Translational control in early sea urchin embryogenesis: initiation factor eIF4F stimulates protein synthesis in lysates from unfertilized eggs of Strongylocentrotus purpuratus

We have used cell-free translation systems from unfertilized eggs and embryos of the sea urchin Strongylocentrotus purpuratus to analyze the mechanisms limiting protein synthesis in early embryogenesis. Unfertilized egg lysates supplemented with nuclease-treated reticulocyte lysate were stimulated 2-4-fold in incorporation of radioactive amino acid into protein. Thirty-minute zygote lysates supplemented in this way were not stimulated. These results suggested that a component limiting translation in the unfertilized egg lysate was provided by the nuclease-treated lysate and that this component was no longer limiting protein synthesis following fertilization. In view of these results, partially fractionated lysates and individual purified translational components from mammalian cells were tested for stimulation of the unfertilized egg lysate. A 1000000g supernatant devoid of ribosomal subunits also stimulated the unfertilized egg lysate. Thus, the stimulation was not due to the addition of active ribosomal subunits but to soluble elements in the reticulocyte lysate. Of the soluble components tested, only the cap-binding protein complex eIF4F caused a dramatic stimulation of the unfertilized egg lysate (2-3.5-fold). The 30-min zygote lysate was not stimulated by eIF4F or by any of the other components tested, supporting the hypothesis that a block in the translational machinery is removed at fertilization. A rabbit reticulocyte shift assay was used to analyze whether mRNA is limiting in early development. When unfertilized egg lysate was added to the shift assay, there was no shift in radioactivity from 43S to 80S complexes, indicating the unfertilized egg mRNA is not available for translation.(ABSTRACT TRUNCATED AT 250 WORDS)     

An assessment of the masked message hypothesis: sea urchin egg messenger ribonucleoprotein complexes are efficient templates for in vitro protein synthesis

The rate of protein synthesis in unfertilized sea urchin eggs is very low, although all components for protein synthesis are present. To determine whether egg messenger RNAs are unavailable for translation because of “masking” by phenol-soluble inhibitors, crude and purified nonpolyribosomal messenger ribonucleoprotein complexes (mRNPs) from eggs of Strongylocentrotus purpuratus were translated in vitro in a wheat germ cell-free system. Crude and purified egg mRNPs were nearly as translatable as the mRNAs extracted from the mRNPs, suggesting that the mRNPs were not masked. No difference in the relative translational activities of mRNPs and their constituent mRNAs was revealed by isolating the mRNPs in buffers of different ionic strength or in the presence of protease and ribonuclease inhibitors. Furthermore, kinetic analysis of the in vitro translation and translation of the mRNPs and mRNAs at several concentrations of K⁺, Mg²⁺, and template all indicate that mRNPs are efficient templates for directing protein synthesis. Separation on polyacrylamide gels of the products of in vitro and in vivo translation demonstrated that both mRNPs and mRNAs extracted from the mRNPs synthesized in vitro high-molecular-weight polypeptides, some of which were also synthesized in vivo. Although sea urchin egg mRNPs may not be masked, there are several alternative mechanisms for regulating translation in the egg.     

Fertilization triggers unmasking of maternal mRNAs in sea urchin eggs.

Fertilization of sea urchin eggs results in a large increase in the rate of protein synthesis which is mediated by the translation of stored maternal mRNA. The masked message hypothesis suggests that messenger ribonucleoprotein particles (mRNPs) from unfertilized eggs are translationally inactive and that fertilization results in alterations of the mRNPs such that they become translationally active. Previous workers have isolated egg mRNPs by sucrose gradient centrifugation and have assayed their translational activity in heterologous cell-free systems. The conflicting results they obtained are probably due to the sensitivity of mRNPs to artifactual activation and inactivation. Previously, we demonstrated that unfractionated mRNPs in a sea urchin cell-free translation system were translationally inactive. Now, using large-pore gel filtration chromatography, we partially purified egg mRNPs while retaining their translationally repressed state. Polysomal mRNPs from fertilized eggs isolated under the same conditions were translationally active. The changes in the pattern of proteins synthesized by fractionated unfertilized and fertilized mRNPs in vitro were similar to those changes observed in vivo. Treatment of egg mRNPs with buffers containing high salt and EDTA, followed by rechromatography, resulted in the activation of the mRNPs and the release of an inhibitor of translation from the mRNPs. Analysis of the inhibitory fraction on one-dimensional sodium dodecyl sulfate gels indicated that this fraction contains a complex set of proteins, several of which were released from high-salt-EDTA-activated mRNPs and not from inactive low-salt control mRNPs. One of the released proteins may be responsible for the repression of egg mRNPs in vitro and be involved in the unmasking of mRNPs at fertilization.     

Inhibitor of translational initiation in sea urchin eggs prevents mRNA utilization

Actively reinitiating cell-free translation systems from sea urchin eggs and embryos have been developed. The extracts retain the overall differences in protein synthetic activity observed in intact eggs and embryos. The effect of combining extracts from eggs and embryos suggests the presence of a dominant inhibitor of translation in the egg. This inhibitor also prevents the initiation of translation in a cell-free system from rabbit reticulocytes. In the reticulocyte system, the egg inhibitor causes the accumulation of 48 S preinitiation complexes, as measured by the accumulation of initiator tRNA and globin mRNA on the small ribosomal subunit. Accumulation of this uncommon intermediate suggests either that the inhibitor prevents the binding of the 60 S ribosomal subunit, or that it prevents migration of the 40 S subunit from the 5' end of mRNA to the first AUG.     

Poly(A)-containing messenger ribonucleoprotein complexes from sea urchin eggs and embryos: polypeptides associated with native and UV-crosslinked mRNPs

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 Cs2SO4 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 embryonic polyribosomal mRNPs were also qualitatively similar to those present in nonpolyribosomal mRNPs, although stoichiometric differences may exist.     

Possible involvement of poly(A) in protein synthesis.

The experiments of this paper have re-evaluated the possibility that poly(A) is involved in protein synthesis by testing whether purified poly(A) might competitively inhibit in vitro protein synthesis in rabbit reticulocyte extracts. We have found that poly(A) inhibits the rate of translation of many different poly(A)+ mRNAs and that comparable inhibition is not observed with other ribopolymers. Inhibition by poly(A) preferentially affects the translation of adenylated mRNAs and can be overcome by increased mRNA concentrations or by translating mRNPs instead of mRNA. The extent of inhibition is dependent on the size of the competitor poly(A) as well as on the translation activity which a lysate has for poly(A)+ RNA. In light of our results and numerous experiments in the literature, we propose that poly(A) has a function in protein synthesis and that any role in the determination of mRNA stability is indirect.     

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

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 Cs₂SO₄ 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.     

Shut-off of actin biosynthesis in adenovirus serotype-2-infected cells

Adenovirus produces a dramatic shut-off of host protein synthesis after infection of HeLa cells. The level of actin messenger RNAs remained relatively unchanged after viral infection, when assayed by in vitro translation and two-dimensional gel electrophoresis analysis of the proteins or hybridization of the total cytoplasmic RNAs to the human actin gene. The distribution of actin mRNA in the polyribosomes is altered after adenovirus infection, with small polyribosomes and monoribosomes of the infected cells occupied by actin messages untranslatable in a rabbit reticulocyte lysate. The large polyribosomes still retain enough functional mRNAs to provide significant levels of actin protein in a rabbit reticulocyte in vitro translation system. In contrast, in homologous infected cell lysates, the translation of exogenous actin mRNA is greatly reduced when compared to uninfected HeLa cell lysates. In nuclease-treated uninfected or infected HeLa cell-free extracts, translation of viral mRNA is equally efficient and higher than that of actin mRNA. Thus, translational regulatory mechanisms which include inactivation of a part of the actin mRNA population accompanied by displacement to small polysomes and/or virus-induced modification of the cellular translational machinery to discriminate against cellular actin mRNA seem to account for the sharp reduction in actin protein synthesis of adenovirus-infected cells.     

'Unmasking' of stored maternal mRNAs and the activation of protein synthesis at fertilization in sea urchins

The isolation and in vitro assay of maternal mRNPs has led to differing conclusions as to whether maternal mRNAs in sea urchin eggs are in a repressed or 'masked' form. To circumvent the problems involved with in vitro approaches, we have used an in vivo assay to determine if the availability of mRNA and/or components of the translational machinery are limiting protein synthesis in the unfertilized egg. This assay involves the use of a protein synthesis elongation inhibitor to create a situation in the egg in which there is excess translational machinery available to bind mRNA. Eggs were fertilized and the rate of entry into polysomes of individual mRNAs was measured in inhibitor-treated and control embryos using 32P-labeled cDNA probes. The fraction of ribosomes in polysomes and the polysome size were also determined. The results from this in vivo approach provide strong evidence for the coactivation of both mRNAs and components of the translational machinery following fertilization. The average polysome size increases from 7.5 ribosomes per message in 15 min embryos to approximately 10.8 ribosomes in 2 h embryos. This result gives additional support to the idea that translational machinery, as well as mRNA, is activated following fertilization. We also found that individual mRNAs are recruited into polysomes with different kinetics, and that the fraction of an mRNA in polysomes in the unfertilized egg correlates with the rate at which that mRNA is recruited into polysomes following fertilization.     

Translation of partially purified poly(A)+ protamine messenger RNA components in wheat germ and rabbit reticulocyte cell-free systems. Evidence for translational control mechanisms.

The coding properties of individual poly(A)+ protamine mRNA subcomponents have been explored by analysis of their translation products in two different cell-free protein synthesis systems, the rabbit reticulocyte lysate and the wheat germ S-30, both of which can translate total protamine mRNA. The products synthesized in the reticulocyte lysate in the presence of total poly(A)+ PmRNA consisted mainly of protamine components CII and CIII with component CI only a minor product. However, in the wheat germ S-30, the same mRNA preparation supported the synthesis of all three protamine components, in approximately equal amounts. In addition a new polypeptide, a putative fourth protamine component, labelled CO, was also synthesized. The translation products of subcomponents of poly(A)+ PmRNA separated as individual bands on polyacrylamide gels were similarly analyzed and it was shown that each of the isolated poly(A)+ PmRNA species could stimulate the incorporation of [3H]arginine into protamines in both translational systems. Although each mRNA band stimulated the synthesis of one particular protamine polypeptide predominantly in a given cell-free system, the same RNA preparation was found to direct preferentially the synthesis of a different protamine component in the second cell-free system. The products synthesized in the rabbit reticulocyte lysate in the presence of the individual mRNA species still showed component CI present as a minor product.     

Studies on a nonpolysomal ribonucleoprotein coding for myosin heavy chains from chick embryonic muscles.

A messenger ribonucleoprotein (mRNP) particle containing the mRNA coding for the myosin heavy chain (MHC mRNA) has been isolated from the postpolysomal fraction of homogenates of 14-day-old chick embryonic muscles. The mRNP sediments in sucrose gradient as 120 S and has a characteristic buoyant density of 1.415 g/cm3, which corresponds to an RNA:protein ratio of 1:3.8. The RNA isolated from the 120 S particle behaved like authentic MHC mRNA purified from chick embryonic muscles with respect to electrophoretic mobility and ability to program the synthesis of myosin heavy chain in a rabbit reticulocyte lysate system as judged by multi-step co-purification of the in vitro products with chick embryonic leg muscle myosin added as carrier. The RNA obtained from the 120 S particle was as effective as purified MHC mRNA in stimulating the synthesis of the complete myosin heavy chains in rabbit reticulocyte lysate under conditions where non-muscle mRNAs had no such effect. Analysis of the protein moieties of the 120 S particle by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows the presence of seven distinct polypeptides with apparent molecular weights of 44,000, 49,000, 53,000, 81,000, 83,000, and 98,000, whereas typical ribosomal proteins are absent. These results indicate that the 120 S particles are distinct cellular entities unrelated to ribosomes or initiation complexes. The presence of muscle-specific mRNAs as cytoplasmic mRNPs suggests that these particles may be involved in translational control during myogenesis in embryonic muscles.     

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

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. © 1994 Wiley-Liss, Inc.     

Inhibition of protein synthesis in reticulocyte lysates by a double-stranded RNA component in HeLa mRNA

Double-stranded RNA (dsRNA) inhibits protein synthesis initiation in rabbit reticulocyte lysates by the activation of a latent dsRNA-dependent cAMP-independent protein kinase which phosphorylates the α-subunit of the eukaryotic initiation factor eIF-2. In this study, we describe a dsRNA-like component which is present in preparations of HeLa mRNA (poly A⁺) isolated from total cytoplasmic RNA. The inhibitory species in the HeLa cytoplasmic mRNA was detected by (a) its ability to inhibit protein synthesis with biphasic kinetics in reticulocyte lysates translating endogenous globin mRNA, and (b) by the inefficient translation of HeLa cytoplasmic mRNA in a nuclease-treated mRNA-dependent reticulocyte lysate. The inhibitory component was characterized as dsRNA by several criteria including (i) the ability to activate the lysate dsRNA-dependent eIF-2α kinase (dsI); (ii) the prevention of both dsI activation and inhibition of protein synthesis by high levels of dsRNA or cAMP; (iii) the reversal of inhibition by eIF-2; and (iv) the inability to inhibit protein synthesis in wheat germ extracts which lack latent dsI. By the same criteria, the putative dsRNA component(s) appears to be absent from preparations of HeLa mRNA isolated exclusively from polyribosomes.     

Poly(A) dependent translation in rabbit reticulocyte lysate

Poly(A) tail has been known to enhance mRNA translation in eukaryotic cells. However, the effect of poly(A) tail in vitro is rather small. Rabbit reticulocyte lysate (RRL) is widely used for studying translation in vitro. Translation in RRL is typically performed in nuclease-treated lysate in which most of the endogenous mRNA have been removed. In this condition, the difference in the translational efficiency between poly(A)⁺ and poly(A)⁻ mRNAs is about two-fold. We studied the effect of poly(A) tail on luciferase mRNA translation in nuclease uncreated reticulocyte lysate, in which endogenous globin mRNAs were actively translated. In the case of capped mRNAs. stimulation of translation by poly(A) addition was about 1.5- to 1.6-fold and the effect of the poly(A) length was small. However, in the case of uncapped mRNAs, the addition of poly(A) tail increased luciferase expression over 10-fold. The effect of the poly(A) tail was dependent on its length. The difference in the translational efficiency was not due to the change of mRNA stability. These data indicate that RRL has the potential to translate mRNA in a poly(A) dependent manner.     

Regulation of in vitro translation by double-stranded RNA in mammalian cell mRNA preparations.

Polyadenylated mRNA has been purified from a variety of human and mouse cell sources. These preparations are actively translated in the wheat germ cell-free system but have only poor ability to stimulate the nuclease-treated reticulocyte lysate. The translation of endogenous and exogenous globin mRNA is strongly inhibited by the poly(A)+ RNA preparations in reticulocyte lysates. Both polysomal and non-polysomal RNA have similar effects but poly(A)+ RNA is almost 2000-fold more inhibitory than poly(A)-RNA on a weight basis. The inhibition is abolished in the presence a high concentration of poly(I).poly(C). Analysis of endogenous eIF-2 in the lysate reveals that the subunit becomes extensively phosphorylated in the presence of the inhibitory poly(A)+ RNA. Prolonged incubation of lysate with poly(A)+ RNA also causes some nucleolytic degradation of polysomal globin mRNA. These characteristics suggest that some eukaryotic cell mRNAs contain regions of double-stranded structure which are sufficiently extensive to activate translational control mechanisms in the reticulocyte lysate.     

Changes in rates of protein synthesis and eukaryotic initiation factor-4 inhibitory activity in cell-free translation systems of sea urchin eggs and early cleavage stage embryos.

The characteristics of cell-free translation systems prepared from unfertilized eggs and early cleavage stage embryos of the sea urchin, Strongylocentrotus purpuratus, closely reflect the developmentally regulated changes in protein synthesis initiation observed in vivo. Cell-free translation systems prepared over the first 0-6 h following fertilization show gradually increasing activities, mimicking the changes observed in vivo. The mechanisms underlying these increases are complex and occur at several levels. One factor contributing to the rise in protein synthetic rate is the gradual increase in eukaryotic initiation factor (eIF)-4 activity. This is correlated with the progressive inactivation of an inhibitor of eIF-4 function, which can be reactivated by in vitro manipulations. The relatively slow activation of eIF-4 follows similar kinetics to the increased utilization of maternal mRNA and ribosomes, in contrast to the rapid rise in maternal mRNA activation, and the increase in eIF-2B activity. This slow release from eIF-4 inhibition following a rapid release from eIF-2B inhibition and increased mRNA availability is reflected in the pattern of initiator tRNA binding to the small ribosomal subunit observed in cell-free translation systems. In translation systems from unfertilized eggs, initiator tRNA is unable to interact with the small ribosomal subunit, consistent with an initial block in both eIF-2B and eIF-4 activity. In translation systems from 30-min embryos, 48 S preinitiation complexes accumulate, reflecting the release from inhibition of mRNA availability and eIF-2B activity, but continued low activity of eIF-4. The accumulation of initiator tRNA in 48 S preinitiation complexes disappears gradually in translation systems from later embryos, as eIF-4 is slowly released from inhibition.     

Nonselective inhibition of messenger RNA translation by highly purified low molecular weight RNA species from ribosomal salt wash of chick embryonic muscle

A low molecular weight RNA species, in the 70–90 nucleotide size range (iRNA), has been purified from the ribosomal salt wash of chick embryonic muscle by a combination of DEAE-cellulose and hydroxyapatite chromatography. This method yields iRNA free from contaminating tRNA and gives better and more reproducible yields than those obtained with our previous method involving lengthy dialysis of the salt wash. The iRNA at a concentration of 20–80 ng range strongly inhibits the translation of homologous and heterologous mRNAs i.e. chick muscle poly(A)⁺mRNA and rabbit globin mRNA; uncapped mRNA; and poly(A)^-mRNA in micrococcal nuclease-treated reticulocyte lysate indicating that inhibition by iRNA is nonselective in nature. The translation of endogenous globin mRNA and polysomes in the lysate is strikingly less sensitive to iRNA suggesting that the initiation step is primarily affected by iRNA. The iRNA does not appear to be double-stranded RNA. It is concluded that iRNA is distinct from other low molecular weight RNA species described in the literature which modulate protein synthesis in cell-free systems.     

Selection of AUG initiation codons differs in plants and animals.

The influence of the nucleotide at position -3 relative to the AUG initiation codon on the initiation of protein synthesis was studied in two different in vitro translation systems using synthetic mRNAs. The four mRNAs, transcribed from cDNAs directed by an SP6 promoter, were identical except for mutations at nucleotide -3. In each case, translation of mRNAs produced a single protein of Mr = 12,600. Relative translational efficiencies showed a hierarchy in the reticulocyte lysate system (100, 85, 61 and 38% for A, G, U and C in position -3, respectively) but no differences in the wheat germ system. Differential mRNA degradation or polypeptide chain elongation were excluded as causes of the differences observed in translation in the reticulocyte lysate. mRNA competition increased the differences observed in translational efficiencies in reticulocyte lysate but showed no effect in wheat germ. Analysis of 61 plant and 209 animal mRNA sequences revealed qualitative and quantitative differences between the consensus sequences surrounding AUG initiation codons. Whereas the consensus sequence for animals was CACCAUG that for plants was AACAAUGGC. Both the structural and functional findings suggest that the factors which select AUG initiation codons in plants and animals differ significantly.     

Putative nuclease-sensitive control element in unfertilized eggs of the sea urchin Lytechinus pictus

Very limited nuclease-treatment of actively translating lysates from unfertilized eggs of the sea urchin Lytechinus pictus results in activation of the lysate. This activation is reflected by the ability of the nuclease-treated (or 'nuclease-activated') lysates to more efficiently utilize exogenous added RNAs. The activation may be reversed by adding small aliquots of untreated lysate to the nuclease-activated lysates. These results suggest that a nuclease-sensitive negative control element may be involved in the translational repression of unfertilized eggs, and that a deactivation of this control element is partially responsible for the dramatic activation of protein synthesis following fertilization in sea urchin eggs.     

A test for masked message: the template activity of messenger ribonucleoprotein particles isolated from sea urchine eggs

The hypothesis that the “masked message” of unfertilized eggs consists of nontranslatable mRNP particles was directly tested by in vitro translation of mRNPs in a system derived from wheat germ. Three classes of mRNPs were tested: particles prepared from sea urchin eggs in buffers containing 0.35 M K⁺, particles prepared from sea urchin eggs in 0.35 M Na⁺, and particles released with EDTA in 0.35 M K⁺ from polysomes of sea urchin embryos cultured in the presence of actinomycin D. The mRNA content of particles was monitored by determination of poly(A) content. The wheat germ system used is quantitatively stimulated by addition of mRNA derived from eggs or from any of the classes of mRNPs used. Particles prepared from eggs with Na⁺ or released from polysomes contain less protein than particles isolated from eggs in K⁺, and as expected these particles are fully translatable in vitro. Particles prepared from eggs in buffers containing 0.35 M K⁺ produce little or no stimulation in the in vitro system. That this lack of translation represents in vivo masking is indicated by several considerations: (1) The nontranslatable particles were prepared in 0.35 M K⁺ and 5 mM Mg²⁺, ion concentrations similar to those found in echinoderm eggs; (2) density and sedimentation rate characteristics of the particles are little changed by isolation; (3) RNA extracted from isolated particles is fully translatable; and (4) particles prepared from polysomes or under conditions which destabilize RNPs are translatable. These data support the masking hypothesis for the protein synthesis repression system of eggs.