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RESEARCH |
Departments of1 , Molecular Anatomy and Medicine2 Surgery for Organ Function and Biological Regulation, Nippon Medical School, 1-1-5 Sendagi, Tokyo 113-8602, Japan
Correspondence should be addressed to To Takizawa who is now at Department of Molecular Anatomy and Medicine, Nippon Medical School, 1-1-5 Sendagi, Tokyo 113-8602, Japan; Email: t-takizawa{at}nms.ac.jp
T Mishima and T Takizawa contributed equally to this work 
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Abstract |
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestAcknowledgementsReferences |
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22 nucleotides (nt) in length that can regulate the expression of complementary mRNA targets (Bartel 2004, Meister & Tuschl 2004). Since the first miRNA, Lin-4, was identified (Lee et al. 1993, Wightman et al. 1993), more than 800 miRNAs have been discovered in animals by using various experimental approaches (e.g., forward genetic methods and sequencing of small RNA libraries), computational predictions, or combined strategies (Berezikov et al. 2006). Although the functions of miRNAs in animals are largely unknown, some are believed to regulate tissue differentiation and the maintenance of tissue identity (Ambros 2004, Wienholds & Plasterk 2005, Kloosterman & Plasterk 2006). Recent evidence also suggests that miRNAs exhibit tissue-specific effects during vertebrate development (Wienholds & Plasterk 2005). Ason et al. (2006) compared miRNA expression among various vertebrate species by in situ hybridization. Their results indicate that the timing and location of miRNA expression are not strictly conserved; instead, miRNA expression may depend on the particular structure and function that is needed. The patterns of gene expression in meiotic and haploid germ cells are repressed by post-transcriptional control (Eddy 1998, Kleene 2001, Grimes 2004). This is partly due to sequestration of mRNAs in translationally inactive free-messenger ribonucleoprotein particles (Eddy & O'Brien 1998). However, recent studies indicate that miRNA is also involved in post-transcriptional repression during spermatogenesis. Yu et al. (2005) reported that Mirn122a down-regulates the expression of transition protein 2 mRNA by mRNA cleavage in the mammalian testis. Moreover, Kotaja et al. (2006) have found that the chromatoid body, a perinuclear cytoplasmic cloud-like structure, in male germ cells serves like a somatic glycine-tryptophan body (GW-body), also known as a mammalian processing body (P-body), which is a cytoplasmic focus involved in the post-transcriptional regulation of gene expression. These findings stimulate us to further studies of post-transcriptional small RNA pathways involved in the reproductive system. Analysis of the expression profiles of miRNAs in reproductive tissues of interest and subsequent identification of tissue-specific miRNAs is the first step toward understanding the biological functions of these molecules. Cloning of miRNAs has contributed greatly to an accelerated advance in miRNA profiling (Lau et al. 2001, Lee & Ambros 2001, Lagos-Quintana et al. 2002), but the total number of clones identified for mouse testis and ovary was relatively small in most previous studies (Yu et al. 2005, Takada et al. 2006, Watanabe et al. 2006, Ro et al. 2007a, 2007b). Here, we sequenced more than 20 000 small RNAs from adult mouse testis and ovary to produce a miRNA expression profile of each reproductive organ and revealed their differences in terms of gender.
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Results |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestAcknowledgementsReferences |
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We also examined paired miRNAs that are the 5'- and 3'-strand miRNAs derived from the same pre-miRNAs. In this small RNA library sequencing analysis, 55 paired miRNA genes (stem-loop sequences) were detected in moue gonads (Table 2). Paired miRNA genes with the ratio of 5'/3'-strand clone count ranging from 1:5 to 5:1 (i.e., 0.2–5.0) were 42 and 34% of all paired miRNA genes in testis and ovary respectively. In these paired miRNA genes, most, if not all, 5'- and 3'-strand miRNAs were almost evenly cloned at least either in testis or in ovary. The other paired miRNA genes were cloned preferentially either 5'-strand or 3'-strand, without sex dependence. Interestingly, four paired miRNA genes (Let7d, Mirn22, Mirn126, and Mirn425) showed sex-dependent expression (Table 2 and Supplementary Table 1). For example, cloning frequency for Let7d (5'-strand) were three times more abundant than that of Let7d* (3'-strand) in testis. On the other hand, Let7d* were four times more abundant than Let7d in ovary. We also found that 12 miRNAs (Let7e-3p, Mirn16-2-3p, Mirn92a-1-5p, Mirn103-2-5p, Mirn107-5p, Mirn195-3p, Mirn328-5p, Mirn350-5p, Mirn351-3p, Mirn361-3p, Mirn-449c-3p, and Mirn670-3p) cloned in this study were the sister miRNAs of the known ‘unpaired’ miRNAs (see miRNAs marked with #3 in Table 2 and Supplementary Table 3).
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G) of the miRNA/miRNA binding-site duplex) are summarized in Table 3.
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestAcknowledgementsReferences |
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miRNA profiling of adult mouse testis and ovary
The most highly cloned miRNA in both testis (13.2%) and ovary (11.7%) was Mirn125b. The expression of Mirn125 in developing mouse embryos and in adult mouse organs has been extensively studied (Lagos-Quintana et al. 2002, Miska et al. 2004, Kloosterman et al. 2006, Takada et al. 2006). These reports indicate that Mirn125 is expressed mainly in the CNS throughout mouse development. We confirmed the ovary-enriched expression of Mirn125b in adult mouse by real-time PCR (Fig. 2). Several researchers cloned miRNAs from adult mouse testis and ovary (Lagos-Quintana et al. 2002, Yu et al. 2005, Takada et al. 2006, Watanabe et al. 2006, Ro et al. 2007a, 2007b). However, the total number of clones identified for each organ was relatively small in those studies. The focus was likely on the discovery of novel miRNAs rather than the organ-specific profiling of miRNA expression. Landgraf et al. (2007) showed an excellent atlas of mammalian miRNA expression including that of mouse testis and ovary by cloning. By comparison between their data and ours, Mirn15b, Mirn16, Mirn21, Mirn29b, Mirn34c, Mirn143, Mirn191, Mirn449, and Mirn741 in testis and Mirn26a, Mirn143, and Mirn322-5p in ovary are common highly cloned miRNAs that are greater than 1% of the entire miRNA clone population. However, there are also differences between the expression profiles obtained by their methods and ours (compare Supplementary Table 1 in this study with Table S6 in Landgraf et al. 2007). These differences may be explained by considering the total number of miRNA clones included in each analysis. They obtained 3075 and 1217 miRNA clones from mouse testis and ovary respectively. Another possible explanation for differences in miRNA profiles between their data and ours is a systematic bias in cloning methods, as indicated by Landgraf et al. (2007). Recently, Ro et al. (2007a) reported PCR-based expression profiles of 122 miRNAs cloned from 15 mouse tissues and two purified spermatogenic cell types using PCR. They found that 24 known miRNAs were preferentially expressed in testis, and that one known miRNA, Mirn469, was exclusively detected in testis. By comparison between our data and those by Ro et al., 6 of the 14 testis-exclusive miRNAs shown in Table 1 in this study (i.e., Mirn465, Mirn468, Mirn470, Mirn470*, Mirn471, and Mirn741) are the miRNAs that were expressed preferentially in testis in their PCR-based study. Yan et al. (2007) observed differential miRNA expression between neonatal (1-week-old) and adult (7-week-old) mouse testes by miRNA microarray analysis. Five miRNAs (Mirn29, Mirn34a, Mirn34b, Mirn34c, and Mirn449) were downregulated in the neonatal mice. Their results are in good agreement with our findings that these miRNAs were highly cloned from adult mouse testis (see Supplementary Table 1). It should be noted that the miRNAs that were cloned only from testis (i.e., testis-exclusive miRNAs) were preferentially derived from the X-chromosome (see Table 1 and Supplementary Table 3). These results are consistent with earlier findings (Landgraf et al. 2007, Ro et al. 2007a). These X-linked, testis-exclusive miRNAs may play specific roles related to spermatogenesis and meiotic sex-chromosome inactivation, as suggested previously (Ro et al. 2007a). In addition, despite great endeavors to sequence several thousands of miRNA clones form testis, no miRNA genes were detected on the Y-chromosome. The Y-chromosome may encode few, if any, miRNA genes.
It has been considered that the thermodynamic stability of the 5'-strand and the 3'-strand in the stem-loop structure of a pre-miRNA is important for preferential selection of the less stable one (designated as the miRNA or guide strand) and obliteration of the other one (designated as the miRNA* or passenger strand; Schwarz et al. 2003). However, Ro et al. (2007a, 2007c) recently reported that the strand selection in pre-miRNAs occurs in a tissue-dependent manner. In certain mouse tissues including the testis, most, if not all, of miRNAs were evenly co-expressed as sister pairs (5'- and 3'-strand miRNAs), whereas in some other tissues either the 5'-strand miRNA or the 3'-strand miRNA were detectable. In the present study, some paired miRNA genes showed the guide strand-biased cloning profiles without sex dependence, others displayed co-cloning profiles or sex-dependent profiles (see Table 2). Although some, but not many, paired miRNAs may not be equally co-expressed in testis and ovary, it is likely that tissue-dependent strand selection occurs in vivo.
Novel miRNAs
In this study, we discovered four different novel miRNA genes using the minimum, operational criteria for miRNA identification (Ambros et al. 2003, Berezikov et al. 2006) followed by analysis of small RNAs associated with Ago2. Ago2 is one of the main components of RNA-induced silencing complex (Hutvagner & Simard 2008). Although it might be hereafter hard to find novel miRNAs that are expressed in low quantities, Ago2-immunoprecipitation would be valuable for discovery of novel miRNAs as well as for profiling of the tissue-specific miRNA expression.
Concluding remarks
We produced detailed miRNA expression profiles for adult mouse testis and ovary by small RNA library sequencing. Acquisition of cDNA clones as many as possible should be critical for the tissue-specific profiling of miRNA expression based on this type of analysis, as mentioned above. Although target mRNAs for the miRNAs revealed in this study are computationally predicted, identification of functional target mRNAs in reproductive organs remains to be elucidated. The miRNA expression profiles presented in this study would provide important information resources for facilitating studies of the reproductive organ-specific functions of miRNAs.
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Materials and Methods |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestAcknowledgementsReferences |
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miRNA cloning
We cloned small RNA by the miRNA cloning protocol of Lagos-Quintana et al. (2002). Briefly, 50 µg of total RNA from mouse testis and ovary were used. Linker ligation, cDNA synthesis, and PCR-amplification of cDNAs were carried out. Then, we concatenated more than 20 cDNAs into a single fragment using a BanI restriction enzyme (New England Biolabs, Ipswich, MA, USA), a DNA ligation kit ver. 2.1 (Takara Bio, Shiga, Japan), and a Geneclean III kit (Qbiogene, Irvine, CA, USA) prior to TA cloning. The concatenated products were then inserted into plasmids and sequenced. Sequencing and the following bioinformatics analysis were supported by Takara Bio DragonGenomics Center (Yokkaichi, Japan).
It was important to avoid contamination from other samples and molecular-weight makers during electrophoresis. Such contaminants considerably diminished the accuracy and efficiency of miRNA cloning. We avoided contamination by performing the cloning procedure separately for each sample, by using a special gel with a small plastic rod that divided the sample and marker lanes, and by using separate vats for each gel for ethidium bromide staining. We made small RNA libraries by excising a portion of a polyacrylamide gel containing species 18–24 nt in length to avoid contaminating our purified RNAs with piRNAs (Kim 2006).
Bioinformatic analysis of the sequence data
We performed a homology search for all cloned small RNAs and a secondary structural analysis for all novel miRNA candidates.
Step 1: extraction of the target sequences
Using Paracel Filtering Package software (Pasadena, CA, USA), the vector sequence, the 5' and 3' linkers, and their coupled sequences (CTGTAGGCACCTGAAA) were removed. Those extracted sequences composed of 16–30 nt were defined as valid small RNAs and were subjected to step 2.
Step 2: comparing the sequences of the clones with those of known RNAs
The small RNA sequences from step 1 were analyzed for homology with known RNAs and mouse genomic DNA sequences, including miRNA (mouse and non-mouse), piRNAs, rRNAs, tRNAs, snRNAs, snoRNAs, mRNA, and genomic DNA. The databases used were: miRNA (mature and pre), Sanger Data Base version 9.1 (http://microrna.sanger.ac.uk/sequences/index.shtml); piRNA, NCBI Entrez Nucleotide database (http://www.ncbi.nlm.nih.gov/entrez/); rRNA, the European rRNA database (http://bioinformatics.psb.ugent.be/webtools/rRNA/); tRNA, the Genomic tRNA database (http://lowelab.ucsc.edu/GtRNAdb/); sn/snoRNA, RNAdb (http://research.imb.uq.edu.au/rnadb/); and NONCODE (http://www.noncode.org); mRNA, NCBI Reference Sequence Release18 (ftp://ftp.ncbi.nih.gov/refseq/); Mouse genome, UCSC Genome Bioinformatics Site (mm8, Build 36, February 2006 Assembly; http://genome.ucsc.edu). In our search, we defined the top-hit results with greater than 90% Mus musculus homology as valid if they met our criteria for sequence error, erroneous PCR amplification, and 3', 5' variation.
Those clones with 100% homology to mouse genomic DNA but that did not match with known RNAs were subjected to step 3. The cloned small RNAs were compared with the above databases on 31 March 2007. Subsequent additions and changes to these databases are not reflected in our analysis.
Step 3: secondary structural analysis
The two-dimensional pre-miRNA configurations of our novel miRNA candidates were predicted as per Mineno et al. (2006). Briefly, 198 nt of genomic sequence were added to the candidate sequences (88 nt at each end). Each candidate sequence was divided into 110-nt windows and subjected to two-dimensional analysis along its entire length, using RNAfold software from the Vienna RNA Secondary Structure Package (Hofacker 2003). Those configurations with the least free energy and that met the following criteria were termed novel miRNA candidates: i) contains a stem-loop configuration, ii) cloned mature miRNA sequence portion consists of more than 16 nt in its double-stranded region, iii) the loop is less than 20 nt long, iv) the internal loop is less than 10 nt long, and v) the bulge is less than 5 nt long. Furthermore, novel sequences with overlapping positions in the genome were grouped together. The remaining candidates were then subjected to PCR analysis described below.
Real-time PCR analysis of known miRNAs
Real-time PCR was performed on an ABI7300 (Applied Biosystems, Foster City, CA, USA) using various mirVana qRT-PCR primer sets (Ambion, Austin, TX, USA) and a SYBR ExScript RT-PCR kit (Takara Bio), or with TaqMan miRNAs assays (Applied Biosystems), a High capacity cDNA archive kit (Applied Biosystems), and Absolute QPCR ROX mix (Abgene, Rochester, NY, USA), according to the manufacturers' instructions. As an endogenous control, 5SrRNA or U6 snRNA was used. The primers used for Let7d* (catalog number: 30208), Mirn15b (30061), Mirn21 (30102), Mirn34a (30168), Mirn99a (30205), Mirn125b (30022), Mirn145 (30047), Mirn191 (30079), Mirn199b (30090), Mirn351 (30266), and 5SrRNA (30302) were purchased from Ambion. The primers used for Mirn124a (part number: 4373295), Mirn204a (4373313), Mirn542-5p (4378110), and U6 snRNA (4373381) were from Applied Biosystems.
Ago2-immunoprecipitation and PCR analysis of novel miRNAs
After bioinformatic analysis of the sequence data, we further validated novel miRNAs by using a combination of Ago2-immunoprecipitation (Azuma-Mukai et al. 2008) followed by PCR-based miRNA detection (Ro et al. 2006). Briefly, 50 µl Dynabeads protein G slurry (Invitrogen) was immobilized with 20 µg mouse anti-mouse Ago2 monoclonal antibody (clone 2D4, Wako Pure Chemical Industries, Osaka, Japan). One hundred fifty micrograms of adult mouse testis were homogenized in 1.5 ml of a cell lysis solution (provided in miRNAs isolation kit, Wako) using a Polytron PT1200C homogenizer (Kinematica AG, Lucerne, Switzerland) for 10 s at 4 °C, and then 1.5 ml of the cell lysis solution was added into the homogenized solution. Following incubation for 15 min on ice, testis lysate was centrifuged at 20 000 g for 20 min at 4 °C and filtered through a 0.8 µm Supor Acrodisc syringe filter (Pall Corporation, Ann Arbor, MI, USA). One milliliter of the filtered lysate was incubated with 25 µl of the anti-Ago2-Dynabead protein G for incubation for 60 min at 4 °C. After immunoprecipitation, Ago2-associated RNAs were isolated from the immunoprecipitate according to the manufacture's protocol (Wako). We confirmed that the immunoprecipitate contained mouse Ago2 protein of 100 kDa in size by western blot (data not shown). Non-immune mouse IgG (Sigma) was used as a control for Ago2-immunoprecipitation. Preparation of the cDNA library using the Ago2-associated RNAs and semi-quantitative PCR analysis of the above-mentioned novel miRNA candidates were performed, as reported previously (Ro et al. 2006). The expected cDNA sizes for mature miRNAs are 120 bp. PCR information (primer sequence, annealing temperature, and PCR cycle) is shown in Supplementary Table 4, which can be viewed online at www.reproduction-online.org/supplemental. Complimentary DNA libraries were also generated from small RNAs isolated from seven mouse organs including heart, lung, liver, kidney, spleen, testis, and ovary. The expression levels of novel miRNAs in the seven organs were examined by semi-quantitative PCR and scored as 4 for high, 3 for medium, 2 for low, 1 for little to none and 0 for none.
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Declaration of interest |
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Funding |
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Acknowledgements |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestAcknowledgementsReferences |
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Received August 13, 2008
First decision September 4, 2008
Accepted September 4, 2008
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestAcknowledgementsReferences |
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