Translational activity of mouse protamine 1 messenger ribonucleoprotein particles in the reticulocyte and wheat germ cell-free translation systems

MOLECULAR REPRODUCTION AND DEVELOPMENT 37:1!2-20 (1994)

Translational Activity of Mouse Protamine 1 Messenger Ribonucleoprotein Particles in the Reticulocyte and Wheat Germ Cell-Free Translation Systems KENNETH C. KLEENE AND JEAN SMITH Department of Biology, University of Massachusetts at Boston, Boston, Massachusetts

ABSTRACT Protamine 1 mRNAs are inactivated by a block to the initiation of translation in early spermatids and are translationally active in late spermatids in mice. To determine whether translation of protamine 1 mRNAs is inhibited by a protein repressor, the translational activity of ribonucleoprotein particles and deproteinized RNAs were compared in the reticulocyte and wheat germ cell-free translation lysates. To isolate RNPs, cytoplasmic extracts of total testes were fractionated by large-pore gel filtration chromatography. Ribonucleoprotein particles in the excluded fractions stimulated synthesis of radiolabeled translation products for protamine 1 about twofold less effectively than deproteinized RNAs in the reticulocyte lysate, but were inactive in the wheat germ lysate. The ability of translationally repressed protamine 1 ribonucleoprotein particles to form initiation complexes with 80s ribosomes in the reticulocyte lysate was also measured. Protamine 1 ribonucleoprotein particles isolated by gel filtration and in unfractionated cytoplasmic extracts of early spermatids were nearly as active in forming initiation complexes as deproteinized mRNAs. The isolation of ribonucleoprotein particles in buffers of varying ionic strength, protease inhibitors, and several other variables had no major effect on the ability of protamine 1 ribonucleoprotein particles to form initiation complexes in the reticulocyte lysate. These results can be explained by artifacts in the isolation or assay of ribonucleoprotein particles or by postulating that protamine 1 mRNAs are inactivated by a mechanism that does not involve protein repressors, such as sequestration. 0 1994 Wiley-Liss, Inc.

Key Words: Spermatids, Translational regulation, mRNPs, Translational initiation

INTRODUCTION The postmeiotic phase of spermatogenesis in mam-

mals includes a series of extensive modifications of the organelles of the haploid developing male germ cells (spermatids). The end product is a highly specialized cell type, the spermatozoon. About midway through the 13 day haploid phase in mice, the nuclei begin a process of condensation, which results in the total inactivation of transcription (reviewed in Meistrich, 1989). Thus mRNAs that encode proteins utilized in remodelling

0 1994 WILEY-LISS, INC.

organelles in late spermatids are necessarily transcribed in early haploid cells (round spermatids) and are stored for a period of several days as translationally repressed messenger ribonucleoprotein particles (mRNPs), becoming translationally active at specific times in late spermatids. Examples of mRNAs that fol- low this pattern of translational regulation include mRNAs encoding basic chromosomal proteins that re- place the histones during nuclear condensation; transition proteins 1 and 2 and protamines 1 and 2; and the mitochondrial capsule selenoprotein, a constituent of the mitochondrial sheath (Kleene et al., 1984; Heidaran and Kistler, 1987; Kleene, 1989).

Mechanisms proposed to explain how the rate of translation of individual mRNAs in eukaryotic cells is controlled include the following. 1) As a well-docu- mented example of the familiar masked messenger hypothesis, the translation of ferritin mRNA is regulated by a protein repressor (reviewed in Klausner et al., 1993). 2) Translational control in Xenopus oocytes is mediated by a change in the length of the poly(A) tail, a modification of the primary structure of the mRNA (McGrew et al., 1989). 3) Large changes in the rate of translation of individual mRNAs can be achieved by interactions of mRNAs levels, a discriminatory initiation factor, and secondary structure in the 5 nontranslated region (Lawson et al., 1986). 4) Translation of histone mRNAs in sea urchin oocytes is blocked by sequestration in the pronucleus (DeLeon et al., 1983).

Relatively little information is available concerning the mechanisms that regulate the timing of translational activity of spermatidal mRNAs. Since translationally inactive spermatidal mRNAs sediment as free mRNPs (Kleene, 1989), translation is blocked a t the initiation step of translation, although there appear to be additional forms of translational control over the translationally active mRNAs in late spermatids (Kleene, 1993). In addition, the poly(A) tracts on translationally repressed spermatidal mRNAs, including

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Received April 16,1993; accepted June 10,1993. Address reprint requests to Kenneth C. Kleene, Department of Biol- ogy, University of Massachusetts at Boston, 100 Morrissey Blvd., Bos- ton, MA 02125-33943.

TRANSLATION OF PROTAMINE 1 mRNPs 13

protamine 1 mRNAs in early and middle spermatids, are 150 bases long and are homogeneous in size, whereas the poly(A) tracts on translationally active mRNAs in late spermatids are shorter and more heter- ogenous, 30-150 bases long (Kleene et al., 1984; Kleene, 1989, 1993). However, it is unclear whether poly(A) shortening is a cause or a consequence of the change in translational activity. The timing of protamine 1 mRNA translation is determined by a 62 base sequence in the 3' nontranslated region (Braun et al., 1989; Braun, 19901, and specific proteins bind to sequence elements in the 3' nontranslated region of the protamine 2 mRNA (Kwon and Hecht, 1989). The activity of a translational repressor is supported by a report that trout protamine mRNP particles are translated inefficiently (Sinclair and Dixon, 1982). High-resolution in situ hybridizations have yielded conflicting evidence regarding whether translationally repressed protamine and transition protein mRNAs are distributed homogeneously throughout the cytoplasm (Morales et al., 1991) or are sequestered in a small region of the cytoplasm (Saunders et al., 1992).

The present study was carried out to determine whether the initiation of translation of protamine 1 mRNAs with 150 base poly(A1 tracts in early and middle spermatids of mice is blocked by mRNP proteins. We isolated mRNPs from testes and purified round spermatids by gentle procedures to minimize artifactual changes in mRNP structure and compared the translational activity of mRNPs and deproteinized mRNAs in the wheat germ and reticulocyte cell-free translation systems.

MATERIALS AND METHODS Preparation and Fractionation of

Cytoplasmic Extracts Sepharose CL-6B and most chemicals were obtained

from Sigma (St. Louis, MO). The water was double deionized and double glass distilled. Buffers were prepared by dissolving the salts, autoclaving, and addition of solid HEPES and KOH to the desired pH. Dithiothre- itol was added to cold buffers just before use. CD-1 mice at least 60 days old were obtained from Charles River Laboratories (Wilmington, MA). All procedures involv- ing the preparation and manipulation of cellular extracts were carried out at 0-C.

For analysis of the distribution of protamine 1 mRNAs in the nonpolysomal and polysomal regions of sucrose gradients, two decapsulated testes were homogenized with five strokes in a prechilled motor-driven glass-Teflon homogenizer in 0.5 ml 100 mM KC1, 1.5 mM MgCl,, 20 mM HEPES (pH 7.6) containing 0.5% Triton-N101; the nuclei were removed by centrifugation at 14,OOOg for 5 min; and the cytoplasmic fraction was sedimented on a 12.5 ml 1540% sucrose gradient (wt/wt) in the same buffer for 3 hr a t 28,000 rpm in a Beckman SW40 rotor. The gradient was collected as fractions, and the RNAs were purified from each frac-

tion with guanidine isothiocyanate as described elsewhere (Kleene, 1993).

For large-pore gel filtration chromatography, four decapsulated testes were homogenized in 1 ml LSB llow-salt buffer: 40 mM KOAc, 1 mM Mg(OAc),, 20 mM HEPES, pH 7.5, 1 mM dithiothreitol] supplemented with 0.5% Triton-N101,300 unitdm1 RNasin (Promega Biotec); the nuclei were removed by centrifugation at 14,OOOg for 5 min; and the supernatant was fractionated on a 0.9 x 50 cm Sepharose CL-6B column in LSB using a flow rate of about 0.5 ml/min. The excluded fractions were identified by absorbance at 260 nm, the peak excluded fractions were pooled, and aliquots were flash frozen in 1.5 ml tubes that had been prechilled in liquid N2. The fractions were stored at -80C or -184"C, thawed, and discarded after one use.

To analyze the translational activity of RNPs in purified spermatogenic cells, round spermatids (steps 1-8) were purified by sedimentation at unit gravity on bovine serum albumin gradients (Romrell et al., 1976; Kleene, 1989). The cells were washed twice in cold RPMI 1640 medium and once in LSB and were lysed at a concentration of 1 4 x lo7 cells/100 p.1 in LSB supplemented with 0.5% Triton-N101, 300 units/ml RNasin, 500 pglml soybean trypsin inhibitor, 500 pglml ovomucoid trypsin inhibitor, 250 pg/ml leupeptin, and 250 pg/ml pepstatin. The nuclei were removed by centrifugation at 14,OOOg for 2 min, and the cytoplasmic extract was flash frozen as described above.

RNA Purification and Northern Blots RNAs were purified with phenol and chloroform, so-

dium dodecyl sulfate (SDS), and proteinase K (Kleene, 1989). Poly(A)+ mRNA for translation was purified by two cycles of chromatography on poly(U) Sepharose (Kleene and Flynn, 1987). To obtain a fraction enriched in mRNAs with short poly(A) tails, poly(A)+ mRNA was fractionated by a third cycle of thermal chromatography on poly(U) Sepharose using an elution tempera- ture of 34C (Kleene et al., 1984). RNAs were precipitated three times with ethanol and dissolved in distilled water before translation.

Protamine 1 mRNAs with poly(A) tracts of differing sizes were separated by electrophoresis for 26 hr a t 35 V in 2% agarose gels containing 2.2 M formaldehyde, transferred to nitrocellulose, and hybridized to 32P-la- beled antisense RNA (Melton et al., 1984) or DNA probes synthesized from cDNAs encoding mouse protamine 1 (Kleene et al., 1985).

Reticulocyte and Wheat Germ Cell-Free Translations

Nuclease-treated reticulocyte cell-free translation lysate was obtained from Promega Biotec and was used according to the directions of the supplier. Wheat germ was purchased from General Mills, and a cell-free translation lysate was prepared in house as described by Anderson et al. (1983). RNasin (300 units/ml) was added to each translation reaction. When cell-free translations were used to synthesize radiolabeled prot-

14

amines; the total volumes were 25 p1; the unlabeled amino acid mix lacked cysteine; 35S-cysteine (New En- gland Nuclear) was included at 0.5 pCi/pl; and RNAs, poly(A)+ mRNAs, and RNPs were added to 25% of the total volume. When the translational activities of RNPs and deproteinized RNAs were compared, the concentration of RNA was estimated from the absorbance at 260 nm in 0.5% SDS to eliminate light scattering by RNPs. Before translation, RNAs extracted from RNPs and poly(A)+ mRNAs (in DH,O) were heated to 65C for 5 min and cooled quickly. Wheat germ and reticulocyte translations were incubated for 60 min at 25C and 30"C, respectively. After translation, the reactions were digested with RNase A (1 pg/25 p1 reaction) for 15 min at 37C; 20 pg polyarginine (Sigma P3892) was added to each reaction; and the reactions were extracted with 0.25 N HC1 and precipitated with 20% trichloroacetic acid, washed with acid-acetone and acetone, dried, and dissolved in acid-urea sample buffer as described previously (Kleene and Flynn, 1987).

The formation of protamine 1 mRNA initiation complexes in the reticulocyte lysate was measured with assays developed by Darnbrough et al. (1973) and Weber et al. (1979). The reticulocyte translations contained lop4 M emetine (Sigma) to prevent elongation and reinitiation. Initiation assays were carried out using 1 pg poly(A)+ mRNA in 50 pl reactions or RNPs in 150-300 pl reactions. In some experiments, the initiation assays were performed by incubating RNPs and W A S with the lysate a t 30C for 5 min (Weber et al., 1979). In other experiments, a modified version of the "shift assay" of Darnbrough et al. (1973) was used. The reticulocyte lysates were preincubated for 5 min at 30"C, and RNAs and RNPs were prewarmed at 30C for 15 sec and then added to the lysate, incubated for 1 or 2 min at 30"C, and chilled quickly. In our experience, the principal difference between these assays is that the "shift assay" permits more precise control over short incubation times, but the total amount of initiation complexes formed by poly(A)+ mRNA in 2 or 5 min incubation is similar. To measure the formation of initiation complexes, the lysates were layered over 3.7 ml 1540% (w/w> sucrose gradients in high-salt buffer (HSB; 0.5 M KC1, 30 mM MgCl,, 20 mM HEPES, pH 7.2; Weber et al., 1979) and centrifuged at 50,000 rpm for 3 hr at 4C in the Beckman SW60 rotor. In some experiments, 10 mM EDTA (pH 7.2) was added after incubation at 30"C, and the assays were incubated for 5 min at O'C, and sedimented on gradients in which 10 mM EDTA replaced the MgC1,. The gradients were collected as seven to nine fractions, the RNAs were deproteinized from each fraction, and the size and amount of protamine 1 mRNA in each fraction were analyzed with Northern blots. It is relevant to note that the protamine 1 mRNA initiates efficiently a saturat- ing concentrations of poly(A)+ mRNA in the reticulocyte lysate (e.g., 40 pg poly(A)+ mRNA/ml) and that reducing the concentration of poly(A)+ mRNA by five- fold increases the efficiency of formation of initiation complexes slightly (Kleene, 1993).

K.C. KLEENE AND J. SMITH

Acid-Urea Polyacrylamide Gel Electrophoresis Radiolabeled cell-free translation products were ana-

lyzed by electrophoresis in acid-urea polyacrylamide gels as described elsewhere (Kleene, 1993). We have previously reported that the translation products for protamine 1 and the precursor for protamine 2 fail to migrate in acid-urea polyacrylamide gels unless carrier protamines are coelectrophoresed with the translation products (Kleene and Flynn, 1987). Carrier protamines were replaced with polyarginine (see above). After electrophoresis gels were stained with Coomasie blue, dried, and autoradiographed.

RESULTS Translational Activity of Protamine mRNPs in

the Reticulocyte and Wheat Germ Cell-Free Translational Systems

The objective of this study was to compare the translational activity of protamine 1 mRNPs and deproteinized mRNAs with 150 base poly(A) tracts in the reticulocyte and wheat germ cell-free translation lysates. This objective is defined by the observation that, when postmitochondrial extracts of the testes of sexually mature mice are sedimented on sucrose gradients, about 85-90% of protamine 1 mRNAs sediment as translationally inactive free-mRNPs and bear homogeneous poly(A) tracts about 150 bases long, and about 10-15% are translationally active on polysomes with heteroge- neous poly(A) tracts 30-150 bases long (Kleene et al., 1984; Kleene, 1989, 1993). The difference in amount and size of protamine 1 mRNAs in the free-mRNP (fractions 2 4 and polysomal regions (fractions 6-8) of sucrose gradients is illustrated in Figure 1. Note that the vast majority of protamine 1 mRNAs with 150 base poly(A) tracts sediment in the free mRNP region of the gradient. In the remainder of this paper, the longest and shortest poly(A) tracts are referred to as poly(A)150 and poly(A)30, respectively.

We began these experiments by fractionating cytoplasmic extracts of testes from sexually mature mice by large-pore gel filtration chromatography on Sepharose CL-GB, a procedure that preserves the translational regulation of mRNPs from sea urchin and surf clam eggs (Grainger and Winkler, 1987; Standart et al., 1990). Figure 2 (upper panel) demonstrates that the cytoplasmic extract elutes from the column in two major peaks, an excluded fraction consisting of mRNPs, ribosomes, and polysomes; and an included fraction containing proteins and other small molecules. To examine the integrity and distribution of protamine 1 mRNA in the eluant, a small aliquot of the first 12 fractions was deproteinized and analyzed by Northern blots. The results in Figure 2 (lower panel) demonstrate that the vast majority of the translationally active and inactive forms of protamine 1 mRNA, i.e., poly(A)30 and poly(A)150 protamine 1 mRNAs, coelute in the excluded fractions and that protamine 1 mRNA is not degraded during fractionation.


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Fig. 1. Postmitochondrial extracts of testes from sexually mature mice were sedimented on sucrose gradients, the gradient was collected as fractions, RNA was purified from each fraction, and the amount and size of protamine 1 mRNA in each fraction was analyzed by Northern blots. The direction of sedimentation is left (top) to right (bottom). The positions of protamine 1 mRNAs with poly(A) tracts 30 and 150 bases long are indicated.

1

Fig. 2. The cytoplasmic fraction from total testes was chromatographed on Sepharose CL-6B and collected as 1.2 ml fractions begin- ning after a 15 ml void. Upper panel: The absorbance at 260 nm (*) and the protein content (A; Bradford, 1976) were measured for each fraction. Lower panel: The size and amount of protamine 1 mRNAs in the first 12 fractions after the void were determined in Northern blots. The positions ofprotamine 1 mRNAs with poly(A) tracts 30 and 150 bases long are indicated.

To compare the translational activity of deproteinized protamine 1 mRNAs and mRNPs, equal amounts of RNAs and RNPs were translated in nuclease-treated reticulocyte lysates using 35S-cysteine, and the radiolabeled translation products were analyzed by acid-urea

polyacrylamide gel electrophoresis and autoradiogra- phy. Figure 3A reveals that RNPs, RNAs extracted from RNPs, and total testis poly(A)+ mRNA direct the synthesis of numerous translation products that are not detected among the translation products directed by endogenous mRNAs in the reticulocyte cell-free translation lysate. Many of the testicular translation products migrate more slowly than the endogenous globin and are not resolved in this acid-urea polyacrylamide gel system, which is designed to separate only small, highly basic proteins. As was reported previously (Kleene and Flynn, 1987; Elsevier et al., 1991), promi- nant translation products comigrate with protamine 1 (Pl) and the precursor for protamine 2 (P2) isolated from sonication-resistant spermatid nuclei (not shown). Comparison of the amounts of protamine translation products stimulated by varying amounts of RNAs and RNPs (Fig. 3A, lanes 3-6, 7-10) demonstrates that RNPs are about twofold less active than deproteinized RNAs in stimulating the synthesis of protamine 1, while RNPs are about four- to eightfold less active than RNAs in stimulating the synthesis of protamine 2. The reasons for the lower translational activity of the protamine RNPs are unclear, since the yield of translation products is determined by the rate of mRNA degradation and by the rates of initiation, reinitiation, and elongation in the reticulocyte lysate. We also do not know why protamine 2 mRNPS are less active than protamine 1 mRNPs.

Unexpectedly, protamine mRNPs were totally inactive in the wheat germ system, as indicated by the failure to stimulate synthesis of radiolabeled testicular translation products, including protamines 1 and 2 (Fig. 3B, lanes 4-6), even though numerous testicular translation products and both protamines were readily detectable when deproteinized RNAs from RNPs were translated (Fig. 3B, lanes 7-10). The inactivity of mRNPs demonstrated in lanes 4-6 appears to be due to a general inhibition of translation, since no testicular translation products are detectable, and comparison with lane 1 demonstrates that all the radiolabeled

16 K.C. KLEENE AND J. SMITH

Fig. 3. Translational activity of protamine mRNPs and mRNAs in the reticulocyte and wheat germ lysates. Sepharose CL-6B fractions, deproteinized RNAs extracted from Sepharose fractions, and poly(A)+ mRNA were translated in the reticulocyte and wheat germ lysates with 35S-cysteine, and the radiolabeled translation products were analyzed by electrophoresis on acid-urea polyacrylamide gels and autora- diography. The translation products for protamine 1 (PI), precursor for protamine 2 (P2), and hemoglobin (Gb) are indicated. The quantity of RNA in the RNP translations applies to the amount of RNA measured by absorbance at 260 nm as described in Materials and Methods. A Reticulocyte lysate. Lane 1, DH,O; lane 2, 1 pg poly(A)' mRNA; lane 3,2 pg R N P lane 4 , l pg R N P lane 5,0.5 pg R N P lane 6,0.25 pg RNP lane 7 , 2 pg RNA, lane 8 , l pg RNA; lane 9,0.5 pg RNA lane 10,0.25 pg RNA. B. Wheat germ lysate. Lane 1, DH,O; lane 2 , l pg poly(A)+ mRNA; lane 3 , l kg poly(A)+ mRNA, lane 4 , 2 pg RNP; lane 5 , l pg R N P lane 6,0 .5 pg RNP; lane 7,2 pg RNA; lane 8,1 pg RNA; lane 9, 0.5 pg RNA; lane 10, 0.25 pg RNA, lane 11, 1 pg poly(A)+ mRNA and 2 pg RNP; lane 12, l pg poly(A)+ mRNA and 1 pg R N P lane 13,l kg poly(A)' mRNA and 0.5 pg RNP; lane 14, l pg poly(A)+ mRNA and 0.25 pg RNP.

translation products in the RNP translations are directed by endogenous wheat germ mRNAs.

Testicular RNPs might fail to stimulate translation because the RNPs contain a nonspecific, dominant in- hibitoir of translation (Hansen et al., 1987). To examine

this possibility, a mixture of RNPs and deproteinized total testis poly(A)+ mRNAs was translated. Both the mixture of poly(A)+ mRNA with RNPs (lanes 11-14) and poly(A)+ mRNA alone (lane 2) stimulate the synthesis of similar amounts of testicular translation products, including protamines, ruling out a dominant inhibitor of translation. conceivably, the failure of testicular mRNPs to stimulate translation in the wheat germ system reflects a constituent common to many testicular mRNPs. We attempted to test the translational activity of RNPs after gel filtration in 0.5 M KC1, 1 mM MgCl,, 20 mM HEPES (pH 7.5) to extract RNP proteins (Grainger and Winkler, 1987; Standart et al., 19901, but a large precipitate formed during dialysis against LSB.

Formation of Initiation Complexes by Poly(A)lBO Protamine 1 mRNPs in the Reticulocyte

Cell-Free Translation System As was pointed out above, yields of translation prod-

ucts in cell-free translation systems are difficult to in- terpret, because the total amounts of translation product are determined by a variety of factors. In the experiments described below, we analyzed the ability of poly(A)150 protamine mRNPs to initiate translation directly by comparing the activity of protamine 1 mRNAs and mRNPs in forming initiation complexes with 80s single ribosomes in the reticulocyte lysate. mRNPs and poly(A)+ mRNAs were incubated for 1-5 min with the reticulocyte lysate, which contained lop4 M emetine, an inhibitor of translational elongation, to prevent elongation and reinitiation. The translation reactions were sedimented on high-salt sucrose gradients, and the amount of protamine 1 mRNA in eight or nine fractions across the gradient was determined by Northern blots to resolve poly(A)30 and poly(A)150 protamine 1 mRNAs. Translationally inactive mRNAs sediment near the top (fractions 1 4 ) , and mRNAs in initiation complexes sediment with 80s single ribosomes near the bottom (fractions 5-8). mRNAs that have formed preinitiation complexes with 40s ribosomal subunits are dissociated by the high salt and sediment with translationally inactive mRNPs at the top of the gradient (Weber et al., 1979). Initiation assays pro- vide a rigorous measure of the translational compe- tence of poly(A)l5O protamine l RNPs, because the sizes of poly(A)150 and poly(Al30 protamine 1 mRNAs can be distinguished in Northern blots (Kleene et al., 1984; Kleene, 1989). In addition, the brief incubation with the cell-free lysate minimizes the period during which mRNPs could be modified by the reticulocyte lysate.

Figure 4 illustrates two findings that are analyzed further below. First, in numerous preparations of RNPs, approximately 45-66% of poly(A)150 protamine 1 mRNAs formed initiation complexes in 2-5 min incubations with the reticulocyte lysate (Fig. 4K,M) even though virtually no poly(A)150 protamine 1 mRNAs are associated with polysomes in the testis (Fig. 1). Second, a large fraction of protamine 1 mRNAs form initiation complexes in the first 1 min (Fig. 4D,E),


Fig. 4. Formation of initiation complexes by protamine 1 mRNAs and mRNPs. RNPs and poly(A)+ mRNAs were incubated with the reticulocyte lysate containing M emetine for 5 min as described by Weber et al. (1979) or for 1 or 2 min as described by Darnborough et al. (1973) and sedimented on high-salt sucrose gradients, and the levels and size of protamine 1 mRNAs were measured in Northern blots. In the column at left, the initiation activities of poly(A)+ mRNA was assayed, whereas, in the column a t right, the initiation activities of RNPs was assayed. A. Poly(A)+ mRNA, 5 min incubation at 30"C,

M aurintricarboxylic acid. B: Poly(A) + mRNA, 5 min incubation at 0C. C: Poly(A) ' mRNA, 5 min incubation at 30C, sedimented on gradients containing 10 mM EDTA. D: Poly(A)+ mRNA, 1 min incubation a t 30C. E: Poly(A)+ mRNA, 2 min incubation at 30C; F: A

whereas the vast majority of poly(A)150 protamine 1 mRNPs form initiation complexes after a lag lasting about 1 min (Fig. 4L,M).

At this point it is important to note several controls demonstrating that the protamine mRNAs and mRNPs that sediment near the bottom of these gradients are in initiation complexes. First, initiation assays were incubated for 2-5 min at 0C or a t 30C with lOW4M aur-

mixture of poly(A)+ mRNA and round spermatid extract containing inhibitors of protein synthesis, 1 rnin incubation at 30C. G A mixture of round spermatid extract containing inhibitors of protein synthesis and poly(A)' mRNA, 2 min incubation at 30C. H: Round spermatid cytoplasmic extract, 2 min incubation, M aurintricarboxylic acid. I Round spermatid cytoplasmic extract, 5 min incubation at 0C. J Round spermatid cytoplasmic extract, 2 min incubation, sedimented on sucrose gradients containing 10 mM EDTA. K Sepharose CL-6B excluded fractions, 5 min incubation at 30C. L: Round spermatid cytoplasm containing inhibitors of protein synthesis, 1 min incubation at 30C. M: Round spermatid extract containing inhibitors of protein synthesis, 2 min incubation a t 30C.

intricarboxylic acid, conditions that block translational initiation (Darnbrough et al., 1973; Weber et al., 1979). Virtually no initiation complexes were formed, as indicated by the absence of protamine 1 mRNAs from the bottom of the gradients (Fig. 4A,B,H,I). Second, initiation assays were incubated for 2 min at 30C and were sedimented on gradients in which 10 mM EDTA replaced the Mg2+. EDTA caused protamine 1 mRNAs


Fig. 5. Formation of initiation complexes by poly(A)30 and poly(A)150 protamine 1 mRNAs. Poly(A)+ rnRNAs from total testes were fractionated by thermal chromatography on poly(U) Sepharose to obtain a fraction enriched in poly(A)30 protamine 1 mRNAs. The ability of this fraction to form initiation corn- plexes in 2 min was assayed as described in Materials and Methods.

and mRNPs to sediment at the top of the gradient, because Mg2' is required for mRNAs to bind to ribosomes (Fig. 4C,J).

The observation that protamine 1 mRNPs are translationally active in the reticulocyte lysate could be explained if the structure of protamine RNPs was altered during isolation. Therefore, we varied numerous parameters in an attempt to isolate poly(A)150 protamine 1 mRNPs in a repressed form; some of these variations are described below. First, we reasoned that gel filtration of Sepharose CL-6B might reduce the concentration of a translational repressor or that an activator of protamine mRNP translation in late spermatids might be present in extracts from total testes. To test both possibilities, round spermatids were purified on bovine serum albumin gradients, yielding a fraction that con- sisted of about 80% round spermatids, 10% late spermatids, and 10% spermatogonia. Total cytoplasmic extracts of the round spermatids were translated in the reticulocyte cell-free system. Figure 4M demonstrates that the amount of initiation complexes formed by the poly(A)150 protamine 1 mRNPs in unfractionated round spermatid cytoplasmic extracts in 2 min is similar to the amount formed by protamine 1 mRNA in poly(A)+ mRNA (Fig. 4E). Second, the buffer used in isolating mRNPs for Figure 2 is similar to the buffer reported to be optimal for binding of mRNP proteins to the 3' untranslated region of protamine 2 mRNA (Kwon and Hecht, 1991) and can be used to prepare a cell-free translation extract from testes that supports high levels of incorporation of radiolabeled amino acids (Kleene, unpublished). However, substantial fractions (at least 45%) of poly(A)150 mRNPs formed initiation complexes when cytoplasmic extracts were prepared andlor chromatographed on Sepharose in buffers containing 40 mM KCl, 100 mM KC1, or 100 mM KOAc (not shown). Third, since proteolytic degradation might activate mRNPs, cytoplasmic extracts of round spermatids were prepared in a buffer containing soybean and ovomucoid trypsin inhibitors, leupeptin, and pepstatin (Fig. 4M) or aprotinin (not shown), but, once again, the poly(A)150 protamine 1 mRNPs were active in forming

initiation complexes. Fourth, it seemed plausible that freezing-thawing extracts might artifactually activate protamine mRNPs, but fresh cytoplasmic extracts from round spermatids were active in forming initiation complexes (not shown).

We also wondered whether the 1 min lag in the formation of initiation complexes by protamine 1 mRNPS might be due to a general inhibitor of initiation of translation. Therefore, poly(A)+ mRNA and the round spermatid cytoplasmic extract used for Figure 4L and M were mixed and assayed. In this mixture, about 90% of the hybridization signal is due to poly(A)+ mRNA, and there was no lag in the formation of initiation complexes (Fig. 4F,G).

Formation of Initiation Complexes by Poly(A)30 and Poly(A)lBO Protamine 1 mRNAs in the

Reticulocyte Lysate The preceding experiments have implied that prota-

mine mRNAs in the testis are not repressed by some feature in the nucleotide sequence of the mRNA. To test this possibility directly, we compared the ability of translationally repressed [poly(A)150] and translationally active [poly(A)301 protamine 1 mRNAs to form initiation complexes. Total testis poly(A)+ mRNAs were fractionated by thermal chromatography on poly(U) Sepharose according to differences in poly(A) length, resulting in an mRNA fraction in which poly(A)3O and poly(A)150 protamine 1 mRNAs are present in approximately equal proportions. The results shown in Figure 5 demonstrate that poly(A)30 and poly(A)150 protamine 1 mRNAs form virtually equivalent amounts of initiation complexes in 2 min assays.

DISCUSSION Previous experiments have demonstrated that prota-

mine 1 mRNAs are initially transcribed in step 7 spermatids, stored as translationally repressed mRNPs for 4 days, and then translated actively in step 12-14 spermatids (Kleene et al., 1984; reviewed in Hecht, 1989; and in Braun, 1990). The poly(A) tails on the active and

TRANSLATION OF PROTAMINE 1 mRNPs 19 inactive forms differ in length, 30-150 bases vs. 150 bases, providing a reliable criterion to identify translationally active and inactive mRNAs (Kleene et al., 1984; Kleene, 1989, 1993). We report here that protamine 1 mRNAs with 30 and 150 base poly(A) tracts are equally efficient in forming initiation complexes in the reticulocyte lysate, indicating that protamine 1 mRNAs are not repressed by some feature of the cova- lent structure of the mRNA, in agreement with findings reported earlier (Kleene and Flynn, 1987).

The remainder of the experiments were designed to test the hypothesis that the initiation of translation of poly(A)150 protamine 1 mRNP is inactivated by transacting protein repressors. Although protamine 1 RNPs were slightly less active in directing the synthesis of radiolabeled protamine translation products in the reticulocyte cell-free translation system that deproteinized RNAs, the lower translational efficiency of RNPs could be explained by a variety of factors, such as in- creased mRNA degradation and lower rates of elongation and/or initiation. However, initiation assays con- sistently reveal that a large fraction, usually over 50%, of poly(A)150 protamine 1 mRNPs form initiation complexes with 80s ribosomes in 2-5 min incubations with the reticulocyte lysate, far higher than the insignifi- cant fraction of translationally active poly(A)150 mRNPs in the testis. We have also found that protamine 2 and mitochondria1 capsule selenoprotein mRNPs are active in forming initiation complexes (Kleene, unpublished).

The implication of our finding that protamine mRNAs are not repressed by transacting factors as judged from initiation assays should be scrutinized carefully since a series of studies of the translational activity of mRNPs in sea urchin eggs led to conflicting conclusions, apparently caused by artifacts in the preparation of the mRNPs (Jenkins et al., 1978; Moon et al., 1982; Grainger and Winkler, 1987). The fractionation procedures employed here preserve the in vivo translational activity of inactive and active mRNPs in marine embryos (Rosenthal et al., 1980; Grainger and Winkler, 1987; Standart et al., 1990). Our procedures are gentle; use low- and moderate-ionic-strength buffers, which are generally believed to preserve mRNP structure; and avoid ultracentrifugation, which can cause aggre- gates (Moon et al., 1982) and dissociate mRNP proteins (Grainger and Winkler, 1987; Drawbridge et al., 1990). In our hands, varying numerous parameters did not enable us to isolate protamine 1 mRNPs that are inactive in forming initiation complexes. The observation that poly(A)150 protamine mRNPs in cytoplasmic extracts from round spermatids isolated with protease inhibitors are active in forming initiation complexes seems particularly convincing. Nevertheless, none of the experiments reported here rules out several artifactual explanations for our observations: 1) an unusual factor is necessary for the integrity of spermatidal mRNPs, 2 ) a transacting repressor of protamine 1 mRNA translation in the testis is not functional in the reticulocyte lysate, or 3) factors in the reticulocyte ly-

sate rapidly unmask protamine mRNPs. These issues are difficult to address rigorously without an actively initiating cell-free translation system that mimics the translation patterns in spermatids.

The finding that mouse protamine 1 mRNPs are active in the reticulocyte lysate conflicts with Sinclair and Dixons (1982) report that trout protamine RNPs are inactive in the same lysate. This discrepancy could be explained by differences in species or RNP isolation techniques, since the trout RNPs were isolated by ultracentrifugation.

Since protamine 1 mRNPs were active in the reticulocyte cell-free system, it was quite unexpected that testicular mRNPs were inert in the wheat germ cell- free system. We have examined the literature to assess whether mRNPs from other systems have radically different translational activities in the wheat germ and reticulocyte cell-free systems. There are reports that mRNPs from sea urchins and mammalian cells have similar translational activities in both systems (Moon et al., 1982; Geoghegan et al., 1979; Bergman et al., 1982) and reports that free mRNPs from reticulocytes are active in the reticulocyte system and inactive in the wheat germ system (Minich et al., 1989). However, since the mRNPs in each study were prepared by a different method, it is impossible to distinguish whether differences in translational activity reflect differences in species, tissues, andlor isolation techniques.

In conclusion, our results do not support the hypothesis that the timing of protamine 1 mRNA translation is regulated by a transacting protein repressor, although we cannot rule out artifactual explanations. Neverthe- less, our observations can be reconciled with a report that high-resolution nonisotopic in situ hybridizations show that translationally repressed transition protein 2 mRNAs are restricted to a discrete region of the cytoplasm of step 7 spermatids (Saunders et al., 1992). The position and size of this region correspond to the chromatoid body, which has been proposed to function in the storage of spermatidal mRNAs (Soderstrom and Parvi- nen, 1976). Since transition proteins and protamines are members of a single gene family (Heidaran et al., 1989; Kleene et al., 1990; Reinhart et al., 1991), it is plausible that translation of protamine mRNAs may be blocked by physically separating the mRNAs and the translational apparatus, without the intervention of a transacting protein repressor. It is interesting to note that a similar mechanism is believed to explain the repression of histone mRNA translation in sea urchin eggs: The histone mRNPs are translationally active and are compartmentalized in the pronucleus (DeLeon et al., 1983; Grainger and Winkler, 1987).

ACKNOWLEDGMENTS This study was supported by NSF grants DCB-

8710485 and DCB-90128486.

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Translational activity of mouse protamine 1 messenger ribonucleoprotein particles in the reticulocyte and wheat germ cell-free translation systems