadotropin-releasing hormone. further expression analysis using double-luciferase reporter genes assay showed that mir- and mir- can downregulate the 3′-utrs of the crab cyclin b gene, indicating that they could inhibit the translation of the cyclin b. western blot analysis confirmed that cyclin b protein is completely disappeared in fertilized egg at the metaphase-anaphase transition of meiosis i, suggesting that mir- and mir- could function in destruction of cyclin b near the end of mi.  a high number of mirnas have been identified from the crab ovarian small rna transcriptom for the first time. mir- and mir- exhibit differential expression during the meiotic maturation of the oocytes and have activity in regulating the 3′-utr of the crab cyclin b gene. this result is inconsistent with recent finding that mirna activity is globally suppressed in mouse oocytes. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords ovarymicrorna transcriptomeoocyte meiosischinese mitten crabissue-copyright-statement© the author 2014 