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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2006 Aug 24;103(36):13415–13420. doi: 10.1073/pnas.0605506103

MIWI associates with translational machinery and PIWI-interacting RNAs (piRNAs) in regulating spermatogenesis

Shane T Grivna *,†, Brook Pyhtila *, Haifan Lin *,‡
PMCID: PMC1569178  PMID: 16938833

Abstract

Noncoding small RNAs have emerged as important regulators of gene expression at both transcriptional and posttranscriptional levels. Particularly, microRNA (miRNA)-mediated translational repression involving PIWI/Argonaute family proteins has been widely recognized as a novel mechanism of gene regulation. We previously reported that MIWI, a murine PIWI family member, is required for initiating spermiogenesis, a process that transforms round spermatids into mature sperm. MIWI is a cytoplasmic protein present in spermatocytes and round spermatids, and it is required for the expression of its target mRNAs involved in spermiogenesis. Most recently, we discovered a class of noncoding small RNAs called PIWI-interacting RNAs (piRNAs) that are abundantly expressed during spermiogenesis in a MIWI-dependent fashion. Here, we show that MIWI associates with both piRNAs and mRNAs in cytosolic ribonucleoprotein and polysomal fractions. As polysomes increase in early spermiogenesis, MIWI increases in polysome fractions. Moreover, MIWI associates with the mRNA cap-binding complex. Interestingly, MIWI is required for the expression of not only piRNAs but also a subset of miRNAs, despite the presence of Dicer. These results suggest that MIWI has a complicated role in the biogenesis and/or maintenance of two distinct types of small RNAs. Together, our results indicate that MIWI, a PIWI subfamily protein, uses piRNA as the major, but not exclusive, binding partner, and it is associated with translational machinery.

Keywords: Argonaute, germ line, ribosomes, Dicer, cap binding

Translational regulation is a major mechanism that controls gene expression in diverse biological processes (1–4). The importance of such regulation is highlighted in the last phase of spermatogenesis, known as spermiogenesis, where translation is uncoupled from transcription as the haploid genome is condensed and repackaged (5–9). Genes involved in spermiogenesis are transcribed during earlier stages of spermatogenesis, and they are translationally repressed for several days until their protein products are needed. This translational repression has been well studied; its mechanisms include alteration of poly(A) tail length (10) and binding of particular sequences in the untranslated regions by regulatory proteins (11, 12).

Recently, microRNA (miRNA)-mediated translational repression has emerged as a distinct mechanism of posttranscriptional regulation (3, 13, 14). Although the precise nature of such regulation remains unclear, the miRNA machinery is targeted to an mRNA by target sequences nearly complementary to an ≈21-nt miRNA (15, 16). The miRNA machinery consists of the Dicer ribonuclease and the effector complex known as RISC, containing a member of the PIWI/Argonaute protein family (17, 18). Evidence is emerging that miRNAs may constitute a major mechanism of translational regulation during spermatogenesis in the mouse. Members of the PIWI protein family are essential for completion of spermatogenesis (19, 20), and miRNAs are expressed in male germ cells (21). In addition, it has been shown in vitro that transition protein 2 (TP2) mRNA, a posttranscriptionally regulated spermiogenic mRNA, is targeted by miR-122a, an miRNA expressed in late germ cells (21).

In addition to miRNAs, we and others have recently discovered a class of noncoding small RNAs termed PIWI-interacting RNAs (piRNAs) that are expressed during spermatogenesis, mostly in spermatids, at much higher levels than miRNAs (22). piRNAs are ≈30 nt in length. They tend to cluster, and they are diversely distributed among exonic, intronic, intergenic, and repeat sequences in the mouse genome, suggesting their potentially diverse roles in regulating gene expression. We also observe that a fraction of piRNAs are associated with polysomes. Interestingly, piRNAs associate with MIWI, a murine PIWI/Argonaute protein expressed exclusively in spermatogenic cells and required for initiating the spermiogeneic program (19). Moreover, piRNAs depend on MIWI for their expression (22).

To explore the functional relationship among MIWI, piRNAs, and miRNAs in spermatogenesis, we have biochemically characterized MIWI. We previously showed that MIWI is a cytoplasmic protein that binds to and maintains the level of its mRNA targets (19), some of which are known to be translationally repressed and involved in spermiogenesis (23–25). Spermatogenesis in miwi-null testes is arrested uniformly at the onset of spermiogenesis, indicating the crucial role of MIWI in initiating this process. Most recently, MIWI was shown to localize in the chromatoid body, a perinuclear structure related to the P body of somatic cells, and it was proposed to serve as an RNA storage and processing center (26, 27). Here, we report that MIWI is associated with translational machinery and piRNAs. MIWI is also required for the expression of a subset of miRNAs. These results imply a potentially complicated role for MIWI in small RNA-mediated processes during spermatogenesis.

Results

MIWI Associates with Polysomes in an RNA-Dependent Manner.

To explore the biochemical function of MIWI, we first examined the subcytoplasmic localization of MIWI in spermatogenic cells by immunofluorescence microscopy. In spermatocytes, MIWI is present throughout the cytoplasm (Fig. 1A). In round spermatids, MIWI concentrates in the chromatoid body as reported by Kotaja et al. (27), but it is also present throughout the cytoplasm (Fig. 1A). The presence of MIWI outside the chromatoid body implies an additional function for the protein beyond its proposed role in RNA storage and/or processing.

Fig. 1.

Fig. 1.

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MIWI is present in both the chromatoid body and the cytosol. (A) Cross-section of a 24-days postpartum (dpp) seminiferous tubule costained for MIWI (red) and DNA (green). In a seminiferous tubule, spermatogonia (Sg) contain DAPI-intense nuclei, and they reside in the basal layer where MIWI expression is absent. Interspersed among the spermatogonia are a small number of somatic supporting cells called Sertoli cells (St), which also reside in the basal layer. The nuclei of Sertoli cells show diffuse DAPI staining but contain two distinct nucleoli (DAPI bright spots). Spermatogonia divide to produce primary spermatocytes (Sc) that contain the largest nuclei and reside mostly in the two to three subbasal layers. These primary spermatocytes are at the prophase of meiosis I. Completion of meiosis then generates round spermatids (Sd) that contain small nuclei and reside in the luminal layers. The round spermatids each contain a chromatoid body that accumulates a high concentration of MIWI (arrowheads). Round spermatids then undergo spermiogenesis to produce mature sperm (not shown). Surrounding the tubule are thin myoid cells (M), which are separated from the tubule proper by basal lamina (not shown). (Scale bar, 20 μm.) (B) Membrane-flotation assay. Testicular extract was centrifuged on a sucrose step gradient for isopycnic separation of membranes from cytosol. rER, rough endoplasmic reticulum. (C) Western blots of individual sedimentation fractions probed for MIWI and TRAPα. RNA was isolated from 20% of each fraction.

Because the chromatoid body is a nonmembranous structure, to determine whether cytoplasmic MIWI is associated with membranes such as endoplasmic reticulum, we subjected testicular extract to a membrane-flotation assay to separate membranous and cytosolic fractions (illustrated in Fig. 1B). MIWI does not cofractionate with membranes, as marked by TRAPα, an endoplasmic reticulum integral membrane protein (fractions 2 and 7, Fig. 1C); but rather, it fractionates to the membrane-free cytosol (fractions 3–6, Fig. 1C), suggesting that cytosolic MIWI does not associate with membranes.

Because some MIWI target mRNAs are known to be translationally regulated, we wanted to determine whether MIWI plays a role in translation. Two Argonaute subfamily proteins, human eIF2C2 and trypanosome TbAgo, are known to associate with polysomes (28, 29). To determine whether MIWI, a PIWI subfamily protein, also associates with polysomes, we subjected adult testicular extracts to sucrose density gradient fractionation. MIWI cosediments with both free ribonucleoproteins (RNPs) and polysomes (Fig. 2A). The addition of EDTA to dissociate ribosomal subunits results in a shift of MIWI to subunit fractions of the gradient, suggesting that a portion of MIWI associates with polysomes. To ensure that MIWI is not in an EDTA-sensitive complex that cosediments with polysomes, we digested testicular extracts with micrococcal nuclease before sedimentation to cleave mRNAs between ribosomes. This treatment results in a complete shift of MIWI to the RNP fractions, away from ribosomal subunits. This result not only confirms the association of MIWI with polysomes, it also reveals that the association of MIWI with ribosomes is mRNA-dependent.

Fig. 2.

Fig. 2.

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MIWI associates with mRNA in both RNPs and polysomes. (A) Untreated (+Mg) and EDTA-treated (+EDTA) or micrococcal nuclease-treated (+MNase) postnuclear testicular extract was fractionated on 15–50% sucrose density gradients and analyzed by UV spectrometry and Western blotting. RNA was isolated from 20% of every other fraction. Five percent of each fraction was immunoblotted for MIWI. (B) RNP and polysome fractions from sucrose gradients were subjected to RNP-capture assay with oligo(dT)-cellulose. Poly(A) sequences were added as a competitor. Twenty-five percent of the input and bound proteins were immunoblotted for MIWI, MSY2, and glycogen synthase kinase 3β (GSK3β).

MIWI Associates with Translationally Inactive mRNAs.

To determine whether MIWI associates with mRNAs in the RNP fractions, we performed an RNP capture assay. RNP and polysomal fractions from sucrose gradients were incubated with oligo(dT)-cellulose to capture poly(A)+ RNA and associated proteins (see Materials and Methods). Both RNP- and polysome-associated MIWI bind to oligo(dT) (Fig. 2B), as does the germ-cell RNA-binding protein MSY2 (30). In contrast, the non-RNA-binding cytosolic protein GSK3β does not bind to oligo(dT). Furthermore, preincubation of the oligo(dT) matrix with poly(A) significantly reduces both MIWI and MSY2 binding in both RNP and polysome fractions, indicating that MIWI binds oligo(dT) through associated poly(A)+ RNAs. Thus, MIWI is associated with mRNAs in both polysomal and RNP fractions.

Global Translation Profile Is Not Altered inmiwi−/− Testes.

Because MIWI associates with polysomes, we examined whether MIWI regulates global translation in spermatogenic cells or plays a unique role in translational regulation. MIWI is first detectable in mid-pachytene spermatocytes, and it becomes much more abundant in diplotene spermatocytes (19). This enhanced expression persists in meiotic spermatocytes and steps 1–3 round spermatids. We therefore compared the polysome profiles of age-matched miwi+/− and miwi−/− testicular extracts isolated at three key time points of MIWI expression during the first wave of spermatogenesis: (i) at 16 dpp, 2 days after MIWI expression begins, when only spermatogonia and primary spermatocytes have formed (31); (ii) at 20 dpp, when MIWI is abundantly expressed in diplotene to secondary spermatocytes, before round spermatid formation; (iii) at 24 dpp, when abundant MIWI expression extends to newly formed steps 1–3 round spermatids. Twenty-four days postpartum is also the age at which spermatogenesis is arrested in miwi mutants (19). Importantly, at all three ages, the equivalent complement of spermatogenic cells is present in both genotypes. The polysome profiles of miwi+/− and miwi−/− testes at all three times are identical (Fig. 3 A–C), suggesting that global translation is not affected in miwi mutants.

Fig. 3.

Fig. 3.

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MIWI association with polysomes corresponds to increased translation during spermatogenesis. (A–C) Postnuclear testicular extracts from miwi+/− and miwi−/− mice at 16 dpp (A), 20 dpp (B), and 24 dpp (C) were fractionated on sucrose density gradients and analyzed by UV spectrometry. Numbers on the left in each panel correspond to A260 maximum and minimum. Five percent of every other fraction was immunoblotted for MIWI, which is shown below the corresponding A260 profile. (D) MIWI expression at 16, 20, and 24 dpp, normalized against CDK5 and set with the expression level at 16 dpp as 1. (E) Effect of cycloheximide (CHX) on polysome profile and MIWI distribution in cultured seminiferous tubules. (F) Immunoblots of fractions from C probed for MIWI, mouse vasa homolog (MVH), and GW182.

The absorbance profiles (Fig. 3 A–C) also reveal a significant increase in both the abundance of polysomes and MIWI association with polysomes at 24 dpp, when round spermatids have formed. To ensure that the increased association of MIWI with polysomes at 24 dpp is not simply the result of increased MIWI expression, we examined MIWI levels in 16-, 20-, and 24-dpp testes, relative to CDK5 (Fig. 3D). MIWI levels increase ≈2-fold from 16 to 20 dpp; however, they remain steady through 24 dpp. Thus, more MIWI becomes associated with mRNAs on polysomes as translational activity increases.

To determine whether MIWI associates with elongating polysomes, we cultured seminiferous tubules under conditions allowing active translation in germ cells (32). Cultures were treated with an elongation inhibitor, cycloheximide, and they were subjected to sucrose density fractionation. As expected, cycloheximide-treated tubules show a decrease in the monosomal peak and a corresponding increase in the polysomal peak compared with untreated tubules (Fig. 3E). Interestingly, MIWI distribution correspondingly shifts to heavier polysomal fractions (Fig. 3E). The accumulation of polysomes after cycloheximide treatment is likely caused by a reduced elongation rate, reduced premature termination, or both (32–34). In any case, our results suggest that MIWI associates with actively elongating polysomes.

The RNP Fraction Contains Chromatoid Body Components.

Because a large portion of MIWI sediments to the RNP fractions of sucrose gradients, we hypothesized that the RNP fractions contain the dissociated elements of the chromatoid body. To test this possibility, we examined the distribution of MVH and GW182 in 24-dpp sucrose gradients from Fig. 3C. MHV is present in the cytoplasm of spermatocytes, and it is concentrated in the chromatoid body of round spermatids (35, 36), whereas GW182, a P body-specific component, also localizes in the chromatoid body (27, 37). In our experiments, MVH and GW182 sediment predominantly in the RNP fractions (Fig. 3F), suggesting that chromatoid body components sediment in the RNP fractions. Hence, it is likely that chromatoid body-associated MIWI also sediments in the RNP fractions. However, we cannot conclude whether a portion of cytosolic MIWI also sediments in the RNP fractions.

MIWI Complexes with piRNAs.

Although the roles of mammalian Argonaute subfamily proteins in RNAi and miRNA pathways are well documented, the roles of PIWI subfamily members have not yet been determined (13, 14). We have recently shown that MIWI complexes with piRNAs and that a fraction of piRNAs cosediment with polysomes (22). To investigate whether MIWI associates with piRNAs on polysomes, we immunoprecipitated MIWI from three pools of testicular extract created by combining sucrose gradient fractions corresponding to the messenger ribopucleoprotein (mRNP), monosomal, and polysomal fractions. Control precipitations were performed with preimmune serum. Coprecipitated RNA was end-labeled and separated on denaturing polyacrylamide gels. Small RNA species of expected piRNA size (≈30 nt) specifically coprecipitated with MIWI in all three pools, suggesting that MIWI complexes with piRNAs in RNP, monosomal, and polysomal fractions (Fig. 4A).

Fig. 4.

Fig. 4.

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MIWI complexes with piRNAs and Dicer, and it is required for the expression of a subset of miRNAs. (A) Gradient fractions from untreated extract (+Mg) in Fig. 2 were combined into RNP, monosomal, and polysomal pools. Immunoprecipitation from each pool was performed with MIWI antibody (αMW) or its preimmune serum (Pre). Coprecipitated RNA was isolated, end-labeled, and separated by 6 M urea/15% PAGE. piRNAs (arrowhead) were specifically pulled down with anti-MIWI from each pool. Lane M, 10-bp DNA ladder, which migrates 10% faster than RNA molecules that contain the same number of nucleotides. (B) Northern blots of miRNAs in miwi−/− testes. RNA isolated from the polysomal pool of fractionated 24-dpp miwi+/− and miwi−/− testes and 20 μg of total RNA from 24-dpp miwi+/− and miwi−/− testes were probed for the indicated miRNAs, with tRNA as a loading control. (C) Northern blots of total RNA from 16- and 24-dpp miwi+/− and miwi−/− testes probed for the indicated miRNAs, with tRNA as a loading control. (D) Immunoprecipitation (IP) of MIWI complexes from adult testicular extract by MIWI antibody or its preimmune serum. Precipitated protein was immunoblotted (IB) for MIWI (arrow) and Dicer. Arrowhead, IgG heavy chain. (E) Western blots of testicular extracts from 24-dpp miwi+/− and miwi−/− mice probed for MIWI and Dicer.

A Subset of Testes-Expressed miRNAs Is Down-Regulated in miwi−/− Mice.

Before the discovery of piRNAs, testis-expressed miRNAs were identified and characterized, some of which are present in late spermatogenic cells (21, 38). Because MIWI is required for piRNA expression, we examined whether MIWI is also required for miRNA expression. Northern blots of total and polysome-associated RNA from 24-dpp miwi+/− and miwi−/− testes reveal that a subset of the known testis-expressed miRNAs, including miR-469, DQ020191, DQ020197, and DQ020201, was absent in miwi−/− testes, whereas many others (e.g., miR-16) are unaffected (Fig. 4B and data not shown). In addition, all miRNAs tested associate with polysomes. Therefore, in addition to piRNAs, MIWI may be required for the function of a subset of miRNAs.

We next examined whether MIWI and its affected miRNAs share overlapping expression patterns. Northern blots of 16- and 24-dpp testes show that both the affected and unaffected miRNAs accumulate from 16 to 24 dpp (Fig. 4C). In miwi−/− testes, the affected miRNAs are undetectable at 16 and 24 dpp. Our data suggest that MIWI is required for the biogenesis and/or stability of a subset of miRNAs expressed in spermatocytes and spermatids.

MIWI Associates with the Ribonuclease Dicer.

It has been shown that PIWI/Argonaute proteins associate with Dicer and miRNAs (17, 39–41). In spermatids, MIWI and Dicer colocalize in the chromatoid body (27). To distinguish whether MIWI is involved in the biogenesis or stabilization of MIWI-dependent miRNAs, we first examined whether MIWI associates with Dicer. Immunoprecipitation reactions with MIWI from testicular extracts coprecipitated Dicer (Fig. 4D), indicating their association. We then determined whether the absence of MIWI-dependent miRNAs in miwi−/− testes is caused by the destabilization of Dicer. Immunoblots for Dicer from 24-dpp miwi+/− and miwi−/− testes show that Dicer levels are similar in the two genotypes (Fig. 4E). Therefore, MIWI is not required for Dicer expression or stability. Furthermore, because some miRNAs are still produced in miwi−/− testes from 16 to 24 dpp (e.g., miR-16; Fig. 4C), it is likely that Dicer is still functional in miwi mutant mice. Therefore, MIWI is likely necessary for the stabilization of a subset miRNAs (see Discussion).

MIWI Associates with the Cap-Binding Complex.

To characterize further the possible role of MIWI in translation, given the proposed 7-methyl-G-cap dependence of miRNA-mediated translational control (42, 43), we examined whether MIWI associates with the cap. We performed a cap-binding assay in which testicular extract was incubated with either immobilized 7-methyl-GTP-cap analog or protein A. MIWI binds to the cap analog matrix, as does the cap-binding protein eIF4E (Fig. 5). This binding is specific because it is not observed for GSK3β. Furthermore, none of the proteins analyzed binds to the protein A matrix. Moreover, MIWI and eIF4E were both significantly dissociated from the matrix by preincubation with free cap analog, indicating a cap-specific association. Together, these results suggest that MIWI is associated with piRNAs, mRNAs, and the mRNA cap-binding complex.

Fig. 5.

Fig. 5.

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MIWI associates with the cap-binding complex. Adult testicular extract was subjected to 7-methyl-GTP-Sepharose and protein A–Sepharose chromatography. Free cap analog was added as a competitor; 2.5% of input and 12% of bound proteins were immunoblotted for MIWI, the cap-binding protein eIF4E, and GSK3β.

Discussion

Recent advancements in small RNA research have shed light on the crucial role of PIWI/Argonaute proteins and miRNAs in diverse biological processes. Although Argonaute subfamily proteins have been implicated in siRNA-mediated mRNA degradation and epigenetic silencing as well as miRNA-mediated translational repression, the precise underlying mechanisms remain unclear (13, 14). Even less is known about the molecular processes mediated by the PIWI subfamily proteins. Recently, we have shown that MIWI binds to a class of small RNAs called piRNAs (22). In addition, MIWI and Dicer were shown to colocalize in the chromatoid body in spermatids, and they were speculated to be involved in mRNA storage and/or processing mediated by miRNAs (27). We show here that MIWI also exists outside the chromatoid body and is associated with the translational machinery. Moreover, MIWI associates with piRNAs in RNPs and on polysomes. In addition, MIWI is also required for the expression of a subset of miRNAs. Therefore, it is tempting to speculate that mammalian PIWI proteins might be involved in translational regulation, possibly through more than one type of small RNA.

Is MIWI Involved in Translational Regulation?

Current analysis indicates that various members of the Argonaute subfamily have distinct biochemical activities (44). Likewise, the siRNA and miRNA pathways are different in that the former leads to either mRNA degradation or epigenetic silencing, whereas the latter is thought to lead to translational repression (13, 14). Despite these differences, a common feature among PIWI/Argonaute proteins, as well as siRNA and miRNA pathways, is that they all negatively regulate gene expression. Our results demonstrate that MIWI becomes associated with polysomes at 20 dpp, when translation is reinitiated in late spermatocytes; and the association is significantly increased at 24 dpp, when round spermatids undergo a major phase of translation (6, 31). MIWI is also associated with both the cap-binding complex and with piRNAs on elongating polysomes, although these associations alone are not sufficient to suggest a role of MIWI in translational regulation. Further functional assay is required to conclude definitively whether MIWI regulates translation or whether piRNAs play an miRNA-like role in translational regulation.

MIWI in Chromatoid Bodies vs. Polysomes.

The presence of MIWI in both the chromatoid body and the free cytosol is consistent with a two-phase distribution of MIWI between RNP and ribosomal fractions in sucrose gradients (Fig. 2). The RNP fractions also contain MVH and GW182, two other chromatoid body components, suggesting that chromatoid body-associated MIWI likely sediments in the RNP fractions. This conclusion is supported by the observation that ribosomes are primarily excluded from the chromatoid body (27), a phenomenon that was also observed in P bodies (45). The association of mRNA with MIWI in the RNP fractions further supports the observations that the chromatoid body contains mRNAs (26) and appears to be related to the somatic P body, a structure involved in mRNA storage/degradation (27).

Does MIWI Partner with piRNA for Its Function?

An exciting finding in our study is that MIWI is associated with piRNAs and mRNAs in both RNP and ribosomal fractions. This discovery raises the possibility that MIWI partners with piRNAs for its function. Such partnership might be essential for initiating spermiogenesis because spermatogenesis in the miwi mutant is arrested at the beginning of spermiogenesis (19).

Although it is accepted that PIWI/Argonaute proteins play conserved roles in small RNA biology, it remains unclear what, if anything, distinguishes the two subfamilies functionally. Our recent study (22) and results reported here demonstrate that MIWI, a mammalian PIWI subfamily member, associates mainly, although not exclusively, with piRNAs, and it is responsible for the expression of piRNAs and a small subset of miRNAs. The association of MIWI with piRNAs on polysomes provides correlative evidence that PIWI subfamily members and piRNAs might be partners in the translational machinery.

Interestingly, a subset of miRNAs known to be expressed in late-stage spermatogenic cells is also down-regulated in miwi mice, which suggests that MIWI is also required, either directly or indirectly, for the expression of these miRNAs. We favor the possibility that MIWI is required to maintain the stability of these miRNAs because Dicer is present at normal levels and is functional in the miwi mutant. It remains possible, however, that MIWI interaction with Dicer or a Dicer-like enzyme is required for the production of piRNAs and particular miRNAs. In any case, because MIWI associates with piRNAs and mRNAs both in RNPs and on ribosomes, it is possible that piRNAs are involved in the regulation of both translation and mRNA stability control by MIWI.

Materials and Methods

Membrane-Flotation Assay.

This assay was performed as described by Lerner et al. (46), with testes from adult CD-1 mice. Fractions (0.5 ml) were collected manually, and 5% of each fraction was immunoblotted for MIWI and TRAPα. RNA was extracted from 20% of each fraction with TRIzol (Invitrogen, Carlsbad, CA).

Immunofluorescence Microscopy of MIWI.

Testes from 24-dpp miwi+/− mice were prepared and sectioned as described by Deng and Lin (19). Fixed sections were blocked in PBS supplemented with 10% cold fish skin gelatin and 10% normal goat serum for 1 h at room temperature. Sections were stained with MIWI antibody (19) and Alexa Fluor 568-labeled secondary antibody (Molecular Probes, Eugene, OR) and counterstained for DNA with DAPI.

Sucrose Density Gradient Fractionation.

Decapsulated testes from adult CD-1 mice were Dounce-homogenized in 150 mM KOAc/50 mM Hepes, pH 7.5/5 mM Mg(OAc)2/2 mM DTT/0.5% Triton X-100/1× protease inhibitors (complete EDTA-free; Roche, Indianapolis, IN)/80 units/ml RNaseOUT (Invitrogen)/0.25 M sucrose. Postnuclear supernatants were loaded onto continuous 15–50% sucrose gradients prepared in lysis buffer without detergent. Gradients were centrifuged for 3 h at 150,000 × g in an SW41 rotor (Beckman, Mountain View, CA). Fractions (0.5 ml) were collected manually, and the A260 was measured. RNA was isolated from 20% of gradient fractions with TRIzol. For EDTA-treated samples, Mg(OAc)2 in all buffers was replaced with 20 mM EDTA. For nuclease treatment, lysates were supplemented with 1 mM CaCl2 and 200 μM cycloheximide and incubated with 30 units/ml Staphylococcus micrococcal nuclease for 15 min at room temperature. Digestion was stopped with 3 mM EGTA. Five percent of each fraction was immunoblotted with MIWI antibody.

For developmental polysome analysis, testes from 16-, 20-, and 24-dpp miwi+/− and miwi−/− mice were prepared and fractionated as above. Four testes were used for 16- and 20-dpp analysis, and two were used for 24-dpp analysis. Five percent of every other fraction was immunoblotted for MIWI, MVH, or GW182.

For total-protein analysis, 50 μg of total testicular extract from 16-, 20-, and 24-dpp miwi+/− mice was probed for MIWI and CDK5 and quantified with ImageQuant TL software (GE Healthcare, Amersham, U.K.).

Seminiferous-Tubule Culture.

Seminiferous-tubule culture of single adult testes was performed as described by Kleene (32), with the following modifications. Cultures were supplemented with 0 or 200 μM cycloheximide for 2 h at 34°C in 5% CO2; they were washed in cold RPMI medium and subjected to sucrose density gradient fractionation as above. Four percent of every other fraction was immunoblotted for MIWI.

mRNP-Capture Assay.

Fractions from sucrose gradient fractions corresponding to the RNP and polysomal pools were subjected to RNP capture with oligo(dT)-cellulose. Briefly, 10 mg of oligo(dT)-cellulose (Sigma, St. Louis, MO) was blocked in 1 ml of binding buffer [50 mM Hepes, pH 7.5/150 mM KOAc/5 mM Mg(OAc)2/2 mM DTT] containing 5% BSA for 1 h at 4°C; 1.5% of four RNP fractions or 2% of 10 polysome fractions was mixed with 0.5 ml of binding buffer and 25 μl of blocked matrix. For competition assays, 200 μg of (A)25 (Integrated DNA Technologies, Coralville, IA) was added 10 min before the addition of extracts. After being rocked for 30 min at 4°C, beads were spun down and washed twice with binding buffer; bound proteins were eluted by boiling in 40 μl of 2× Laemmli buffer. Twenty-five percent of input and bound protein was immunoblotted for MIWI, MSY2, and GSK3β.

Small RNA Immunoprecipitation.

Fractions from sucrose gradients were combined to create mRNP, monosomal, and polysomal pools. One-half of each pool was combined with an equal volume of NT2 buffer (50 mM Tris, pH 7.4/150 mM NaCl/1 mM MgCl2/0.05% Nonidet P-40) and incubated with protein A–Sepharose beads coated with either anti-MIWI or preimmune serum. To recover coprecipitated RNA, 200 μl of TE (10 mM Tris/1 mM EDTA, pH 7.5)/1% SDS was added to the precipitate and heated to 95°C for 3 min, followed by phenol/chloroform extraction and isopropyl alcohol precipitation. Precipitated RNA was dephosphorylated with calf intestinal phosphatase (New England Biolabs, Ipswich, MA) and 5′-end-labeled with [γ-32P]ATP and T4 polynucleotide kinase (New England Biolabs). An end-labeled 10-bp DNA ladder (Invitrogen) was used as a marker. Labeled RNA and marker were separated on denaturing 15% polyacrylamide gels and exposed to a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

miRNA Northern Blotting.

RNA from 24-dpp mouse testes and pooled polysome fractions was extracted with TRIzol. Twenty micrograms of total testicular RNA and one testis equivalent of polysomal RNA were separated by 6 M urea/15% PAGE and transferred to a Hybond-N+ membrane (Amersham Biosciences, Piscataway, NJ). Hybridizations were performed at 42°C in hybridization buffer (1× SSC/7% SDS/20 mM Na2HPO4, pH 7.2/1× Denhardt's solution/0.1 mg/ml salmon sperm DNA). Blots were probed with radiolabeled DNA oligonucleotides (Integrated DNA Technologies) complementary to mature miRNA sequences. Probes were labeled with [γ-32P]ATP and T4 polynucleotide kinase. Blots were washed twice in 1× SSC/0.1% SDS at 42°C for 10 min and exposed a PhosphorImager. Two duplicate blots were stripped and reprobed as necessary.

Dicer-MIWI Coimmunoprecipitation.

Immunoprecipitations from 2 mg of adult testis extract were performed as described by Deng and Lin (19). Ten percent of each pellet was immunoblotted for MIWI, and the remainder was probed with Dicer antibody (Imgenex, San Diego, CA). For total-protein analysis, 200 μg and 50 μg of total testis extract from 24-dpp mice was immunoblotted for Dicer and MIWI, respectively.

Cap-Binding Assay.

Adult CD-1 testes were homogenized in binding buffer [50 mM Hepes, pH 7.4/100 mM KOAc/2 mM Mg(OAc)2/1 mM DTT/0.1% Triton X-100/1× protease inhibitors (complete EDTA-free; Roche)]. Fifty microliters of a 1:1 suspension of either 7-methyl-GTP-Sepharose 4B (Amersham Biosciences) or protein A–Sepharose 4B (Sigma) was blocked in binding buffer containing 3% BSA for 1 h at 4°C. Two milligrams of postnuclear extract was added to blocked beads in 500 μl of binding buffer for 2 h at 4°C with rocking. For competition assays, 200 μg/ml m7G(5′)ppp(5′)G cap analog (Ambion, Austin, TX) was added to the extract and incubated for 1 h at 4°C before the addition of beads. Beads were washed with binding buffer, and bound proteins were eluted by boiling in Laemmli buffer; then 2.5% of input and 12% of eluted proteins were immunoblotted for MIWI, eIF4E (Santa Cruz Biotechnology, Santa Cruz, CA), and GSK3β.

Acknowledgments

We thank Dr. C. Nicchitta for advice and help; members of the Lin and Nicchitta laboratories for stimulating discussions; Dr. N. Hecht (University of Pennsylvania, Philadelphia, PA) for MSY2 antibody; Dr. T. Noce (Mitsubishi Institute, Japan) for MVH antibody; Dr. M. Fritzler (University of Calgary, Alberta, Canada) for GW182 antibody; and Drs. B. Hogan, J. Keene, A. Fedoriw, S. Findley, and Y. Unhavaithaya as well as Ms. H. Megosh and Mr. H. Yin for valuable comments on the manuscript. This work was supported by National Institutes of Health (NIH) Grant HD42012 (to H.L.) and NIH Predoctoral Training Grant T32 CA59365 (to S.T.G).

Abbreviations

dpp

days postpartum

GSK3β

glycogen synthase kinase 3β

miRNA

microRNA

mRNP

messenger RNP

MVH

mouse vasa homolog

piRNA

PIWI-interacting RNA

RNP

ribonucleoprotein.

Footnotes

Conflict of interest statement: No conflicts declared.

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