Poly(A)-Containing Messenger Ribonucleoprotein Complexes from Sea Urchin Eggs and Embryos: Polypeptides Associated with Native and UV-Crosslinked mRNPs

Differentiat ion

Differentiation (1983) 24: 13-23

0Springer-Verlag 1983

Poly(A)-Containing Messenger Ribonucleoprotein Complexes from Sea Urchin Eggs and Embryos: Polypeptides Associated with Native and UV-Crosslinked mRNPs Randall T. Moon Department of Zoology, University of Washington, Seattle, WA. 98195 and Marine Biological Laboratory, Woods Hole, MA. 02543,USA

Abstract. Fertilization of sea urchin eggs results in the rapid recruitment of stored messages into polyribosomes. Whether translational control in sea urchin eggs is mediated by macromolecules associated with the stored messages remains unknown, since preparations of messenger ribonucleoprotein complexes (mRNPs) were active in protein synthesis in a rabbit reticulocyte lysate. To facilitate the study of mRNPs, chromatography on oligo(dT)-cellulose was used to purify poly(A)containing mRNPs from eggs and embryos of the sea urchin Strongylocentrotus purpuratus. Nonpolyribosomal mRNPs purified from eggs had a similar sedimentation in sucrose to unpurified mRNPs, a peak buoyant density in metrizamide of 1.22 g/cm3, and peak buoyant densities in Cs2S04 in 1.42 g/cm3 after fixation with glutaraldehyde and 1.46 g/cm3 without fixation. Nonpolyribosomal mRNPs from eggs and zygotes contained 5-10 major proteins on sodium dodecylsulfate (SDS) polyacrylamide gels, and numerous minor bands. UV-irradiation of living eggs of the sea urchin Arbacia punctulata produced cross-linked mRNPs which contained a similar pattern of polypeptides to noncross-linked mRNPs. The polypeptides associated with embyronic polyribosomal mRNPs were also qualitatively similar to those present in nonpolyribosomal mRNPs, although stoichiometric differences may exist.

Introduction

The rapid post-fertilization increase in protein synthesis in sea urchin eggs [7,9, 14, 30) is an easily detected yet complex event. Our partial understanding of the dynamic molecular interactions controlling this phenomenon has been facilitated by the discovery that both intracellular Ca" and intracellular pH increase after fertilization [S, 19, 24, 39, 461. Significantly, artificial manipulations of the intracellular pH and Ca' concentrations elicit increased rates of protein synthesis which mimic the response to actual fertilization 18, 10, 45, 501. In order to determine how these ionic changes modulate protein synthesis, recent investigations have been made of the initiation and elongation steps, and of ribosome activity. Hille et al. [13] have shown that the availability of recycling initiation factors is not ratelimiting in the egg, and that the major block in translation +

Address for correspondence: Division of Biology 156-29, California Institute of Technology, Pasadena, CA.91125, USA

must be at some other step in initiation. Other investigations have shown that although the rate of polypeptide chain elongation increases approximately 2-3-fold after fertilization [4, 121, this increased rate is clearly insuficient to account for the rapid kinetics of the post-feritilization increase in protein synthesis [14, 351. A recently discovered difference in the in vitro activities of egg and embryo ribosomes [6] may, however, be of great importance in regulating translation. The early hypothesis that message availability is the primary level of translational control in sea urchin eggs [reviewed in 341 has remained attractive, if largely untested. One potential mechanism for reducing the pool of translatable messages in the egg is for the stored mRNPs of the egg to be 'masked'by association with an inhibitor of translation [25, 441. The post-fertilization loss or modification of such an inhibitor would enable the translation of the message. Supporting the masking hypothesis are data suggesting that the in vitro translational activity of crude mRNPs may be less than that of RNA purified from the mRNPs [16, 181. However, a subsequent study has shown that egg mRNPs are translatable in vitro, which reopens the possibility that masking is not the principal mechanism of translational control in eggs [27]. The positive reports of translationally inactive mRNPs, cited above, while consistent with masking, are not a rigorous proof. Our negative report on the in vitro translational repression of mRNPs suffers from the recognized potential artifact that any masking factors may be lost or inactivated during mRNP isolation or translation [27]. A complementary approach to in vitro translation for studying translational control is do determine whether stored mRNPs or ribosomes undergo changes in associated polypeptides or small RNAs after fertilization, and to determine whether any developmentally regulated macromolecules also resemble known inhibitors or facilitators of translation. Direct investigations of the macromolecules associated with sea urchin messages have been difficult because of the cosedimentation of ribosomes with the poly(A)containing mRNPs. It has recently been demonstrated that oligo(dT)tellulose chromatography can be used to purify poly(A)-containing mRNPs from sea urchin eggs [2q. In the present study1 describe improved conditions for the isolation of sea urchin mRNPs, and compare the polypeptides associated with native mRNPs with those polypeptides which can be cross-linked to poly(A)-containing RNA in vivo by UV irradiation.

14

Methods

10 min in the presence of EDTA the solution was diluted 10-fold with HB-20.Samples of intact dissolved polyribo-

Solutions Buffers had the following compositions: Homogenization buffer (HB) contained 220 mM K + , 40 mM Na', 5 mM M g + + , 80 mM C1-, 67 mM PO;, 180 mM OAc-, 1 mM dithiothreitol, and 0.1 mM phenylmethylsulfonylfluoride, pH 6.8 (0"C). HB-20 was prepared to the above salt concentrations except that PO,' was 20 mM. Isolation buffer (IB) consisted of 220 mM K ', 5 mM Mg' +,80 mM C1-, 140 mM OAc-, 20 mM Piperazine-N,N'-bis(2-ethanesulfonic acid) (Pipes), 1 mM dithothreitol, pH 6.8 (0"C) for eggs and 7.2 for embryos. Column wash buffer (CWB) contained 220 mM K + , 80 mM C1-, 140 mM OAc-, 5 mM EDTA, 20 mM Pipes, and 1 mM dithiothreitol, pH 7.2 (0"C). Elution buffer (EB) contained 15 mM KCl, 5 mM Mg (OAc), ,and 20 mM Pipes, pH 6.8 (45" C). To eliminate ribonuclease activity, solutions were treated, prior to addition of dithiothreitol, with 0.01 YOdiethylpyrocarbonate followed by autoclaving or heating overnight at 60" C, then readjustment of the pH. Glassware was baked at 200" C for 8 h. Preparation of'Egg Nonpolyribosomal mRNPs Eggs of the sea urchin Strongylocentrotus purpuratus were washed twice by centrifugation through 30 volumes of filtered sea water (12" C), then twice with HB (-2" C). The washed cells were resuspended in 6-8 volumes of HB and homogenized by passing through a 22 gauge needle. Nuclei and yolk granules were then pelleted by centrifugation at 5,OOO g,,, for 5 min (- 2" C). The resulting supernatant was centrifuged at 27,000 g,,, for 6 min (-2" C) to pellet mitochondria and cellular debris. Up to 8 ml of the post-mitochondrial supernatant were layered onto each 30 ml 10-30 (w/v) linear sucrose gradient made to the final salt concentration of HB-20. Generally the gradients were centrifuged in two Beckman SW-27 rotors at 93,000 g,,, for 3.5 h (1" C) and RNP sedimenting at approximately 30 to 90 S was collected and pooled [26]. The earlier finding that PO; increased the extraction of mRNPs from eggs [26] was not confirmed after repeating the experiments later in the spawning season of the sea urchin. Preparation of' Embryo RNPs To prepare mRNPs from early stages of embryos, the eggs were fertilized in the presence of 1 mM 3-amino,l,2,4-triazole to prevent the enzymatic hardening of the fertilization layer. Embryos were cultured and washed using conditions designed to maximize the isolation of polyribosomes [26]. Post-mitochondria1supernatants were then prepared as described for eggs. Polyribosomes were separated from nonpolyribosomal material by layering 10 ml of the post-mitochondria1 supernatant onto 27 ml 10-30% (w/v) linear sucrose gradients, which were then centrifuged in two Beckman SW27 rotors at 93,000 gave for 3.0 h (0' C). Material sedimenting at approximately 30 to 90 S was collected and used for the purification of embryo nonpolyribosomal mRNPs. The pelleted polyribosomes in each centrifuge tube were dissolved in 10 ml of HB-20 to a final concentration of approximately 15 A260nm units/ml. The dissolved polyribosomes were dissociated by addition of EDTA from a 0.5 M stock soluLion to a final concentration of 5 mM. After approximately

somes were also centrifuged on analytical gradients to monitor polyribosome size distribution and separation from monoribosomes. Oligo( d T )-Cellulose Chromatography of Noncross-linked mRNPs

Poly(A)-containing mRNPs were purified by binding to oligo(dT)cellulose as described by Moon et al. [26] followed by an improved elution procedure. Typically 150-500 A260nm units of dissociated polyribosomes or sucrose gradient fractions containing nonpolyribosomal RNP, in up to 250m1, were added per 1 g of oligo(dT)cellulose (P-L Biochemical Co., type 7) which had been washed with 0.1 N NaOH, then HB-20. Specific adsorption of the poly(A)containing mRNP was accomplished by mixing the total RNP with oligo(dT)-cellulose by 1-2 h (0"C) on a rotary shaker at 25 rpm. The oligo(dT)-cellulose was then washed by settling through two 20-volume changes of HB-20, followed by a third wash with either HB-20 or CWB (0"C), as indicated. To further remove unbound material, the oligo(dT)-cellulose was poured into a 15 x 1 cm jacketed column and washed with 50 ml of either HB-20 or CWB per g oligo(dT)-cellulose at a flow rate of approximately 1 ml/min. Regardless of whether the final wash contained M g + + (HB-20) or EDTA (CWB), the column was then washed with approximately 10 ml of buffer containing M g + + (IB) until no change in the baseline absorbance at 254 nm could be observed. The bound mRNPs were then eluted by raising the temperature of the jacketed column to 34" C and simultaneously adding 50% (v/v) IB (34" C). Further elution with a buffer containing 15 mM K + (EB at 45" C) [25] yielded only negligible amounts of material absorbing at 254 nm, so was not done in this series. For the analysis of mRNP proteins and in vitro translation, mRNPs eluted at 34°C were concentrated by one of two methods: (1) concentration by negative pressure dialysis against IB (0"C) for 5-8 h in a MicroProdicon (BioMolecular Dynamics, Beaverton, Ore.) to 10% or less of the original volume and (2) concentration by pelleting, achieved by layering the mRNPs over 1 ml of 10% (w/v) sucrose, made to the final salt concentration of IB, and then pelleting in a Beckman SW50.1 rotor at 60,OOOg,,, for 8 h (1" C). The duration of centrifugation was calculated to pellet material sedimenting at greater than 30 S, yet leave smaller material, such as free protein, in the supernatant. The clear pellet of mRNPs dissolved overnight in a small volume of IB.

In vivo Cross-linking of Proteins to RNA Polypeptides associated with poly(A)containng RNA were cross-linked in vivo to the RNA using UV irradiation at 254 nm as described by Mayrand et al. [23]. Briefly, the jelly layer of A. punctulata eggs was removed by a short exposure to pH 5 filtered sea water. Some eggs were then fertilized and cultured at 23" C to the desired stage. Living eggs and embryos in sea water, at a concentration of 1% (v/v) or less, were then poured in a thin layer into ice-cold petri dishes or trays lined on the inside with aluminum foil. The cells were then irradiated for 10 min, with constant agitation, using a 15 W germicidal lamp at a distance of 5 cm, and a dose of approximately 1.2 x lo4 pW/cm2. Fol-

lowing irradiation, the fertilization membrane was removed from zygotes by pouring the embryos through a 56 micron Nitex mesh. The irradiated zygotes, eggs, and hatched blastulae, and non-irradiated cells for controls, were then washed as described above, homogenized with a Dounce homogenizer in HB, and centrifuged on 10-30% sucrose gradients as described above. Material which pelleted was dissolved in 1 mM EDTA, 50 mM Tris-HC1, 0.5% sodium dodecylsulfate (SDS), pH 7.6. RNP sedimenting at 30-90 S in the sucrose gradients was pooled and centrifuged at 81,500 g,,, for 12 h in a Beckman Type 50.2 Ti rotor. The pelleted RNP was then dissolved as above. A. punctulata were used for cross-linking because of their availability; whether they are the species best suited for cross-linking is unknown. Oligo(dT)-Cellulose Chromatography of Cross-linked mRNPs

Procedures similar to those described by Mayrand et al. [23] were used to dissociate noncross-linked protein from the poly(A)-containing RNA. Cross-linked material and non-cross-linked controls in the above Tris-EDTA-SDS buffer were brought to 0.5 M NaCl with a 5.0 M stock, deionized formamide was added to 60% (v/v), and samples were heated at 60"C for 3 min before being precipitated with ethanol. Precipitates were collected by centrifugation, and dissolved in 1 mM EDTA, 14 mM 2-mercaptoethanol, 0.01 M Tris, 0.5% SDS, pH 7.6. NaCl was added to 0.5 M and the samples were heated to 60" C for 3 min, then cooled and applied to oligo(dT)-cellulose. Unbound material was removed by extensive washing with the same buffer, and poly(A)-containing mRNPs were eluted with 1 mM EDTA, 14 mM 2-mercaptoethanol, 0.01 M Tris, 0.5% SDS, pH 7.6. Eluted material was brought to 0.5 M NaC1, again bound to oligo(dT)-cellulose, and then eluted and precipitated with ethanol. Precipitates were collected by centrifugation, dissolved in sterile water, and lyophilized. To release cross-linked protein from the RNA, lyophilized samples were digested for 1 h (37" C) with a ribonuclease solution containing 50 pg/ml of pancreatic ribonuclease A and 1,500 units/ml of TI ribonuclease, in 1 mM EDTA, 10% glycerol, 1 mM dithiothreitol, 0.5% sarkosyl, 0.05 M Tris, pH 8.4. The ribonucleases were first prepared as a 1OX stock in sterile water, and heated at 90" C for 20 min. An equal volume of 2X gel electrophoresis sample buffer was added to the digested RNP before separation of the polypeptides on polyacrylamide gels.

To determine the bouyant density of unfixed poly(A)containing mRNPs in metrizamide, step gradients were prepared according to the method of Jagus and Kay [ l q . The stock solutions of metrizamide contained 220mM K + , 80 mM C1-, 20 mM Pipes, 5 mM MgCl or EDTA as indicated, and 1 mM dithiothreitol, pH 6.8. The 1 ml samples in a buffer containing 5 mM MgCl or 10 mM EDTA were layered over the 4 ml of metrizamide containing, respectively, MgCl or EDTA. The gradients were then centrifuged in a Beckman SW50.1 rotor at 189,000 g,,, for 12 h (4" C). The density of every second fraction was determined from its refractive index [36]. [3HJpoly(U) Hybridization

Sucrose and density gradient fractions were digested with proteinase K (E.C. 3.4.21.14, obtained from EM Laboratories) then hybridized to [5,6-3H]poly(U)(New England Nuclear) as previously described [26]. Preparation and Electrophoresis of mRNP Proteins

Proteins were dissociated from noncross-linked mRNPs with 2 M LiCl and 4 M urea, dialyzed against 0.2% acetic acid, then lyophilized [26]. The lyophilized proteins were weighed, dissolve in sample buffer, then subjected to sodium dodecylsulfate (SDS)/lOo/, polyacrylamide slab gel electrophoresis [2q. The RNA precipitated from LiCl and urea was itself suspended in sample buffer, followed by heating to 95" C, repelleting the RNA, and electrophoresis. Since protein was present in the RNA fraction, the described analyses of the mRNP proteins are qualitative. In vitro Translation of mRNA and mRNP

Gradient fractions from 33 ml SW27 10-30% sucrose gradients, containing A. punctulata mRNPs and ribosomes sedimenting at 30-90 S, were concentrated against IB containing 5% glycerol by negative pressure dialysis in a ProDiCon model 625 as previously described [27. RNA was extracted from the concentrated RNP by CsCl gradient centrifugation [27]. Intact mRNPs and mRNAs purified from the mRNPs, both in IB constituting 7.5% of the volume of the lysate, were translated for 120min (30" C) in a message-dependent rabbit reticulocyte lysate [15] at three concentrations of RNA, and at two concentrations of Mg". Five microliters of lysate were then diluted to 50 pl with SDS gel sample buffer and applied to 10% polyacrylamide gels without heating the samples. Gels were then processed for fluorography [27].

Density Analyses

The bouyant density of poly(A)containing mRNPs in Cs,SO, was determined with and without prior fixation of the mRNP with glutaraldehyde. Linear gradients were preformed using 2.0 ml of a 1.8 g/cm3 Cs,SO, solution and 2.7 ml of a 1.2 g/cm3 solution [20,26] both made to a final concentration of 40 mM 4-(2-hydroxyethyl)-l-piperazine ethanesulfonic acid (Hepes), 15 mM KCI, and 1 mM dithiothreitol, pH 6.8. Some of the samples were fixed with 5% glutaraldehyde (Tousimis, EM grade) for 1 h (0" C). Samples of 0.4 ml were layered over the gradients and centrifuged at 142,OOOg,,, in a Beckman SW50.1 rotor for approximately 40 h at 4°C. The density of every fourth fraction was determined by weighing in a precalibrated micropipette [26].

Results Sedimentation of Purified Noncross-linked Egg mRNPs

In this study I use moderate oligo(dT)-celluloseelution conditions to improve upon the methods previously described [26] for purifying mRNP complexes from sea urchin eggs. The proteins associated with egg and embryo mRNPs were then compared, with and without prior cross-linking of proteins to the RNA with UV-irradiation. Poly(A)-containing mRNPs were purified from sea urchin eggs and embryos by homogenizing the cells in buffers containing near-physiologic ionic concentrations of 220mM K f and 5 mM Mg++ [26] enriching for mRNPs and ribosomes by centrif-

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Fig. 1. Sedimentation of crude and purified S. purpuratus poly(A)containing mRNPs in sucrose gradients. Poly(A)-containing mRNPs sedimenting at 30-90 S in preparative sucrose gradients were bound to oligo(dT)-cellulose, then washed with a buffer containing EDTA (CWB). The EDTA was then removed by washing the column with a buffer containing M g + + (IB) before elution with 50% IB (34°C).The eluted mRNPs and a sample of the preparative sucrose gradient were then concentrated overnight by negative pressure dialysis. The concentrated crude (0-0) and purified (0-0) mRNPs were then centrifuged on 13 ml 10-30% linear sucrose gradients in a Beckman SW40 rotor at 390,000 x g,,, for 3 h (1" C). Fractions were digested with proteinase K then hybridized to ['H]poly(U). The direction of centrifugation was from left to right, with the position of the monoribosomes indicated by the arrow at 80 S

ugation of the post-mitochondria1 supernatant on sucrose gradients, and selectively binding poly(A)-containing mRNPs to oligo(dT)-cellulose (see Methods). The homogenization conditions released into the post-mitochondria1 supernatant 85-95% of the [3H]poly(U)-hybridizable material present in the egg (data not shown). As up to 97% of the sea urchin poly(A) present in the 30 to 90 S region of sucrose gradients readily binds to oligo(dT)-cellulose [26], the described procedures enable the purification of most of the poly(A)-containing material present in the egg and, as described below, these purified mRNPs have sedimentation rates and bouyant densities similar to those of unfractionated mRNPs. The sedimentation positions of purified mRNPs in sucrose gradients were determined by hybridizing the gradient fractions to [3H]poly(U). Egg mRNPs eluted from oligo(dT)-cellulose with a buffer containing 110 mM K + (50% IB) at 34" C had a sedimentation in sucrose gradients similar to that of the unpurified poly(A)-containing mRNPs present in the 30 to 90 S region of preparative sucrose gradients (Fig. 1). Density of Noncross-linkedEgg in mRNPs in Metrizamide

Purified and unpurified egg mRNPs were banded in metrizamide to demonstrate that the higher ionic strength and the lower temperature of the oligo(dT)-cellulose elution buffer used in the present study produced purified mRNPs with bouyant densities resembling unfractionated mRNPs. For comparison with the purified mRNPs, unpurified egg mRNPs collected from the 30 to 90 S region of preparative sucrose gradients were adjusted to 5 mM Mg" or 10 mM EDTA and then centrifuged in metrizamide. Both Mg + +

Fig. 2A, B. Bouyant density in mctrizamide of crude and purified S. purpuratus poly(A)-containing mRNPs. A Total undialyzed RNP sedimenting at 3&90 S in preparative sucrose gradients was pooled and diluted with an equal volume of IB, which contained 5 mM Mg". EDTA was added to a sample of the diluted RNP to a final concentration of 10mM. Poly(A)-containing mRNPs in the pooled sucrose gradient fractions were also bound to oligo(dT)-cellulose, washed with a buffer containing EDTA (CWB),then eluted with 50% IB (34"C). The unpurified M g + + treated (t-o), unpurified EDTA-treated ( 0 - o ) , and purified mRNP (A-A) were then centrifuged on identical metrizamide gradients as described in Methods. Fractions were digested with proteinase K then hybridized to ['H]poly(U). B Poly(A)-containing mRNPs in the 30-90S region of preparative sucrose gradients were bound to oligo(dT)cellulose and washed with a buffer containing either M g + + (IB) (M) or a buffer containing EDTA (CWB) ( 0 - o ) , then eluted with EB (45"C). KCI was added to the eluted mRNP to a final concentration of 0.1 M before centrifugation. Fractions were digested with proteinase K then hybridized to ['H]poly(U) as in A. The density of all gradients was determined and found to be identical as indicated - and EDTA-treated unpurified mRNPs had peak bouyant

densities of approximately 1.22 g/cm3 in metrizamide, and a range of 1.20 to 1.25 g/cm3 (Fig. 2A). Thus, the absence of M g + + does not change the densities of unfractionated mRNPs. This peak density of 1.22 g/cm3 is similar to the density of nonpolyribosomal myosin mRNPs, and is dissimilar to the 1.17 g/cm3 density of deproteinized RNA in metrizamide [36]. To isolate purified mRNPs, poly(A)-containing mRNPs were bound to oligo(dT)-cellulose, washed consecutively with a buffer containing EDTA and a buffer containing Mg", then eluted with 50% IB (containing 110 mM K+)at 34" C. These purified mRNPs had a major peak at 1.22 g/cm3 in metrizamide, as well as less dense material identical to that of the unpurified mRNPs (Fig.2A). By contrast, when the mRNPs bound to oligo(dT)-cellulose were eluted with the very low ionic strength buffer previously used for eluting sea urchin mRNPs [2q (EB, containing 15 mM KC1) at 45" C, the

17 Fig. 3A, B. Bouyant density in Cs,SO, of S. purpuratus poly(A)-containing mRNPs purified by oligo(dT>cellulose chromatography. A The mRNPs shown in Fig. 1. which had been purified by oligo(dT)-cellulose chromatography, were concentrated by either pelleting or by negative pressure dialysis (dialyzed mRNPs) as described in Methods. These mRNPs were fixed with glutaraldehyde and centrifuged in Cs,SO, to determine the bouyant density of the poly(A)containing mRNPs. The Cs,SO, gradient fractions of the dialyzed (04) or pelleted (-0) purified mRNPs were digested with proteinase K and hybridized to [3H]poly(U) as described in Methods. B The dialyzed, purified mRNPs shown in A were also centrifuged without fixation on Cs,SO, either or without (0-0) a 1 h (Oo C) with (-0) exposure to 10 mM EDTA. The density of fractions from all gradients was determined and found to be identical as indicated

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density of the mRNPs in metrizamide was variable, with a larger fraction of the [3H]poly(U)-hybridizable mRNP banding at decreased densities, indicative of deproteinized RNA (Fig. 2B). Based on these comparisons of bouyant densities I conclude that elution of mRNPs with 50% IB (34" C) from oligo(dT)-cellulose yields a population of mRNPs with heterogeneous densities resembling the densities of unfractionated mRNPs, whereas the previously described procedure for elution with EB (45" C) yields partially deproteinized mRNP [26]. Density of Noncross-linked Egg mRNPs in Cs,SO,

Poly(A)-containing mRNPs eluted from oligo(dT)-cellulose needed to be concentrated for analysis. Since the experimental manipulations may have affected the physical integrity of the mRNPs, the mRNPs were concentrated both by dialysis and by pelleting, as described in Methods. As shown in Fig. 3A both pelleted and dialyzed mRNPs from the 50% IB-eluted fraction of the oligo(dT)-cellulose column displayed a similar peak bouyant density of 1.42 g/cm3 when they were fixed with glutaraldehyde prior to centrifugation in Cs,SO,. However, negative pressure dialysis is the preferable method for concentrating mRNPs, since pelleting results in aggregation of the mRNPs [27l. Since the 1.42 g/cm3 density of the fixed purified mRNPs in Fig. 3A was slightly less than the 1.46 g/cm3 density reported for fixed unpurified egg mRNPs [20], I also determined the bouyant density in Cs,SO, of the same purified mRNPs without fixation with glutaraldehyde, to eliminate the possible cross-linking of free protein to the mRNP. As shown in Fig. 3 B, the unfixed purified mRNPs centrifuged in the presence of M g + + had a peak bouyant density of 1.46 g/cm3, identical to that reported for fiied unpurified mRNPs [20]. From the similarity of the bouyant densities in all of these studies, the mRNPs do not appear to be substantially altered by the purification procedure. To demonstrate that the purified mRNPs were resistant to conditions which dissociate ribosomes, purified, unfixed, mRNPs were reexposed to EDTA before centrifugation in Cs,SO,. As shown in Fig. 3B, the majority of the EDTA-treated mRNPs had a peak bouyant density of approximately 1.50 g/cm3, similar to the 1.46 g / m 3 density of the untreated mRNPs in Fig. 4B, and dissimilar to the

Fig. 4. Translation of A. punctulata mRNPs and mRNP-RNA in a message-dependent rabbit reticulocyte lysate. Sucrose gradient fractions containing mRNPs, and mRNA extracted from the mRNPs, were prepared as described in Methods and translated in vitro. Aliquots were then separated on an SDS 10% polyacrylamide gel which was processed for fluorography and exposed to X-ray film for 36 h. Lane a : Endogenous activity in the lysate not supplemented with mRNA. Lmes h i:Lysate programmed with 254,127, and 64 pg/ml of mRNP, respectively, and translated at 100 mM added K+,0.4 mM added Mg". Lanes eg: Lysate programmed with 274,137, and 69 pg/ml of mRNP-RNA, respectively, and translated at 100mM K + , 0.4mM Mg++. Luneh: Lysate supplemented with 127 pg/ml of mRNP and 137 pg/ml of mRNP-RNA, and translated at 100 mM K + and 0.4 mM Mg++. Lanes Ck: Serial dilutions of mRNP as in lanesbd, translated at 100 mM added K + , 0.8 mM added Mg++. LMes 1-n: Serial dilutions of mRNP-RNA as in lanes e-g, translated at 100mM K + , 0.8 mM Mg". The polypeptide present only in lysates programmed with mRNP-RNA is indicated by a dot ( 0 )

1.66 g/cm3 density of deproteinized RNA [51]. However, the appearance of a small peak of poly(A)-containing material at 1.60g/cm3 indicates that some of the mRNP had been partially deproteinized upon re-exposure to EDTA. Translational Activity of mRNPs

We have recently reported that S. purpuratus egg mRNPs present in preparative sucrose gradient fractions, or purified

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by oligo(dT)-cellulose, are as active as their constituent mRNAs in directing protein synthesis in a cell-free wheat germ lysate 1271. Since a different species of sea urchin, A. punctulala, was used for the UV-crosslinking experiments described in this report, an investigation of the translational activity of A . punctulata mRNPs was undertaken. Unpurified mRNPs cosedimenting with ribosomes in preparative SW27 sucrose gradients were concentrated by negative pressure dialysis, and aliquots were then deproteinized by CsCl gradient centrifugation. Both mRNPs and mRNAs purified from the mRNPs (mRNP-RNA) were then translated in a nuclease-treated, message-dependent rabbit reticulocyte lysate, and the translation products separated by polyacrylamide gel electrophoresis. The most important result is that, using a different species of sea urchin and a different cell-free system, I have confirmed our recent rcport [27] that egg mRNPs are translationally active in vitro. The mRNPdirected translation of trichloroacetic acid-precipitable 35S methionine was about 50% of the incorporation directed by mRNP-RNA (data not shown). Analysis of the products of in vitro translation (Fig. 4) reveals that mRNPs direct the synthesis of a broad spectrum of polypeptides similar to that directed by mRNPRNAs, as previously noted with S. purpuratus mRNPs translated in wheat germ lysates [27l. There is one fascinating difference, however, in the polypeptides synthesized by rnRNPs and mRNP-RNAs. One prominent polypeptide synthesized by mRNP-RNAs (Fig. 4, lanes e, f, g, 1, m, n) was not present in lysates programmed with mRNPs (Fig. 4, lanes b, c, d, i, j, k) even when mRNPs were translated at three mRNP concentrations and two Mg" concentrations. A mixing experiment of lysate containing 127 pg/ml of mRNP and 137 pg/ml of mRNP-RNA (Fig. 4, lane h) shows that the level of synthesis of most polypeptides was increased relative to similar concentrations of mRNP (lane c) or mRNP-RNA (lane f) translated independently, as expected if the concentration of translationally active template were doubled. The polypeptide specific to the mRNP-RNA lanes was present after translation in the mixing experiment at a reduced level (lane h) relative to the level expected from translating mRNP-RNA alone (lane f). This observation clearly merits further attention, as it is consistent with the presence of an inhibitor of translation. More trivial explanations, such as specific degradation of this translation product by a protease in the mRNP preparation, cannot yet be excluded.

Iden tijkation of m RNA-Associated Proteins in Noncross-linked mRNPs To facilitate the identification of putative mRNP proteins, S . purpuratus mRNPs bound to oligo(dT)-cellulose were washed with a buffer containing either EDTA or M g + + before elution with 50% IB (34" C), then some of each eluted peak was pelleted in the ultracentrifuge. The rationale for this experiment was that EDTA, which does not greatly affect the protein content of mRNPs as monitored by density gradient centrifugation, nevertheless removes some low molecular weight proteins, presumably ribosomal in origin [26]. The pelleting step would then separate mRNPs from any free nonaggregated proteins in the sample. Thus, proteins in the EDTA-washed pelleted mRNP preparation were subjected to the more stringent criteria for the identification of noncross-linked mRNP pro-

Fig. 5. Effects of pelleting and EDTA on the polypeptides of nonpolyribosomal mRNPs of S. purpurarus eggs and zygotes. Nonpolyribosomal egg mRNPs present in the 30-90 S region of preparative sucrose gradients were bound to oligo(dT)cellulose, washed with the buffer indicated, and then eluted with a buffer containing Mg' (50% IB) at 34" C. Lanes 1 and 3: Unpelleted egg mRNPs washed with IB and concentrated by negative pressure dialysis. Lanes 2 and 4: Unpellcted egg mRNPs washed with a buffer containing EDTA (CWB) and concentrated by negative pressure dialysis. Lane 6: Egg mRNPs were washed with IB, eluted at 3 4 C, and pelleted through a sucrose pad as described in Methods. Lane 7: Egg mRNPs were washed with CWB, eluted at 34' C, then pelleted as in lane 6. Nonpolyribosomal mRNPs of the zygote (30 min, 14" C aftcr fertilization) were similarly bound to oligo(dT>cellulosc, washed with a buffer containing EDTA (CWB), eluted with 50% IB (34" C), and dialyzed ( h n e 5 , overloaded relative to other lanes) or pelleted ( h e 8 ) . Ten percent polyacrylamide SDS slab gel electrophoresis and staining with Coomassie blue R were as described in Methods. Proteins discussed in the text are indicated by molecular weight x +

teins. At least seven of the approximately 10 more abundant polypeptides in the 50% IB (34" C) fraction were resistant to EDTA and pelleted with the mRNPs through a sucrose pad (apparent M, of 140,000; 118,000; 105,000; 73,000; 69,000; 40,000; and 39,000 in Fig. 5, lanes 7 and S), and consistently met our criteria for mRNP-associated proteins (see Discussion for a description of the criteria). Similar proteins were previously found associated with the unfertilized egg mRNPs eluted from oligo(dT)-cellulose with EB (45" C) [26], conditions which do not maintain the integrity of the rnRNPs (Fig. 2), except that the 105,000 M, protein was previously reported to have a M, of 98,000. In addition to these seven major bands, more than 20 less-abundant proteins co-eluted or were associated with pelleted egg mRNPs in the 50% IB (34" C) fraction, including several proteins with M, between 40,000 and 61,000 (Fig. 5, lanes 1-4). As expected, mRNPs isolated under less stringent con-

19

ditions (Mg" and no pelleting, Fig. 5, lanes 1 and 3; Mg+ and pelleting, lane 6) contained several proteins reduced in mRNP preparations which had been washed with EDTA and then pelleted. The possible association of some proteins with mRNPs via weak or Mg++dependent interactions is unresolved. Most strikingly, an abundant protein of 61,000 M, was associated with both Mg++-and EDTA-washed mRNPs, yet did not pellet with the mRNPs (Fig. 5, lane 7). As described below, however, polypeptides with M, of about 61,000 could be cross-linked to the poly(A)-containing RNA with UV-irradiation. +

Polypeptides Cross-linked to Poly ( A )-Containing RNA

The in vivo covalent cross-linking of proteins to poly(A)containing RNA with UV-irradiation at 254 nm is a rigorous means of identifying those proteins directly in contact with the RNA [23, 371. Since only about 10% of the total hnRNP proteins in tissue culture cells can be cross-linked to hnRNA, this type of experiment indicates only whether particular proteins are present in an RNP, and not the absolute amount of these proteins in the mRNP [23]. A major result of the present study is the demonstration that this technique can be applied to large cells such as eggs. The densitometric tracing presented as Fig. 6 C shows that UV-irradiation cross-links 15-20 proteins to the poly(A)containing RNA of A. punctulata. Control RNPs subjected to the same denaturing isolation conditions, but without prior cross-linking, contain no detectable proteins (Fig. 6D), demonstrating that the UV-irradiation was necessary for the proteins to copurify on oligo(dT)-cellulose with the poly(A)-containing RNA. The higher M, crosslinked proteins migrate similarly on SDS gels to proteins of noncross-linked mRNPs of A. punctulata (Fig. 6B) and S. purpuratus (Fig. 6A) which had been isolated under nondenaturing conditions (Fig. 6). In this experiment the crosslinked proteins of less than about 60,000 M, had an electrophoretic mobility slightly less than the noncross-linked mRNP proteins. As good correspondence between crosslinked and noncross-linked mRNP proteins in this M, range was observed in another experiment, this suggests that the altered mobilities observed in Fig. 6 may have been due to incomplete removal from the protein of cross-linked RNA. Although the relative abundance of different proteins after cross-linking is strikingly different, particularly in the lower molecular weight range, this result need not reflect the relative proportions of these proteins in the native mRNP because, as discussed by previous investigators [23, 371, only a small percentage of the total RNP protein is cross-linked. In addition, preparations of noncross-linked mRNPs may contain low molecular weight polypeptides, presumably ribosomal in origin (Fig. 5). A Comparison of Polypeptides of Noncross-linked Nonpolyribosomal mRNPs from Unfertilized Eggs and Zygotes

An important question is whether the stored nonpolyribosoma1 mRNPs of the egg undergo changes in their constituent proteins following fertilization. To investigate this problem I compared the proteins of purified nonpolyribosomal mRNPs obtained from unfertilized S. purpurafus eggs and from zygotes (30 min, 14" C, after fertilization). I detected no qualitative differences in the most prevalent proteins of EDTA-washed zygote mRNPs that were unpelleted

L

4 D

Fig. 6A-D. Polypeptides associated with UV-crosslinked mRWs of A . punctulata eggs. The proteins of EDTA-washed, pelleted, noncross-linked nonpolyribosomal egg mRNPs of S. purpurarus (A) and A . puncrulara (B) are shown for comparison with proteins cross-linked in vivo to nonpolyribosomal egg mRNPs of A . puncruluto (C).Noncross-linked mRNPs subjected to the same denaturing conditions as the cross-linked mRNPs were used as controls for cross-linking 0). All RNPs were digested with ribonuclease then separated on a 7-12% exponential polyacrylamide gradient SDS slab gel which was stained with Coomassie blue. Numbers designate major polypeptides discussed in the text (M, x

(Fig. 5, lane 5 ) or pelleted (Fig. 5, lane 8) compared to proteins of EDTA-washed egg mRNPs that were unpelleted (Fig. 5, lane 4) or pelleted (Fig. 5, lane 7), although qualitative and quantitative differences in the less prevalent proteins between 40,OOO and 61,000 M, were evident. Within the limits of protein separation of one-dimensional sodium dodecylsulfate/polyacrylamide gel electrophoresis I conclude that fertilization per se does not result in qualitative changes in the most abundant proteins associated with the nonpolyribosomal mRNPs in this species. However, this has yet to be extensively investigated using UVcrosslinking, and it remains possible that less prevalent mRNP proteins are physiologically important. A Comparison of Polypeptides of Noncross-linked Nonpolyribosomal and Polyribosome-Derived mRNPs

Since mRNA-associated polypeptides may affect the utilization of the message, I investigated whether nonpolyribosoma1 and polyribosomal mRNPs contain similar polypeptides. mRNPs were thus purified from eggs and from polyribosomes of hatched blastula embryos. Nonpolyribosomal

20

preted cautiously, as it is always possible that, despite all precautions, some nonpolyribosomal mRNPs may copurify with the polyribosomes. The use of purified hatched blastula polyribosomal mRNPs for comparisons, however, argues against extensive contamination of polyribosomes with nonpolyribosomal mRNPs, since there are very few 30-90 S nonpolyribosomal mRNPs at this stage which bind to oligo(dT)-cellulose (data not shown), and since in some experiments only the largest polyribosomes were isolated.

Discussion

Fig. 7A-C. Polypeptides associated with polyribosomal and nonpolyribosomal mRNPs of S.purpurutus. Polyribosomes of hatched blastulae (A) and nonpolyribosomal mRNPs of eggs (B) were exposed to sufficient EDTA to dissociate ribosomes from the polyribosomes, then isolated on oligo(dT)cellulose as described in Methods. The egg material sedimenting at 3&90 S in preparativesucrose gradients which does not bind oligo(dT)-cellulose is shown in C. The poly(A)-containing mRNPs were eluted from the oligo(dT)cellulose with 50% IB (34" C), ethanol precipitated, the proteins dissociated with LiCl and urea, and then subjected to electrophoresis on a 10% acrylamide SDS slab gcl and stained with Coomassie blue. Numbers designate the M, ( x or major polypeptides discussed in the text

mRNPs from S.purpuratus eggs contain about seven major proteins (Fig. 7B). In contrast, polyribosomal mRNPs of hatched blastula embryos contain one major protein of 73,000 M, and, relative to the 73,000M, protein, less but detectable amounts of the six other major polypeptides present in nonpolyribosomal mRNPs (Fig. 7A). These mRNP polypeptides are not major constituents of the ribosome-enriched material which does not bind to oligo(dT)cellulose (Fig. 7C). Two additional lines of evidence also encourage the conclusion that polyribosomal and nonpolyribosomal mRNPs contain qualitatively, if not quantiatively, similar proteins. First, all of the above-mentioned major proteins in nonpolyribosomal mRNPs were also detected, though again at decreased levels relative to the 73,000 M, protein, in polyribosomes from 16-cell embryos, which contain primarily messages synthesized during oogenesis (data not shown). Second, in the one experiment to date, identical polypeptides were cross-linked with UVirradiation to nonpolyribosomal mRNPs of the zygote and to polyribosomal mRNPs of hatched blastulae (data not shown). The data in Fig. 7 do suggest, however, that there may be at least stoichiometric differences, if not qualitative differences, between the proteins associated with polyribosoma1 and nonpolyribosomal mRNPs. Stoichiometric differences may arise by the addition of poly(A)-binding protein to the poly(A) region of messages concomitant with lengthening of the poly(A) tail after fertilization [33]. Stoichiometric differences could also arise by the selective loss of proteins. This comparison of nonpolyribosomal and polyribosome-derived mRNPs has, however, been inter-

The post-fertilization activation of protein synthesis in sea urchin eggs is mediated by pleiotropic responses to ionic signals. Since message availability may be a major level of translational regulation which can be controlled by mRNA-associated proteins, I have identified polypeptides associated with stored mRNPs and compared them with polypeptides associated with polyribosomal mRNPs. Poly(A)-containing mRNPs from sea urchin eggs and embryos were purified both after homogenization and isolation using near-physiologic ionic conditions which maintained the structural integrity of the mRNP, and after irradiation of living cells with UV light to cross-link in vivo those proteins associated with poly(A)-containing RNA. The proteins associated with the mRNPs were then analyzed by sodium dodecylsulfate/polyacrylamidegel electrophoresis. I have used several criteria to identify proteins as integral components of mRNPs to further investigations of the functions of these proteins. The first three criteria show that the isolated particles are complexes of RNA and protein and are free of contaminating ribosomes. The next three criteria show that under several conditions the putative mRNP proteins copurify with mRNPs, and the last criterion shows that the RNAs of the mRNPs contain translatable mRNAs. 1) Density analyses of the putative mRNPs were used to establish that the RNA and protein are present as a complex. The presence of mRNPs with characteristic bouyant densities in metrizamide and Cs,SO, was demonstrated for mRNP complexes eluted with 50% IB ( 3 4 C) from the oligo(dT)-cellulose column. 2) Specific binding of the mRNP to the oligo(dT)moiety was demonstrated by the absence of binding of any proteins to unmodified cellulose (data not shown). 3) The absence of ribosomes and ribosomal subunits was clearly demonstrated by the absence in sucrose and Cs2S0, gradients of any material absorbing at 260nm which was characteristic of ribosomes (data not shown). 4) At least seven putative mRNP proteins of S.purpurafus (apparent M, of 140,000; 118,000; 105,000; 73,000; 69,000; 61,000; and 40,000) were detected on SDS gels and shown to copurify with the mRNA since they were thermally-eluted with approximately 80% of the poly(A)-containing RNA applied to the oligo(dT)-cellulose. However, two-dimensional gel electrophoresis (data not shown) clearly suggests that there may be a greater number of mRNP proteins. Since it is difficult to develop a model in which poly(A)-containing RNAs all contain this many proteins, it is possible that there is heterogeneity in the proteins associated with messages of different metabolic fate, or with messages which differ in sequence or the timing of their synthesis during oogenesis. It is also possible that

21

some of the putative mRNP proteins are specific to mitochondrial RNAs which may contaminate these preparations WI. 5 ) Putative mRNP proteins cosedimented with the purified mRNPs when layered over a sucrose pad and centrifuged to separate mRNPs from free protein. Since most of the above putative mRNP proteins, as well as approximately 20 less prevalent proteins, were present in the clear pellet of mRNPs, this suggests that these proteins were an integral part of the mRNP. A prevalent S. purpuratus polypeptide of 61,000 M, did not pellet with the mRNPs, nor did some of the less prevalent proteins. However, A . punctufuta polypeptide(s) of approximately 61,000 M,were crosslinked in vivo to RNA with UV-irradiation, which suggests that it is an mRNP protein, though perhaps loosely associated. The presence of loosely-associated mRNP proteins could have important consequences for translation in vivo, which may not be reflected by translating mRNPs in vitro. 6) The least ambiguous criterion for identifying RNAassociated proteins is cross-linking with UV-irradiation, a technique not previously applied to embryonic cells. The results clearly showed that many of the putative mRNP proteins which were associated with mRNPs isolated under physiologic ionic conditions were also cross-linked in vivo to the poly(A)containing RNA by UV-irradiation. The limitation of cross-linking, however, is that only some proteins in direct contact with the mRNA are cross-linked [23]. Thus, stoichiometric differences in mRNP proteins cannot readily be determined, and any mRNP proteins which rely on protein-protein interactions rather than protein-RNA interactions would not be cross-linked. 7) mRNA extracted from purified mRNPs was competent in directing protein synthesis in a cell-free system derived from wheat germ, suggesting that the mRNA had not been degraded during purification of the mRNP [27l. There is at present some ambiguity in the literature as to whether or not the same polypeptides are associated with nonpolyribosomal and polyribosomal mRNPs. The use of non-denaturing mRNP isolation conditions has led to the conclusion that nonpolyribosomal and polyribosomal mRNPs of chick muscles [2], duck erythroblasts [48], and the acellular slime mold Physarum [l] contain qualitatively distinct sets of proteins. In contrast to these results, UVirradiation of mouse L cells cross-linked qualitatively similar proteins to both nonpolyribosomal and polyribosomal mRNPs [37]. Our earlier study provided preliminary evidence that several proteins were decreased or absent in sea urchin polyribosomal mRNPs [26]. This comparison of proteins associated with nonpolyribosomal and polyribosoma1 sea urchin mRNPs has been repeated several times and, in the present study, I found that preparations of polyribosomal mRNPs had one prevalent protein of 73,000 MI. However, detectable amounts of the same proteins found in preparations of nonpolyribosomal mRNPs were also present in polyribosomal mRNPs. Since most of these proteins, relative to the 73,000 MI protein, were decreased in polyribosomal mRNPs as compared with nonpolyribosomal mRNPs, it would be easy to miss, or dismiss, the presence of these minor proteins on the gels. In preliminary experiments, however, UV-crosslinking of proteins to RNA in living sea urchin embryos yielded a result consistent with the findings of Setyono and Greenberg [37] that nonpolyribosomal and polyribosomal mRNPs contained the same set of prevalent proteins. Therefore, both

nonpolyribosomal mRNPs and polyribosomal mRNPs of sea urchins appear to contain similar polypeptides, though in different proportions. Quantitative comparisons and continued investigation using UV-crosslinking are, however, still required. Supportively, Peters and Jeffery [33] reported that the pattern of sea urchin egg proteins associated with the poly(A) tract of messages did not undergo changes in response to fertilization, and Young and Raff [51] showed that maternal mRNPs did not undergo changes in bouyant density after entering into polyribosomes. Three caveats for comparisons of different populations of sea urchin mRNPs are that (1) nonpolyribosomal mRNPs may contaminate polyribosome preparations, (2) the mRNPs are selected for purification on the basis of their poly(A) tail, and it is possible that some poly(A)containing mRNPs undergo developmental changes in their poly(A) tail [reviewed in 341, such that they no longer bind to oligo(dT)cellulose, and (3) about 70% of the poly(A)-containing RNA of eggs contain interspersed repetitive sequences, and it is unresolved whether much of this RNA becomes functional polyribosomal mRNA [47]. Since preparations of nonpolyribosomal mRNPs were translationally active in vitro in the present study, however, it may not be surprising that nonpolyribosomal and polyribosomal mRNPs contain qualitatively, if not quantitatively, similar polypeptides. Since both Artemia gastrulae [ll] and sea urchin eggs [34] contain large pools of nonpolyribosomal mRNPs which subsequently enter into polyribosomes, it is useful to compare the proteins associated with the stored mRNPs of these two organisms. Slegers et al. [41] have recently purified the poly(A)-containing mRNPs from Artemia by chromatography on oligo(dT)-cellulose in the presence of moderate (250 mM) salt concentrations. Since the mRNPs from both Artemia and sea urchins have been purified using the same affinity chromatography procedure and similar salt concentrations (220-250 mM), it is significant to note that very similar mRNP proteins have been found in both organisms. Six to nine major mRNP proteins were purified from Artemia, with MI of 115,000; 87,000; 76,000; 65,000; 50,000; 45,000; 38,000; 23,500; and 21,000. Of the sea urchin mRNP proteins detected by staining SDS gels with Coomassie blue, those with M, of 118,000; 73,000; 40,000; and 39,000 are very similar in MI to the 115,000; 76,000; and 38,000 MI proteins of Artemia mRNPs. In addition, Slegers et al. [41] have shown that cytoplasmic Artemia proteins of 112,000 and 68,000 M, are RNA-binding proteins. These two Artemia RNA-binding proteins are similar in M,to the 105,000 and 69,000 sea urchin nonpolyribosomal mRNP proteins. Therefore, many of the sea urchin mRNP proteins are similar in MI to mRNP or RNA-binding proteins of Artemia. with the possible exception of the sea urchin 340,OOO M, protein. Marvil et al. [22] and Nowak et al. [31] have recently reported the purification of a 40,OOO M, helix destablizing protein (HD40) from ribosome preparations of Artemia. These investigators have shown that HD40 preferentially binds single-stranded nucleic acids, protects against nuclease digestion, and inhibits in vitro translation under some conditions. As one might expect similar activity from some proteins associated with nonpolyribosomal mRNPs, it is interesting to speculate that the 40,000 MIprotein found on mRNPs in the present study may be similar to HD40. However, HD40 (generously provided by Dr. J.O. Thomas) does not comigrate on SDS gels with the sea urchin pro-

22

teins. nor does polyclonal anti-HD40 antibody (also provided by Dr. Thomas) cross-react with any sea urchin mRNP proteins (data not shown). These results may not be too surprising, however, considering the evolutionary divergence of these species. It has recently been suggested that one initiation factor, eIF-3, melts the secondary structure of messages, though this activity may be due to the presence of cap-binding proteins [42, 431. Since message secondary structure may be an important mechanism of translational control [21, 321, it is interesting that eIF-3 contains polypeptides similar in M, to polypeptides of sea urchin nonpolyribosomal mRNPs. eIF-3 from rabbit reticulocytes is a large complex consisting of four major Polypeptides with M, of 140,000; 120,000; 110,OOO; and 69,000, and five minor polypeptides with M, of 47,000; 45,000; 37,000; 31,000; and 28,000 M, [3]. The four major eIF-3 polypeptides are very similar or identical in M, to the 140,000; 118,000; 105,000; and 69,000 M, proteins associated with the sea urchin nonpolyribosomal mRNPs, and the minor eIF-3 polypeptides are similar in M, to less prevalent mRNP proteins. The possibility that eIF-3 may be associated with mRNA is supported by evidence that eIF-3 may bind to RNA in the absence of the 40 S subunit [38]. Although the 2- to 3-fold increase in the rate of elongation after fertilization [4, 121 serves to multiply the magnitude of the increased rate of protein synthesis, there is at present no consensus on the mechanism(s) leading to an approximately 25-fold increase in the number of ribosomes in polyribosomes within 2 h after fertilization [14]. The unmasking of repressed mRNPs has frequently been invoked as the primary level of control. In the first study to directly test this hypothesis, however, S.purpuratus mRNPs isolated in a K + and EDTA buffer, or a Na+ buffer containing either Mg' + or EDTA, translated at 30-100% of the rate of mRNA extracted from the mRNPs, and only mRNPs isolated in K + and Mg' were much less translatable than their mRNP-RNA [18]. In a direct reanalysis of these important experiments, crude and oligo(dT)-cellulose-purified S. purpuratus mRNPs, prepared in buffers containing K and Mg+', always synthesized polypeptides in a wheat germ lysate at 50-100% of the rate of mRNA extracted from the mRNPs [27. Similarly, in the present study, crude preparations of A. punctulata mRNPs synthesized polypeptides in a rabbit reticulocyte lysate at about 50% the rate of their constituent RNA. In a previous study with A. punctulata, Murray and Sosnowski [29] reported that mRNPs in a post-mitochondria1 supernatant translated at 25-50% of the rate of RNA extracted from the supernatant, when either the supernatant or supernatant RNA was added to a rabbit reticulocyte lysate. Taken together, these studies on mRNP translation suggest that there may be about a 2-fold difference between the translational activity of mRNPs and their constituent mRNAs, at least in vitro. While these studies do not prove that protein synthesis in eggs is limited by some mechanism other than message availability, they do raise reasonable doubts. A long overlooked but potentially important contribution to the activation of protein synthesis is made by egg ribosomes, which are less active than ribosomes derived from embryonic polyribosomes [6]. Finally, the asymmetric spatial distribution of histone sequences in eggs [40], and the increased association during development of mRNA with a detergent-insoluble cytoskeleton [28], serve as reminders that cell structure +

+

may contribute to translational control. The further analysis of mRNA- and ribosome-associated macromolecules is but a partial step to our understanding the activation of protein synthesis in eggs. Acknowledgements. I thank Dr. Merrill B. Hille for support and helpful comments on the manuscript, Ms. Sandra Mayrand for technical advice on cross-linking, Dr. Tim Hunt for providing a rabbit reticulocyte lysate, Dr. Michael V. Danilchik for collaborating on the in vitro translation experiments, and Drs. Thoru Pederson, W.R. Jeffery, and Joel Rosenbaum for enabling portions of this work to be conducted at the Marine Biological Laboratory. This work was supported by NIH grants HD-11070 (MBH), GM07784, and HD-07098 (Physiology and Embryology courses, Marine Biological Laboratory, Woods Hole), NRSA HD-07183 (RTM), and American Cancer Society Institutional Grants IN-26T and IN-26U (RTM).

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