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Center for Research on Reproduction and Women's Health, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gametogenesis, gene regulation, spermatid, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the mammalian testis, major cellular changes, including chromatin remodeling as well as axoneme and tail growth, occur as germ cells differentiate en route to becoming functional spermatozoa. In nuclei following meiosis, histones are replaced by the transition proteins (TNP1 and TNP2), which in turn are replaced by the protamines (PRM1 and PRM2). These cellular changes are strongly dependent on posttranscriptional regulatory processes because of the early termination of transcription in this haploid phase of germ cell maturation. The mRNAs encoding these nuclear proteins are part of a large group of mRNAs that are synthesized in round spermatids and stored as mature mRNAs in the cytoplasm for 3–7 days, until they become activated for translation toward the end of spermatogenesis [16, 17]. The mechanisms regulating their degradation are unknown.
To begin to define how miRNAs could be involved in the posttranscriptional regulation of gene expression in the mammalian testis, we have identified by cloning many of the miRNAs expressed in the meiotic and postmeiotic germ cells from mouse testes. Computational predictions using sequence alignment and BLASTN analysis indicate that Mirn122a, a polyribosome-bound miRNA in late-stage germ cells, shares complementarity with a highly conserved sequence in the 3'-UTR of the Tnp2 mRNA. We found that Mirn122a negatively regulates, by endonucleolytic cleavage, the expression of a reporter construct by specific base-pair interactions with a complementary binding sequence in the 3'-UTR of Tnp2 mRNA.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Total RNA from tissues of 17-day, 22-day, and adult (age, 8 wk) CD-1 mice was extracted with guanidine thiocyanate and phenol as described previously [18]. The miRNAs were isolated and cloned as described by Elbashir et al. [19]. For sequence searches and analyses, we used the Ensemble database (http://www.ensemble.org), National Center for Biotechnology Information online resources (http://www.ncbi.nlm.nih.gov), and the University of California at Santa Cruz Genome Bioinformatics web site (http://genome.ucsc.edu). All experiments using animals were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.
Annotation of miRNAs
Mouse testis miRNAs were identified using criteria such as secondary structure of miRNA precursor and Northern blot analysis [20]. Secondary structures of putative precursors of miRNAs were predicted by assessing the folding of the 5'- and 3'-flanking region genomic sequences of each miRNA using the mfold web server (http://www.bioinfo.rpi.edu/applications/mfold/old/rna/). Novel miRNAs have been submitted to GenBank and the miRNA Registry web site (http://www.sanger.ac.uk/Software/Rfam/mirna) for official annotation [21].
Northern Blot Analysis
Total RNAs (20 µg/lane) were loaded on a 15% denaturing polyacrylamide gel and electophoresed at 200 V until the bromophenol blue approached the bottom. The RNA was transferred from the gel to Hybond-N+ membrane (Amersham, Piscataway, NJ) using a Semi-Dry Transfer Apparatus (Bio-Rad Laboratories, Hercules, CA). The DNA oligonucleotide probes (20–26 nucleotides [nt]) were 5'-end labeled with [32P]
-ATP, and hybridization was carried out using Rapid-Hyb buffer following the manufacturer's instructions (Amersham, Piscataway, NJ). Sequences of each probe were complementary to the corresponding miRNA sequence.
Plasmid Constructs, Cell Culture, Transfection, and Luciferase Assays
The 3'-UTR sequences containing putative binding sites for miRNAs were inserted into the XbaI-FseI site immediately downstream of the stop codon in the pGL3 Firefly Luciferase reporter vector (Promega, Madison, WI). The miRNAs were synthesized as double-stranded duplexes with two nt overhangs at the 3'-ends of both strands (Dharmacon, Chicago, IL). The sequences for constructs and miRNAs used in the present study are described in the Supplemental data (available online at http://www.biolreprod.org).
The NIH 3T3 cells were cultured in 10% fetal calf serum in Dulbecco modified Eagle medium. On the day before transfection, growing cells were trypsinized and plated into 12-well plates at a density of 1 x 105 cells/well in an antibiotic-free medium. The next day, cells were cotransfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) with 0.4 µg of pGL3 plasmid construct, 0.1 µg of pRL-TK control vector (coding for Renilla luciferase), and 15 pmol of miRNA in a final volume of 1.0 ml. Twenty-four hours after transfection, firefly and Renilla luciferase activities were measured using the dual-luciferase assays (Promega).
Preparation of Ribonucleoproteins and Polysomes
Testis extracts of adult CD-1 mice were prepared, and ribonucleoprotein (RNP) and polysome fractions were separated into 17 fractions using 10–30% sucrose gradients as described previously [22]. The RNA was purified from pooled RNP (tubes 4–6) and polysomal (tubes 11–13) fractions and then analyzed by Northern blotting as described above.
Real-Time Reverse Transcription-Polymerase Chain Reaction and Ribonuclease Protection Assay
Using aliquots of cells that were assayed for luciferase activity, total RNA was extracted with Trizol reagent (Invitrogen) from six combined wells. For the real-time reverse transcription-polymerase chain reaction (RT-PCR) assays, the ABI 7900 HT Sequence Detection system (Applied Biosystem, Foster City, CA) was used. Primers were designed using Primer Express 1.5/Taqman Primer Design software (Applied Biosystem). One single band of predicted size was obtained. The primer sequences are described in the Supplement.
For the ribonuclease protection assays, a firefly luciferase sequence corresponding to nt 1142–1439 of the mRNA and a Renilla luciferase sequence corresponding to nt 1068–1294 were cloned into PCR 2.1-Topo vector (Invitrogen, Carlsbad, CA). Following in vitro transcription (MAXIscript; Ambion, Austin, TX), radiolabeled probes were hybridized to 15 µg of total RNA from each sample, and ribonuclease protection assays were performed following instructions in the RPA III manual (Ambion).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To start defining the population of miRNAs present in male germ cells, we isolated and randomly cloned 18- to 28-nt RNAs from adult mouse testes. To distinguish miRNAs from degraded mRNAs and rRNAs, the upstream and downstream genomic sequences of each putative miRNA were analyzed for the ability to fold into potential hairpin miRNA precursors. Based on forming the hairpin precursor structure and phylogenetic conservation of the hairpin fold, we considered some of the cloned RNAs to be miRNAs [3, 4, 20].
A total of 58 RNA clones were sequenced, of which 29 were identified as miRNAs (Table 1). Of the 29 testis miRNAs, 14 were identical to known miRNAs from mammalian tissues or cell lines, and the remaining 15 appeared to be novel. Six of the 14 known miRNAs, Mirnlet7c, Mirn15b, Mirn16, Mirn27a, Mirn143a, and Mirn201c, were reported previously to be expressed in the mammalian testis [5].
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The cloning frequency of the testicular miRNAs varied greatly. The miRNAs such as Mirn16 and Mirn34b were cloned multiple times (10 and 7 times, respectively), whereas 19 of the 29 miRNAs were cloned once (Table 1). The frequency of cloning and the robust hybridization signals for Mirn16 and Mirn34 in Northern blots suggest that both are abundant in mouse testis (Fig. 1B). Mirn16 also was expressed in brain, but it was not detectable in liver (data not shown), which is consistent with a previous technical report [23]. Mirn143a, which was reported to be undetectable in either brain or liver of mouse but to be enriched in colon [6], also was abundant in testis (Fig. 1). All the miRNAs localized to one or more sites in the mouse genome and were highly conserved in rat and human, with zero- to two-nt differences (Table 1). The miRNAs that we identified from the testis mapped to most of the mouse autosomes, and five mapped to the X chromosome.
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Testicular miRNAs Show Different Developmental Patterns of Expression
To relate testicular cell types and the temporal expression of RNAs, in situ hybridizations and assays of RNA isolated from purified germ cell types generally are used. Because this is not feasible for most miRNAs because of their low abundance, we have taken a developmental approach, analyzing miRNAs from testes of prepubertal mice undergoing the first wave of spermatogenesis. In the testes of 17-day-old mice, spermatogenesis has proceeded to meiosis, with pachytene spermatocytes present. Haploid round spermatids become abundant in 22-day-old mice, and sexually mature adult testes contain all the germ cell types [24, 25]. Using Northern blot analysis, we examined the expression patterns of the testicular miRNAs as the germ cells differentiate (Fig. 1). Twelve of the 29 miRNAs showed a relatively constant expression level in mice from 17 days of age to adulthood, suggesting that these miRNAs may be present in both germ and somatic cell types (Fig. 1A). Four miRNAs, Mirn465, Mirn100, Mirn101, and Mirn466, were enriched in prepubertal stages of mouse testicular development and decreased in adult testis, suggesting that their presence was primarily in early stage germ cells and/or in the somatic cells of the testis (Fig. 1C). Nine miRNAs, including Mirn122a and Mirn34, increased as the mice matured from 17 days to adulthood, which is consistent with expression in the postmeiotic germ cells, where many mRNAs undergo posttranscriptional regulation (Fig. 1B). The mature form of Mirn122a was detected only in adult testes (Fig. 1B, open arrow).
To establish whether the age-related increase of miRNAs shown in Figure 1B reflects a selective enrichment in later-stage germ cells, we hybridized specific probes for several of the miRNAs to total RNA prepared from the testes of wild-type and mutant Kit mice (WBB6F1/J-kit; Jackson Laboratory). The Kit mice carry a mutation causing germ cell differentiation to arrest at spermatogonia [26]. Notably, Mirn122a and Mirn34b were substantially more abundant in testes from wild-type mice, which is consistent with expression in germ cells (Fig. 2A). Quantitation of the blots failed to detect any Mirn122a in the absence of later-stage germ cells (see Kit lane), and Mirn34b was enriched approximately 16-fold in wild-type testes, which is consistent with the loss of germ cells in the Kit mice. In contrast, miRNAs such as Mirn201c and Mirn465, which show no change or decrease of hybridization intensities in adult testes (Fig. 1), were enriched several-fold in the RNA prepared from adult Kit mice (Fig. 2A).
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TNP2 Is a Target of Mirn122a
Computational and bio-informational approaches have been used to predict successfully potential mRNA targets of miRNAs [27–30]. To identify potential mRNA targets for the testicular miRNAs, we searched for complementarity to cDNAs in testis gene libraries, concentrating on mRNAs known to undergo posttranscriptional regulation in male germ cells. Four putative mRNA targets for the testicular miRNAs were identified by these criteria. The 3'-UTR of Prm2 mRNA contains two potential binding sites for Mirn201c and two for Mirn34b. The 3'-UTR of Vamp2 mRNA contains five putative binding sites for Mirn34b, and the 3'-UTRs of Tnp2 and Tnp1 mRNAs each have one potential site for Mirn122a and Mirn465, respectively. Notably, all four mRNAs are strongly expressed in testis, and PRM2, TNP1, and TNP2 are under posttranscriptional regulation [16, 17].
Of the four possible target mRNAs, the Tnp2 mRNA was especially target, because its 3'-UTR contains the critical binding site at nt 2–9 (5'–3' of the miRNA) of Mirn122a [28, 29]. This sequence in the 3'-UTR is conserved among human, mouse, and rat mRNAs, despite major differences in the open-reading frame sequences in human and mouse Tnp2 mRNAs (Fig. 3A). Moreover, Tnp2 mRNA is first detectable in step-7 spermatids and disappears by step 14 [31], suggesting an active degradation process for this mRNA in postmeiotic cells. Each of the other putative target mRNAs, however, proved to be less attractive. First, Tnp1 was excluded from analysis, because Mirn465 is threefold enriched in Kit mice, suggesting a somatic or early germ cell localization for this miRNA. Second, because of the many binding sites of Mirn201c in the firefly and Renilla luciferase reporter vectors, we could not use this assay to assess the effect of Mirn201c on Prm2 translation. Third, no specific inhibition was seen with Mirn34b in luciferase assays of a reporter gene containing the 3'-UTR of the Vamp2 mRNA, although Mirn34b was very abundant in testes of wild-type adult mice (Fig. 1) and was complementary to a sequence in the 3' UTR of Vamp2 mRNA. Thus, based on its enrichment in wild-type testes (Figs. 1 and 2) and its highly conserved sequence complementarity to the postmeiotic, translationally delayed Tnp2 mRNA (Fig. 3), we focused our efforts on Mirn122a and Tnp2 mRNA interactions.
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To test whether Mirn122a can alter the expression of Tnp2, we cloned a fragment of the 3'-UTR of the Tnp2 mRNA containing the putative miRNA-binding sequences into a firefly luciferase (RLU-1) reporter vector and cotransfected RLU-1 and a control Renilla luciferase (RLU-2) reporter vector and Mirn122a into NIH 3T3 cells (NIH 3T3 cells do not contain Mirn122a; data not shown). As negative controls, plasmids lacking the 3'-UTR of Tnp2 and an miRNA with a scrambled sequence were assayed at the same time. Quantitating the levels of the normalized luciferase activities in the presence of 15 pmol of Mirn122a, we repeatedly observed an approximately 50% decrease in luciferase activity with the construct bearing the 3'-UTR of Tnp2 mRNA (Fig. 3C). Similar results were seen with 20 pmol of Mirn122a, but higher concentrations proved to be toxic to the cells. No decreases in luciferase activity were seen when Mirn122a was replaced with a scrambled miRNA, constructs lacked the 3'-UTR of Tnp2, or constructs containing the 3'-UTR from Prm2, a sequence that does not have the Mirn122a-binding site.
To investigate the specificity of interaction between Mirn122a and the Tnp2 target mRNA sequence, we created two mutations in the Tnp2-binding site for Mirn122a. In mutant 1 (mu-1), three nt (5' at nt 3, 5, and 6 3') were altered to create mismatches with the 5'-region of Mirn122a. In mutant 2 (mu-2), three nt (5' at nt 16, 17, and 19 3') were changed to create mismatches with the 3'-region of Mirn122a (Fig. 3). The mu-1 did not decrease luciferase activity, but mu-2 did, suggesting that near-perfect complementarity between nt 2–9 (from the 5' end) of Mirn122a and its target sequence in the Tnp2 mRNA is required but that base pairing between the 3'-region of Mirn122a and its target sequence is not as critical, which is consistent with reports concerning other miRNAs [30, 32].
Mirn122a Is Predominantly on Polysomes
To determine whether the effect of Mirn122a on Tnp2 expression would be at the RNP particle or polysome level, polysomal gradients of testes extracts from sexually mature mice were prepared, and RNP and polysome fractions were pooled separately. Northern blot analysis of the fractions revealed that Mirn122a was predominantly on polysomes, whereas miRNAs, such as Mirn34b, Mirn201c, and Mirn16, were detected in both RNP and polysome fractions (Fig. 2B).
Mirn122a Degrades Its Tnp2 Target
To determine whether the loss of luciferase activity in these assays is caused by a direct translational inhibition or by degradation of the target mRNA, two approaches, real-time RT-PCR and ribonuclease protection assays, were taken. When total RNA, isolated from control and experimental aliquots of luciferase assays, was quantitated by real-time RT-PCR, Mirn122a reduced luciferase mRNA levels in the construct containing the Tnp2 target sequence by approximately 60% after normalization with 18S rRNA, whereas no luciferase mRNA decrease was seen for a control construct lacking the Tnp2 3'-UTR (Fig. 4A). A similar decrease to 46% of control levels was seen when ribonuclease protection assays were used to compare luciferase mRNAs level of Tnp2 and control constructs in the presence and absence of Mirn122a (Fig. 4B). We propose from these assays that Mirn122a could posttranscriptionally regulate Tnp2 expression in the mammalian testis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Short RNAs (e.g., miRNAs or small interfering RNAs) generally regulate gene expression by one of three mechanisms [3]. First, transcription can be inhibited by either chromatin modification or DNA methylation. Second, translation can be directly inhibited by the pairing of an miRNA to its complementary sequence in a target mRNA. Third, expression of a gene can be silenced by mRNA degradation. Although the mechanisms of miRNA action are just beginning to be understood, examples of translational repression [9–13] and mRNA degradation have been reported [14, 34]. In the present study, we found that Mirn122a inhibits Tnp2 mRNA translation in an in vitro assay by degrading the mRNA. In the presence of 15 pmol of Mirn122a, a statistically significant reduction of both the transcript encoding luciferase and luciferase activity is seen (Figs. 3C and 4). Higher concentrations of Mirn122a do not reduce the relative luciferase level beyond approximately 50%. This apparent plateau for luciferase reduction may partly reflect the percentage of cells being transfected, because similar transfections with green fluorescent protein constructs reveal a 60–80% transfection rate. Alternatively, preliminary studies indicate that Mirn122a selectively binds to translin, an RNA-binding protein involved in the posttranscriptional regulation of many mRNAs transcribed by CREM-tau, including Tnp2. Based on interactions of RNA-binding proteins such as FMR1 with the RNA interference pathway [35], miRNAs such as Mirn122a in male germ cells likely would function in association with one of the many male germ cell RNA-binding proteins. Because Mirn122a is predominantly localized to polyribosomes (Fig. 2B), Mirn122a would serve as a polysomal regulator of its target genes during spermatogenesis. This finding is consistent with those of miRNA studies concerning neurons, in which all the miRNAs tested cofractionated with polyribosomes and were inferred to mediate the posttranscriptional control of target mRNA at the polyribosome level [36]. In Caenorhabditis elegans, Mirnlin4 and its target Lin14 mRNA both associate with polyribosomes, even after Mirnlin4 downregulates the translation of Lin14 mRNA, suggesting that the repression occurs after the initiation of translation [37]. Moreover, its presence on polyribosomes and the ability of Mirn122a to degrade one of its target mRNAs (Fig. 4) raises the possibility that miRNAs may facilitate turnover of some highly stable germ cell transcripts in the postmeiotic spermatids.
Considering the large number of transcripts requiring posttranscriptional regulation in male germ cells [38], miRNAs such as Mirn122a likely have numerous target mRNAs and multiple functions. The mRNA encoding the high-affinity cationic amino acid transporter, SLC7A1, may be a second target, because Mirn122a has been demonstrated to downregulate translation of SLC7A1 in liver [34] and RT-PCR assays have revealed that Slc7a1 mRNA also is detected in mouse meiotic and postmeiotic male germ cells (data not shown). Our in vitro assays suggest one possible function for only one miRNA in male germ cells. Defining the targets and mechanisms whereby miRNAs, likely in association with many of the numerous RNA-interacting testicular proteins, modulate posttranscriptional gene expression in the testis promises to reveal a new level of cellular regulation in gamete differentiation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Norman B. Hecht, University of Pennsylvania School of Medicine, 1310 Biomedical Research Building II/III, 421 Curie Boulevard, Philadelphia, PA 19104. FAX: 215 573 5408; nhecht{at}mail.med.upenn.edu
Received: 14 February 2005.
First decision: 9 March 2005.
Accepted: 5 May 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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