Proc. Natl. Acad. Sci. USA Vol. 88, pp. 3584-3588, May 1991 Developmental Biology

Cytoplasmic protein binding to highly conserved sequences in the 3' untranslated region of mouse protamine 2 mRNA, a translationally regulated transcript of male germ cells (spermatogenesis/stored mRNA/gel retardation assay/UV-crosslinklig/postmeiotic genes)

YUNHEE K. KWON AND NORMAN B. HECHT* Department of Biology, Tufts University, Medford, MA 02155

Communicated by Liane B. Russell, January 22, 1991 (received for review November 22, 1990)

The expression of the protamines, the preABSTRACT dominant nuclear proteins of mammalian spermatozoa, is regulated translationally during male germ-cell development. The 3' untranslated region (UTR) of protamine 1 mRNA has been reported to control its time of translation. To understand the mechanisms controlling translation of the protamine mRNAs, we have sought to identify cis elements of the 3' UTR of protamine 2 mRNA that are recognized by cytoplasmic factors. From gel retardation assays, two sequence elements are shown to form specific RNA-protein complexes. Protein binding sites of the two complexes were determined by RNase T1 mapping, by blocking the putative binding sites with antisense oligonucleotides, and by competition assays. The sequences of these elements, located between nucleotides +537 and +572 in protamine 2 mRNA, are highly conserved among postmeiotic translationally regulated nuclear proteins of the mammalian testis. Two closely linked protein binding sites were detected. UV-crosslinking studies revealed that a protein of about 18 kDa binds to one of the conserved sequences. These data demonstrate specific protein binding to a highly conserved 3' UTR of translationally regulated testicular mRNA.

tional control (12-14). Their genes are transcribed solely in round spermatids, and their mRNAs are stored in the spermatid cytoplasm from 3 days to 7 days before being translated. The studies of Braun et al. (4) have demonstrated that in transgenic mice the translation of the transcript of a protamine 1 (Prm-1) fusion construct is controlled by the 3' UTR of Prm-1 mRNA. When the Prm-1-hGH (human growth hormone) fusion construct contains the 3' UTR of hGH, transcription and translation both occur in the round spermatid. Replacement of the hGH 3' UTR with that of Prm-1 delays translation to the time when the endogenous protamine mRNAs are translated in elongated spermatids. Thus, cis elements of the 3' UTR of Prm-1 mRNA act to delay translation of the fusion construct transcript. To understand the regulatory mechanisms controlling the translation of the protamine mRNAs during spermiogenesis, we have sought to identify essential cis-acting elements ofthe 3' UTR of Prm-2 mRNA that are recognized by cytoplasmic factors. Here, we show that conserved sequence elements in the 3' UTR of Prm-2 mRNA form specific RNA-protein complexes, and a protein of about 18 kDa binds to one of the conserved sequences.

The utilization of functional mRNAs in the cytoplasm of eukaryotic cells can be regulated by controlling the stability of individual mRNAs or by altering their ability to bind ribosomes and be translated. There is growing evidence that the 5' and 3' untranslated regions (UTRs) of mRNAs play important roles in modulating mRNA translation (1-4). One of the best-studied examples of translational regulation mediated by protein-UTR interactions involves cellular iron metabolism in eukaryotic cells (2, 3). Sequences called ironresponsive elements (IREs) have been identified within the UTRs of ferritin and the transferrin receptor mRNAs. The binding of a protein to an IRE represses translation when the IRE is located within the 5' UTR of the ferritin mRNA (7, 8). Binding of the same protein to the IREs in the 3' UTR of the transferrin receptor mRNA increases the utilization of this mRNA by inhibiting its degradation (9). During spermatogenesis, male germ cells differentiate from a population of diploid stem cells, spermatogonia, to haploid spermatozoa. The developing male germ cell undergoes meiosis and enters spermiogenesis, the haploid phase of spermatogenesis, where there are massive changes in cell structure as the round spermatid transforms into the speciesspecific shaped spermatozoon (10, 11). Since transcription ceases during midspermiogenesis in mammals, many of the spermatid and spermatozoan proteins are encoded by mRNAs that are stored as ribonucleoproteins (mRNPs). The protamines and transition proteins, structural DNA-binding proteins, are among the proteins synthesized under transla-

MATERIALS AND METHODS Preparation of Tissue Extract. S100 cytoplasmic extracts were prepared from the testes of adult male CD-1 mice by the procedure of Dignam et al. (15). Preparation of Plasmid Constructs and RNA Transcripts. The following 32P-labeled RNAs containing various lengths of the 3' UTR of Prm-2 were transcribed from pGem plasmids: (i) transcript a, 161 nucleotides (nt) consisting of 41 nt of polylinker sequence, 20 nt of coding region, and the first 100 nt of the 3' UTR of Prm-2; (ii) transcript b, 133 nt consisting of 83 nt of 3' UTR, 17 nt of poly(A)+, and 33 nt of polylinker; (iii) transcript c, 67 nt consisting of 42 nt of 3' UTR and 25 nt of polylinker; and (iv) transcript d, 84 nt consisting of 48 nt of the 3' UTR, 17 nt of poly(A)+, and 19 nt of polylinker (Fig. L4) . The 3' UTR of hGH mRNA consists of a 130-nt transcript with 92 nt of 3' UTR, 9 nt of poly(A)+, and 29 nt of polylinker. A control pGem RNA of 172 nt was prepared by transcribing the Riboprobe positive control template (Promega). Single-stranded templates containing the phage T7 promoter were used to synthesize transcripts Y and H (16). Labeled and unlabeled transcripts were generated in vitro from the above templates with SP6 or T7 RNA polymerase by using the protocol of the supplier (Promega). Abbreviations: Prm-1 and Prm-2, mouse protamines 1 and 2; UTR, untranslated region; hGH, human growth hormone; nt, nucleotide(s); RNP, ribonucleoprotein. *To whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 3584

Proc. Natl. Acad. Sci. USA 88 (1991)

Developmental Biology: Kwon and Hecht Full-length transcripts were isolated from urea/6-20% polyacrylamide gels. Assays of RNA-Protein Complexes. Binding assays were performed by a modified procedure of Leibold and Munro (7). After elution from a polyacrylamide gel, isolated RNAs were heated at 70'C for 15 min and cooled slowly to room temperature. This produced uniform secondary structures in the RNA and yielded one major RNA band in a nondenaturing polyacrylamide gel. Radiolabeled RNAs (3 x 104 cpm/0.5 ng) were incubated with 40-100 pug of S100 cytoplasmic extract in 20 mM Hepes, pH 7.6/3 mM MgCl2/40 mM KCI/2 mM dithiothreitol/5% (wt/vol) glycerol in a volume of 25 ,gl for 20 min at 23TC. The samples were then digested for 10 min at 230C with RNase T1 (0.6-1.0 unit) and incubated with heparin (5 mg/ml) for an additional 10 min at 230C. RNAprotein complexes were resolved in 4% nondenaturing polyacrylamide gels run at 16 V/cm for about 3 hr at 4°C (17). Isolation of RNA-Protein Complexes and RNase T1 Mapping. RNase T1 mapping was performed as described by Leibold and Munro (7) with the following modifications. After protein binding and RNase T1 digestion, the protected RNA fragments were isolated by electroelution from the native gel, followed by extraction with phenol and, after addition of tRNA (2.5 ,ug/ml), precipitation with ethanol. The

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FIG. 1. Formation of specific RNA-protein complexes between the 3' UTR of Prm-2 mRNA and mouse testicular cytoplasmic extracts. (A) Map of the 3' region of the Prm-2 mRNA with specific transcripts indicated (a-d). Restriction enzyme sites, the termination codon (TAA), the polyadenylylation signal (PolyA), and the start of polyadenylylation (A") are marked on the diagram. Y, H, and Z represent the conserved sequences of the 3' UTR, while Y' and Z' represent sequences homologous to Y or Z. (B) Gel retardation assays of 32P-labeled transcripts (a-d) incubated with cytoplasmic extract. Free transcript is seen in the left lane (R) of each binding assay. The right lane (E) contains RNA incubated with extract. Transcript c migrates faster after annealing, probably because of a change in secondary structure. U, upper complex; L, lower complex. The reduction of RNA amount in the RNA-protein lanes (lanes E), especially seen with the pGEM and hGH 3' UTR RNAs, is the result of the digestion of the uncomplexed RNAs by RNase T1. The digested RNA fragments of the hGH 3' UTR RNA ran off the gel.

3585

isolated RNAs were digested to completion with 10 units of RNase T1, boiled in formamide buffer, and analyzed by electrophoresis in a urea/25% polyacrylamide gel. Crosslinking of RNA-Protein Complexes. Binding reactions were performed as described above except that the amounts of RNA and RNase T1 were changed to 105 cpm/0.5 ng and 0.2-0.7 unit, respectively (7). The reaction products were irradiated on ice in a UV Stratalinker 1800 (Stratagene) with a 254-nm, 8-W UV bulb (maximum intensity, 3.6 mV/cm2) and were resolved in a SDS/12.5% polyacrylamide gel (19).

RESULTS RNA-Protein Binding of the 3' UTR of Prm-2. The 3' UTRs of mRNAs encoding mammalian protamines and transition proteins sequenced to date contain several conserved sequences. These sequences in Prm-2 mRNA are denoted Y, H, and Z (Fig. L4). To investigate the binding of cytoplasmic trans-acting factors to the Y, H, and Z sequences, 32P-labeled transcripts for subclones a, b, c, and d of the 3' UTR of Prm-2 mRNA were incubated with a S100 cytoplasmic testicular extract, and RNA-protein complexes were resolved in a nondenaturing polyacrylamide gel. RNA-protein complexes were detected with transcripts from clones a, b, and c (Fig. 1B). These complexes were not found when the protein extract was previously digested with proteinase K or heat-denatured (data not shown). No complexes were detected with transcript d, with control pGem RNA, or with the 3' UTR of hGH mRNA. To remove proteins bound nonspecifically to the RNA, heparin (Fig. 2, lane 3) or RNase T1 and heparin (Fig. 2, lane 4) were added to the incubation mixture. The concentrations of MgCI2 and KCl were adjusted for maximal RNA-protein binding. Two distinct complexes, U and L, were resolved when radiolabeled transcripts b or c were incubated with cytoplasmic extract (Fig. 1). These transcripts contain both the Y and H homology sequences of the 3' UTR of Prm-2 mRNA. The formation of both complexes was rapid, occurring within 1 min (data not shown). A small amount of RNA-protein complex with an electrophoretic mobility similar to the U complex was also formed with transcript a. It may be a result of protein binding to Y', a sequence similar to Y (Fig. 1A). To determine the specificity of the U and L complexes, RNA competition assays were performed (Fig. 2). Protein binding to radiolabeled transcript c was abolished with a 15-fold molar excess of unlabeled transcript c (Fig. 2, lane 6), whereas nonspecific competitors such as pGem RNA (Fig. 2, lanes 10-13) or yeast tRNA (data not shown) at concentrations up to 450-fold molar excess did not reduce binding. These results show that cytoplasmic proteins bind to specific sequences of the 3' UTR of Prm-2 mRNA. Identification of Protected RNA Sequences by RNase T1 Mapping. To define the regions in the 3' UTR of Prm-2 mRNA protected by cytoplasmic proteins, RNase T1 mapping was used (7). The protein-protected fragments of 32P_ labeled transcript b were isolated from the complexes, digested with RNase T1, which cleaves 3' of guanosine residues, and analyzed on 25% polyacrylamide gels. Protected RNA fragments of about 35-45 nt were detected (Fig. 3, lane 2). Digestion of control full-length transcript b with RNase T1 generated oligonucleotides of the following sizes: two of 10 nt, one of 7 nt, seven of 5 nt, five of 4 nt, four of 3 nt, and six of 2 nt (Fig. 3, lane 3). Complete RNase T1 digestion of the protein-protected fragments yielded a subset of these oligonucleotides, with the missing ones presumably not protected by protein (Fig. 3, lane 4). Densitometric analysis revealed nine missing oligonucleotides: two of 10 nt, one of 7 nt, three of 5 nt, one of 4 nt, and two of 2 nt. In the entire 133 nt of transcript b, there was only one possible 7-nt fragment

Developmental Biology: Kwon and Hecht

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Proc. Natl. Acad. Sci. USA 88 (1991)

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FIG. 2. The protein binding to the 3' UTR of Prm-2 mRNA is specific. 32P-labeled transcript c was incubated with cytoplasmic extract, followed by the sequential addition of RNase T1 and heparin. Lanes: 1, free RNA; 2, RNA and cytoplasmic extract; 3, RNA and cytoplasmic extract plus heparin; 4, RNA and cytoplasmic extract plus sequential treatment with RNase T1 and heparin. Unlabeled transcript c at a molar excess of 5- to 150-fold (lanes 5-9) or unlabeled pGem RNA at a molar excess of 15- to 450-fold (lanes 10-13) was incubated with cytoplasmic extract before the labeled transcript c was added. U, upper complex; L, lower complex.

generated by RNase Ti. It is located within the Z sequence (Fig. 4). Adjacent to this sequence are one fragment of 10 nt, one of 4 nt, and several of 5 nt. These oligonucleotides were absent in the digest generated from the protein-protected RNA fragments (Fig. 4). Since the only other 10-nt fragment is in the polylinker region of transcript b, a region that we know does not bind protein, we believe that the protected RNA fragments of transcript b contain the Y and H sequences and about 20 nt upstream of the Y sequence. RNase Ti mapping of transcript c revealed a protected region of about 42 nt also containing the Y and H sequences (data not shown). We were not able to determine the exact boundaries of the protected fragment because transcripts b and c contain many guanosine residues, resulting in a large number of oligonucleotides of 3, 4, or 5 nt after RNase Ti digestion and, more importantly, because we were mapping a mixed population of two RNA-protein complexes (U and L). We can conclude, however, that protein binds to the conserved Y and H elements but not the Z element. Determination of RNA Elements in the U and L Complexes. To identify the precise RNA binding elements of the U and L complexes, we utilized several 32P-labeled oligonucleotides for binding studies (Fig. 5A). In addition to the Y and H conserved sequences, we used Y and H elements (Y29 and H28) that also contain the 6 nt between the Y and H regions. Gel retardation assays showed that the Y29 sequence bound protein to form the U complex, whereas the H sequence formed the L complex (Fig. SB, lanes 1 and 2). Transcripts containing the Y23 or H22 sequences only formed reduced amounts of the complexes (data not shown). To confirm the RNA sequences required for the U and L complex formation, we hybridized specific antisense oligonucleotides to 32P-labeled transcript c to block putative protein binding sites (Fig. 5B). No protein complexes formed with RNA-DNA hybrids of the Prm-2 3' UTR. When transcript c was hybridized with an antisense oligonucleotide to the H sequence (aH22), only the U complex was detected (Fig. SB, lane 4). When an antisense oligonucleotide to the Y sequence (aY23) was hybridized, only the L complex was

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FIG. 3. RNase T1 mapping of Prm-2 3' untranslated RNA seprotected by protein. Protected transcript b labeled with [32P]GTP was isolated from RNA-protein complexes after the binding assay. The protected and intact transcripts b were digested completely with RNase T1 and resolved on a urea/25% polyacrylamide gel. Oligonucleotide size markers are indicated in bases. Lanes: 1, intact transcript b; 2, protected transcript b; 3, complete RNase T1 digestion of intact transcript b; 4, complete RNase T1 digestion of protected transcript b.

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seen (Fig. 5B, lane 6). When antisense H and Y oligonucleotides containing the 6 nt between the Y and H sequences (aY29, aH28) were hybridized to transcript c, the formation of both complexes was prevented (Fig. 5B, lanes S and 7). Hybridization of control sense oligonucleotides Y and H with transcript c did not affect the formation of either complex (Fig. 5B, lanes 8 and 9). In the absence of the U complex, the L complex often migrated slowly on native gels. This was seen when the L complex was formed with the H28 transcript (Fig. SB, lane 2) or when the formation of the U complex was blocked with antisense oligonucleotide aY23 (Fig. SB, lane 6). Additional support for two distinct complexes was provided by oligonucleotide competition assays (Fig. SC). When unlabeled Y29 transcript was added to the binding assay, the amount of U complex formed with transcript c was substantially diminished (Fig. 5C, lanes 5-7). When unlabeled transcript H28 was similarly used as competitor, the L complex disappeared (Fig. SC, lanes 2-4). These data indicate that the U and L RNA-protein complexes are derived from closely linked but distinct protein-binding sites. Characterization of Binding Proteins by UV-Crossling. To identify the protein components of the complexes formed with the 3' UTR of Prm-2 mRNA, we used UV-crosslinking to covalently bind proteins to labeled RNAs. Using our standard binding conditions with transcript c, we detected increasing amounts of a 32-kDa RNA-protein complex with

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Proc. Natl. Acad. Sci. USA 88 (1991)

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UV doses up to 10 min (Fig. 6, lanes 2-6). When the complexes were incubated with proteinase K before UVcrosslinking, no RNA-protein complexes were seen (Fig. 6, lane 12). The UV-crosslinked complex was also diminished by prior addition of unlabeled transcript c (Fig. 6, lanes 7 and 8) or unlabeled transcript Y29 (Fig. 6, lane 10). However, a 500-fold excess of unlabeled nonspecific pGem RNA (Fig. 6, lane 9) did not substantially reduce the amount of complex detected. Since we estimate a protected region of about 42 nt (about 14 kDa) in transcript c, the contribution of protein to the 32-kDa RNA-protein complex is about 18 kDa. This is consistent with the estimated size of the protein crosslinked to transcript Y29 forming a more rapidly migrating RNAprotein complex of about 27 kDa (Fig. 6, lanes 15 and 16). Even though transcript H28 forms the L complex, no

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DISCUSSION We have demonstrated that cytoplasmic testicular proteins bind to two conserved sequences in the 3' UTR of Prm-2 mRNA. The RNA-protein binding is specific to the 3' UTR sequences because a 15-fold excess of unlabeled 3' UTR prevents formation of the complexes and no reduction in binding is seen with nonspecific competitor RNAs. The binding protein(s) is cytoplasmic because no complexes are formed with nuclear testicular extracts (data not shown). We do not know whether cytoplasmic extracts from other tissues contain one or more similar proteins. The RNA-protein binding sites of the 3' UTR of Prm-2 mRNA are localized to two conserved sequence elements, Y and H. RNase Ti mapping reveals no complex formation with the Z sequence, a third highly conserved protamine sequence adjacent to the polyadenylylation signal, that contains a sequence present in an immunoglobulin enhancer (21). UV (min)

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FIG. 5. Determination of protein-binding sites of the U and L complexes. (A) Map ofthe 3' region of the Prm-2 mRNA with specific transcripts Y23, H22, and H28 and transcript c indicated. Y and H represent the conserved sequences. The Y and H transcripts also contain 6 nt from the T7 promoter. (B) Control RNA-protein complexes formed with Y29 (lane 1), H28 (lane 2), and transcript c (lane 3). Antisense and sense oligonucleotides were hybridized to 32P-labeled transcript c at molar ratios of 1.0-1.2, and binding assays were performed. Lanes 4-9 contain antisense oligonucleotides to H22, H28, Y23, and Y29 (aH22, aH28, aY23, and aY29) (lanes 4-7) and sense oligonucleotides to H22 and Y29 (sH22 and sY29) (lanes 8 and 9). (C) Competition assays with unlabeled H28 and Y29 transcripts. Lanes: 1, control transcript c-protein complex; 2-7, 32P-labeled transcript c (3 x 104 cpm; 0.5 ng) incubated with cytoplasmic extract in the presence of 30- to 300-fold molar excess of unlabeled H28 (lanes 2-4) or Y29 (lanes,5-7).

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FIG. 6. UV-crosslinking of proteins to transcripts from the 3' UTR of Prm-2. 32P-labeled RNA-protein complexes were formed and crosslinked with UV-irradiation and resolved by SDS/PAGE. Lanes: 1, free 32P-labeled transcript c; 2-6, transcript c-protein complexes irradiated with UV light for 0-15 min; 7-11, transcript c (0.5 ng) crosslinked for 5 min in the presence of unlabeled transcript c (lanes 7 and 8), in the presence of nonspecific competitor (lane 9), in the presence of unlabeled Y28 (lane 10), and in the presence of unlabeled H28 (lane 11); 12, proteinase K (100 ,ug/ml) incubated with the RNA-protein complex for 30 min at 37°C before UV irradiation for 5 min; 13 and 14, 32P-labeled H28 and protein UV-irradiated for 5 or 10 min; 15 and 16, 32P-labeled Y29 and protein UV-irradiated for 5 or 10 min. Molecular mass markers are indicated in kDa.

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Binding assays with Y or H transcripts reveal that the two complexes U and L are derived from two distinct binding elements, Y and H. The formation of the U complex is abolished with antisense oligonucleotides to Y23, Y29, and H28 but not to H22 alone. Formation of the L complex is blocked with antisense oligonucleotides to H22, H28, and Y29 but not to Y23 alone. These results imply that two binding sites are closely linked and likely overlap a common 6-nt sequence. Thus, the RNA-protein interactions of one element may sterically interfere with protein binding to the adjacent RNA element. Computer predictions of RNA secondary structure suggest that the protein binding sites of the U and L complexes contain a potential stem-loop structure in which the 6 nt between Y and H regions are a part of'the stem. Although we know that the U and L complexes of Prm-2 RNA contain different elements, we do not know whether more than one protein binds to the two sequences. We have

shown from UV-crosslinking that the Y element binds a protein of about 18 kDa. However, we could not detect any protein bound to the H element after UV-irradiation. We postulate that specific binding conformations of the H RNAprotein complex may not allow UV-crosslinking or that RNA-protein interaction is sufficiently weak so that little protein crosslinks under the conditions used. Our competition studies with unlabeled Y and H transcripts suggest that the U and L complexes contain different protein components, since Y or H sequences only compete with the formation of U or L complexes, respectively. However, we cannot exclude the possibility that the 18-kDa binding protein assumes different conformations as a result of posttranslational modifications or interaction with additional factors and could bind to the H element. Modulation of translation by protein binding to the 5' or 3' UTR of mRNAs has been implicated as a general cellular regulatory mechanism for many genes including ferritin (2224), transferrin receptor (9, 25), and creatine kinase (26). Although the proteins and RNA elements of the ferritin and Prm-2 mRNAs differ, comparison of the RNA-protein complexes of ferritin and Prm-2 mRNAs reveals the strikingly similar interactions with cytoplasmic proteins (7, 22, 23). Ferritin mRNA forms two complexes, B1 and B2, with similar electrophoretic mobilities and high binding affinities as we found for the U and L complexes with Prm-2 mRNA. Furthermore, UV-crosslinked protein was detected only with the slower migrating complex for either mRNA. The proteinbinding elements of ferritin mRNA are also capable of forming a stem-loop structure, which appears essential for the RNA-protein interactions. 'Whether the 18-kDa protein can inhibit translation of Prm-2 mRNA in a cell-free system as has been shown for the 90-kDa protein that binds to the ferritin B1 complex (24) is not known. Translational control and RNA-protein interactions are particularly evident in early development (1). In oocytes mRNPs are sequestered for months before moving onto polysomes. These stored mRNAs exist as mRNA-protein complexes in which the protein component seems to act as a repressor of translation (27). In trout, polysomal protamine mRNAs can be readily translated in vitro while protamine mRNPs do not translate in vitro unless treated with high salt (18). The expression of the mammalian protamines and transition proteins may be similarly regulated. Their mRNAs are transcribed during spermiogenesis and stored as mRNPs until translation days later. Upon translation, their polysomal mRNAs shorten as a result of partial deadenylylation (12, 28). We know from transgenic studies that the temporal expression of Prm-1 mRNA is controlled by its 3' UTR (4). Although the coding regions of the protamine and transition protein mRNAs differ substantially, the Y, H, and Z elements in their

Proc. Natl. Acad. Sci. USA 88 (1991) 3' UTRs share great homology among many mammalian species (5, 6, 10, 20). Not only are the sequences of these elements conserved, but their order, 5'-Y-H-Z-3', adjacent to the poly(A) addition signal, is also maintained. We have detected RNA-protein complexes that have similar mobilities to the ones presented here with Prm-1 and mouse transition protein 1 (data not shown). These interactions between the 3' UTRs and cytoplasmic proteins are likely to control translational regulation during spermiogenesis. We are grateful to Drs. C. Moore and S. Ernst for helpful discussions. We thank L. Hake and Dr. D. Bunick for encouragement and careful reading of this manuscript and Drs. E. Leibold and H. Munro for helpful advice about the gel retardation assay. We also thank Dr. H. Goodman for the kind gift of the hGH cDNA clone. This work was supported by National Institutes of Health Grant GM 29224. 1. Jackson, R. J. & Standart, N. (1990) Cell 62, 15-24. 2. Aziz, N. & Munro, H. N. (1987) Proc. Natl. Acad. Sci. USA 84, 8478-8482. 3. Hentze, M. W., Caughman, S. W., Rouault, T. A., Barriocanal, J. G., Dancis, A., Harford, J. B. & Klausner, R. D. (1987) Science 238, 1570-1573. 4. Braun, R. E., Peschon, J. J., Behringer, R. R., Brinster, R. L. & Palmiter, R. D. (1989) Genes Dev. 3, 793-802. 5. Heidaran, M. A., Kozak, C. A. & Kistler, W. S. (1989) Gene 75, 39-46. 6. Krawetz, S. A., Connor, W. & Dixon, G. H. (1988) J. Biol. Chem. 263, 321-326. 7. Leibold, E. A. & Munro, H. N. (1988) Proc. Natl. Acad. Sci. USA 85, 2171-2175. 8. Rouault, T. A., Hentze, M. W., Caughman, S. W., Harford, J. B. & Klausner, R. D. (1988) Science 241, 1207-1210. 9. Mullner, E. W., Neupert, B. & Kuhn, L. C. (1989) Cell 58, 373-382. 10. Hecht, N. B. (1990) J. Reprod. Fertil. 88, 679-693. 11. Hecht, N. B. (1986) in Experimental Approaches to Mammalian Embryonic Development, eds. Rossant, J. & Pedersen, R. (Cambridge Univ. Press, New York), pp. 151-193. 12. Kleene, K. C., Distel, R. J. & Hecht, N. B. (1984) Dev. Biol. 105, 71-79. 13. Heidaran, M. A., Showman, R. M. & Kistler, W. S. (1988) J. Cell Biol. 106, 1427-1433. 14. Yelick, P. C., Kwon, Y. K., Flynn, J. F., Borzorgzadeh, A., Kleene, K. C. & Hecht, N. B. (1989) Mol. Reprod. Dev. 1, 193-200. 15. Dignam, J. D., Lebovitz, R. M. & Roeder, R. G. (1983) Nucleic Acids Res. 11, 1475-1489. 16. Milligan, J. F., Groebe, D. R., Witherell, G. W. & Uhlenbeck, 0. C. (1987) Nucleic Acids Res. 15, 8783-8798. 17. Konarska, M. M. & Sharp, P. A. (1986) Cell 46, 845-855. 18. Sinclair, G. D. & Dixon, G. H. (1982) Biochemistry 21, 18691877. 19. Moore, C. L., Chen, J. & Whoriskey, J. (1988) EMBO J. 7, 3159-3169. 20. Maier, W. M., Adham, I., Klemm, U. & Engel, W. (1988) Nucleic Acids Res. 16, 11826. 21. Johnson, P. A., Peschon, J. J., Yelick, P. C., Palmiter, R. D. & Hecht, N. B. (1988) Biochim. Biophys. Acta 950, 45-53. 22. Barton, H. A., Eisenstein, R. S., Bomford, A. & Munro, H. N. (1990) J. Biol. Chem. 265, 7000-7008. 23. Leibold, E. A., Laudano, A. & Yu, Y. (1990) Nucleic Acids Res. 18, 1819-1824. 24. Walden, W. E., Daniels-McQueen, S., Brown, P. H., Gaffield, L., Russell, D. A., Bielser, D., Bailey, L. C. & Thach, R. E. (1988) Proc. Natl. Acad. Sci. USA 85, 9503-9507. 25. Koeller, D. M., Casey, J. L., Gerhardt, E. M., Chan, L. N., Klausner, R. D. & Harford, J. B. (1989) Proc. Natl. Acad. Sci. USA 86, 3574-3578. 26. Ch'Ng, J. L., Shoemaker, D. L., Schimmel, P. & Holmes,

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Cytoplasmic protein binding to highly conserved sequences in the 3' untranslated region of mouse protamine 2 mRNA, a translationally regulated transcript of male germ cells.

The expression of the protamines, the predominant nuclear proteins of mammalian spermatozoa, is regulated translationally during male germ-cell develo...
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