Expression of alpha- and beta-tubulin genes during development of sea urchin embryos

Mature unfertilized eggs of the sea urchin Lytechinus pictus contain multiple alpha-tubulin mRNAs, which range in size from 1.75 to 4.8 kb, and two beta-tubulin mRNAs, 1.8 and 2.25 kb. These mRNAs were found at similar levels throughout the early cleavage stages. RNA gel blot hybridizations showed that prominent quantitative and qualitative changes in tubulin mRNAs occurred between the early blastula and hatched blastula stages. The overall amounts of alpha- and beta-tubulin mRNAs increased two- to fivefold between blastula and pluteus. These increases were due mainly to a rise in a 1.75-kb alpha RNA and a new 2.0-kb beta RNA. Other, minor changes also occurred during subsequent development. All size classes of alpha- and beta-tubulin RNAs in early and late embryos contained poly(A)+ translatable sequences. As reported earlier, some of each of the alpha RNAs, but neither of the beta RNAs, are translated in the egg and a small portion of each of the stored alpha and beta RNAs is recruited onto polysomes within 30 min of fertilization. In the work described here, subsequent development up to the morula stage was accompanied by a gradual recruitment of tubulin mRNAs into polysomes. By the early blastula stage, most of the maternal tubulin sequences were associated with polysomes. In contrast to the gradual recruitment of maternal sequences throughout cleavage, the tubulin mRNAs which appeared at the blastula stage showed no delay in entering polysomes. The exact fraction of each mRNA that was translationally active at later stages varied somewhat among the individual mRNAs. From the differential hybridization patterns of egg, embryo, and testis RNAs to various tubulin cDNA and genomic DNA probes, it is concluded that at least one gene producing maternal alpha mRNA is different from a second one which is expressed only in testis. Each of the three embryonic beta RNAs is encoded by a different beta gene; at least two of these different beta genes are also expressed in testis.     

Poly (A) binding protein is bound to both stored and polysomal mRNAs in the mammalian testis

RNA-binding proteins that bind to the 3′ untranslated region of mRNAs play important roles in regulating gene expression. Here we examine the association between the 70 kDa poly (A) binding protein (PABP) and stored (RNP) and polysomal mRNAs during mammalian male germ cell development. PABP mRNA levels increase as germ cells enter meiosis, reaching a maximum in the early postmeiotic stages, and decreasing to a nearly nondetectable level towards the end of spermatogenesis. Most of the PABP mRNA is found in the nonpolysomal fractions of postmitochondrial extracts, suggesting that PABP mRNA is either inefficiently translated or stored as RNPs during spermatogenesis. Virtually all of the testicular PABP is bound to either polysomal or nonpolysomal mRNAs, with little, if any, free PABP detectable. Analysis of several specific mRNAs reveals PABP is bound to both stored (RNP) and translated forms of the mRNAs. Western blot analysis and immunocytochemistry indicate PABP is widespread in the mammalian testis, with maximal amounts detected in postmeiotic round spermatids. The presence of PABP in elongating spermatids, a cell type in which PABP mRNA is nearly absent, suggests that PABP is a stable protein in the later stages of male germ cell development. The high level of testicular PABP in round spermatids and in mRNPs suggests a role for PABP in the storage as well as in the subsequent translation of developmentally regulated mRNAs in the mammalian testis. © 1995 Wiley-Liss, Inc.     

Small nuclear ribonucleoprotein antigens are absent from 10S translation inhibitory ribonucleoprotein but present in cytoplasmic messenger ribonucleoprotein and polysomes

A cytoplasmic 10S ribonucleoprotein particle (iRNP), which is isolated from chick embryonic muscle, is a potent inhibitor of mRNA translation in vitro and contains a 4S translation inhibitory RNA species (iRNA). The iRNP particle shows similarity in size to the small nuclear ribonucleoprotein (snRNP) particles. Certain autoimmune disease patients contain antibodies directed against snRNP antigenic determinants. The possibility that iRNP may be related to the small nuclear particles was tested by immunoreactivity with monospecific autoimmune antibodies to six antigenic determinants (Sm, RNP, PM-1, SS-A (Ro), SS-B (La), and Scl-70). By Ouchterlony immunodiffusion assays, the cytoplasmic 10S iRNP did not show any immunoreactivity. Also, a more sensitive hemagglutination inhibition assay for detecting Sm and RNP antigens failed to show reactivity with the 10S iRNP. Thus, the 10S iRNP particles are distinct from the similarly sized snRNP. However, free and polysomal messenger ribonucleoprotein (mRNP) particles and polysomes also isolated from chick embryonic muscle and analyzed by Ouchterlony immunodiffusion and hemagglutination inhibition for the presence of the antigenic determinants showed reactivity to Sm and RNP autoantibodies, but were not antigenic for the other four antibodies. Some of the Sm antigenic peptides of mRNP particles and polysomes were identical to those purified from calf thymus nuclear extract, as judged by Western blot analysis. The association of Sm with free and polysomal mRNP and polysomes suggests that Sm may be involved in some cytoplasmic aspects of mRNA metabolism, in addition to a nuclear function in mRNA processing.     

Cytogenetic analysis of oocytes and early preimplantation embryos from XO mice.

The cytoplasm of early sea urchin embryos contains nonribosomal, high molecular weight RNA both associated with ribosomes in polysomes and free of ribosomes in particles termed free RNP. In a 1-hr labeling period, 50% of the newly synthesized RNA enters the pool of ribosome-free RNP particles during the cleavage stages, and this percentage decreases until less than 20% of the new RNA in the mesenchyme blastula stage is found in the free RNP. mRNA from both polysomes and free RNP contain poly(A)(+) and poly(A)(?) species. During the cleavage stages only 8–10% of the RNA from each fraction is polyadenylated; however, in the blastula, 40–50% of the nonhistone polysomal RNA is polyadenylated while only 22–30% of the free RNP RNA is polyadenylated. At any developmental stage, the poly(A)(+)RNA from the free RNA and polysomes have identical sedimentation profiles; this is also the case for the poly(A)(?)RNA except for the absence of the 9 S histone mRNA from the free RNP. Changes in poly(A)(+)RNA content and sedimentation profiles during development occur simultaneously in the free RNP and the polysomes. Kinetic studies of these two RNP populations as well as nuclear RNP show that the bulk of the free RNP are not unusually stable cytoplasmic components. The free RNP decay with a half-life of about 40 min while nuclear RNA and polysomal RNA display half-lives of about 12 and 65 min, respectively. Further, the rate of synthesis of the free RNP is not consistent with their being the only precursors for polysomes. Our estimates of the rates of synthesis for nuclear RNA, polysomes, and free RNP are, respectively, 1.1 × 10^?15, 2.2 × 10^?16, and 5.0 × 15^?17 g/min/nucleus. The data on free RNP is discussed in terms of translational regulation of protein synthesis in the developing sea urchin.     

Arterial smooth muscle cells in vivo: relationship between actin isoform expression and mitogenesis and their modulation by heparin

Quiescent smooth muscle cells (SMC) in normal artery express a pattern of actin isoforms with alpha-smooth muscle (alpha SM) predominance that switches to beta predominance when the cells are proliferating. We have examined the relationship between the change in actin isoforms and entry of SMC into the growth cycle in an in vivo model of SMC proliferation (balloon injured rat carotid artery). alpha SM actin mRNA declined and cytoplasmic (beta + gamma) actin mRNAs increased in early G0/G1 (between 1 and 8 h after injury). In vivo synthesis and in vitro translation experiments demonstrated that functional alpha SM mRNA is decreased 24 h after injury and is proportional to the amount of mRNA present. At 36 h after injury, SMC prepared by enzymatic digestion were sorted into G0/G1 and S/G2 populations; only the SMC committed to proliferate (S/G2 fraction) showed a relative slight decrease in alpha SM actin and, more importantly, a large decrease in alpha SM actin mRNA. A switch from alpha SM predominance to beta predominance was present in the whole SMC population 5 d after injury. To determine if the change in actin isoforms was associated with proliferation, we inhibited SMC proliferation by approximately 80% with heparin, which has previously been shown to block SMC in late G0/G1 and to reduce the growth fraction. The switch in actin mRNAs and synthesis at 24 h was not prevented; however, alpha SM mRNA and protein were reinduced at 5 d in the heparin-treated animals compared to saline-treated controls. These results suggest that in vivo the synthesis of actin isoforms in arterial SMC depends on the mRNA levels and changes after injury in early G0/G1 whether or not the cells subsequently proliferate. The early changes in actin isoforms are not prevented by heparin, but they are eventually reversed if the SMC are kept in the resting state by the heparin treatment.     

Modifiers of solid RNP granules control normal RNP dynamics and mRNA activity in early development

Ribonucleoproteins (RNPs) often coassemble into supramolecular bodies with regulated dynamics. The factors controlling RNP bodies and connections to RNA regulation are unclear. During Caenorhabditis elegans oogenesis, cytoplasmic RNPs can transition among diffuse, liquid, and solid states linked to mRNA regulation. Loss of CGH-1/Ddx6 RNA helicase generates solid granules that are sensitive to mRNA regulators. Here, we identified 66 modifiers of RNP solids induced by cgh-1 mutation. A majority of genes promote or suppress normal RNP body assembly, dynamics, or metabolism. Surprisingly, polyadenylation factors promote RNP coassembly in vivo, suggesting new functions of poly(A) tail regulation in RNP dynamics. Many genes carry polyglutatmine (polyQ) motifs or modulate polyQ aggregation, indicating possible connections with neurodegenerative disorders induced by CAG/polyQ expansion. Several RNP body regulators repress translation of mRNA subsets, suggesting that mRNAs are repressed by multiple mechanisms. Collectively, these findings suggest new pathways of RNP modification that control large-scale coassembly and mRNA activity during development.     

Translation of an mRNA in rat L6 muscle cells is regulated within the cell cycle

In muscle cells two populations of mRNA are present in the cytoplasm. The majority of mRNA is associated with ribosomes and active in protein synthesis. A small population of cytoplasmic mRNA occur as free mRNA-protein complex and is not associated with ribosomes. This apparently repressed population of mRNA from rat L6 myoblast cells was used to construct a cDNA library. Radioactively labeled cDNA preparations of polysomal and free (or repressed) mRNA populations showed that at least ten recombinant clones preferentially annealed to the cDNA from repressed mRNA. One of these clones was extensively studied. The DNA from a recombinant plasmid D12 hybridized to a 1.3-kb poly(A)-rich mRNA. In proliferating myoblast cells, the 1.3-kb mRNA was more abundant in the polysomal fraction and mostly free in the non-dividing myotubes. In contrast to this mRNA, 90% of alpha and beta actin mRNAs were translated in both myoblasts and myotubes. Further analysis of distribution of the 1.3-kb RNA in the polysomal (active) and free (repressed) fractions in fusion-arrested postmitotic myotubes suggested that fusion of myoblasts was not necessary for the control of translation of this mRNA. Withdrawal of muscle cells from the cell cycle appeared to be involved in regulating translation of this mRNA. The presence of this mRNA was not, however, limited to muscle cells. This mRNA was also present in the repressed state in rat liver and kidney cells. These results, therefore, suggest that the 1.3-kb mRNA is probably translated during a particular phase of the cell cycle and is not translated in terminally differentiated non-dividing cells. Messenger RNA homologous to the 600-base-pair insert of the recombinant plasmid D12 was isolated by hybrid selection procedure from both polysomal mRNA of myoblasts and free mRNA of myotubes. Translation of the hybrid selected mRNAs from both myoblasts and myotubes in rabbit reticulocyte lysate cell-free system synthesized a 40-kDa polypeptide. These results suggest that the repressed population of 1.3-kb mRNA can be translated in vitro. The hybridization pattern of DNA from the recombinant plasmid D12 with rat genomic DNA suggested that the 1.3-kb mRNA is derived from moderately repetitive rat DNA with a repetition frequency of approximately 100 copies per haploid genome.     

Isolation of Undegraded Free and Membrane-Bound Polysomal mRNA from Rat Brain

A new procedure is described for the isolation of undegraded free and membrane-bound polysomal mRNA from rat brain in which polysomes are simultaneously separated from soluble components of the cell and slowly sedimenting ribosome species and concentrated by rate-zonal centrifugation on short linear sucrose gradients. This avoids the problem encountered in conventional procedures of pelleting polysomes along with membranous materials that are not solubilized by detergents and that appear to contain traces of nucleases. It also permits a more thorough analysis of the mRNA populations actively engaged in protein synthesis, since both polyadenylated and nonpolyadenylated mRNAs are isolated together. Moreover, the likelihood of sedimenting nonpolysomal mRNP particles along with polysomes is reduced by using rate-zonal rather than pelleting centrifugation. The translational activity in vitro of free and membrane-bound polysomal RNA prepared in this way is high and is about 1.5 times that of RNA prepared by a conventional pelleting technique. The difference is attributable to better preservation of large mRNAs, as inferred from two-dimensional gel electrophoretic analysis of translation product abundance. The recovery of both classes of polysomal RNA is about 90%. The method is simple, efficient, and adapted for isolation of small amounts of polysomal RNA.     

Poly(A+)mRNA sequences in free mRNP and polysomes of mouse ascites cells

The poly(A⁺)RNA of the free mRNP of mouse Taper ascites cell contains a very reduced number of different mRNA sequences compared to the polysome poly(A⁺)RNA. By the technique of mRNA:cDNA hybridization we have determined that the free mRNP contains approximately 400 different mRNA sequences while the polysomes contain about 9000 different mRNAs. The free mRNP poly(A⁺)RNA sequences are present in two abundance classes, the abundant free mRNP class containing 15 different mRNA sequences and the less abundant free mRNP class containing 400 different mRNAs. The polysome poly(A⁺)RNA consists of three abundance classes of 25, 500 and 8500 different mRNA sequences.Despite its intracellular location in RNP structures not directly involved in protein synthesis the poly(A⁺)RNA purified from the free RNP of these cells was a very effective template for protein synthesis in cell-free systems. Cell-free translation products of free mRNP and polysome poly(A⁺)RNAs were analyzed by two-dimensional gel electrophoresis. This analysis confirmed the hybridization result that the free mRNP poly(A⁺)RNA contained fewer sequences than polysomal poly(A⁺)RNA. The abundant free RNP-mRNA directed protein products were a subset of the polysome mRNA-directed protein products. The numbers of more abundant products of cell-free protein synthesis directed by the free RNP-mRNA and polysomal mRNA were in general agreement with the hybridization estimates of the number of sequences in the abundant classes of these two mRNA populations.     

Mouse kidney nonpolysomal messenger ribonucleic acid: metabolism, coding function, and translational activity

To elucidate the distribution and function of mRNA in mouse kidney cytoplasm, we compared mRNA isolated from polysomal (greater than 80S) and native postpolysomal (20--80S) ribonucleoproteins with respect to synthesis and lifetime, sequence content, and translational activity. The 20--25% of cytoplasmic mRNA recovered from postpolysomal ribonucleoprotein is similar to polysomal mRNA in size (20--22S), in apparent half-life (11--13 h), in major products of cell-free translation, and in nucleotide complexity (approximately 4 x 10(7) nucleotides). The labeling kinetics of polysomal and postpolysomal mRNA suggest these mRNA populations are in equilibrium. [3H]cDNAs transcribed from polysomal and from postpolysomal poly(A)-containing mRNAs react with template mRNA and with the heterologous mRNA at the same rate (Cot1/2 approximately 6.3 mol.s/L) and to the same extent (95%). Therefore, these mRNAs are equally diverse and homologous and occur at similar relative frequencies. Postpolysomal mRNA directs cell-free protein synthesis at only approximately 30% of the rate of polysomal mRNA and to only 30% of the extent of mRNA from polysomes. Postpolysomal mRNA is approximately 3-fold less sensitive than polysomal mRNA to inhibition of translation by m7GMP, suggesting postpolysomal mRNA contains a greater fraction of molecules deficient in 5'-terminal caps. Postpolysomal mRNA may derive from renal mRNAs that initiate translation inefficiently and thus accumulate as postpolysomal ribonucleoproteins.     

RNA synthesis in unfertilized sea urchin eggs

We have examined the synthesis of messenger-like RNA in unfertilized sea urchin eggs. Most of the RNA synthesized is restricted to the nucleus and sediments from 16 to 30S. A small fraction can be isolated from the postmitochondrial supernatant and displays a sedimentation profile typical of embryonic mRNA with peaks at 9 and 18S. This cytoplasmic RNA is largely present as free RNPs and we estimate that less than 20% of the RNA is in polysomes. The RNA made in the egg is unstable and reaches a steady state with a half-time of about 30 min. We have examined the accumulation of RNA in the egg and have calculated a rate of synthesis of 1.4 × 10^?14 g of RNA/min/egg which is similar, on a per-nucleus basis, to that found in the just-fertilized egg and very early embryo. It is approximately 10 times greater than the rate of RNA synthesis in the blastula nucleus. We estimate that the RNA synthesized by the unfertilized egg amounts to a maximum of 3 × 10^?13 g of potential mRNA at the time of fertilization, or 10–15% of its immediate needs. This RNA cannot account for the increase in protein synthesis that occurs after fertilization, which must be the result of the translation of another population of more stable egg or oogenic mRNA that is kinetically distinct from the RNA we have measured. The steady-state level of labeled RNA present in the egg does not change upon fertilization until after the first cleavage, at about 2.5 hr after fertilization. Thus the RNA synthesis that occurs in the just-fertilized zygote appears to be merely a continuation (at least quantitatively) of the RNA synthesis taking place in the egg.     

Messenger ribonucleoprotein particles in developing sea urchin embryos

Messenger RNA entering polysomes during early development of the sea urchin embryo consists of both oogenetic and newly transcribed sequences. Newly transcribed mRNA enters polysomes rapidly while oogenetic mRNA enters polysomes from a pool of stable, nontranslatable messenger ribonucleoprotein particles (mRNPs) derived from the unfertilized egg. Protein content may relate to differences in the regulation of newly transcribed and oogenetic mRNAs. Oogenetic poly(A)⁺ mRNA was found to be present in both polysomal and subpolysomal fractions of cleavage stage and early blastula stage embryos. This mRNA was found to be present in subpolysomal mRNPs with a density of 1.45 g/cm³ in Cs₂SO₄. Poly(A)⁺ mRNPs released from polysomes of embryos cultured in the presence of actinomycin D sedimented in a broad peak centered at 55 S and contained RNA of 21 S. The density of these particles was sensitive to the method of release; puromycin-released mRNPs had a density of 1.45 g/cm³, while EDTA caused a shift in density to 1.55 g/cm³, indicating a partial loss of protein. The results with newly synthesized mRNAs contrast sharply. Newly transcribed mRNA in subpolysomal mRNPs had a density of 1.55–1.66 g/cm³, a density approaching that of deproteinized RNA. Messenger RNA released from polysomes either by EDTA or puromycin was examined to determine the possible existence of polysomal mRNPs. When [³H]uridine-labeled mRNA was released from late cleavage stage embryo polysomes by either technique, and centrifuged on sucrose gradients, two broad peaks were found. One peak centered at 30 S contained 21 S mRNA while the other at 15 S contained 9 S histone mRNA. When these fractions were fixed with formaldehyde, they banded on Cs₂SO₄ gradients at a density of 1.60–1.66 g/cm³, very similar to that of pure RNA. We conclude that the newly transcribed mRNA may be present in stable mRNPs containing up to 10% protein in either subpolysomal or polysomal fractions. These mRNPs are clearly distinguishable from the protein-rich mRNPs containing oogenetic mRNAs.     

Stored Messenger Ribonucleoprotein Particles in Differentiated Sclerotia of Physarum polycephalum

Starvation induces vegetative microplasmodia of Physarum polycephalum to differentiate into translationally-dormant sclerotia. The existence and the biochemical nature of stored mRNA in sclerotia is examined in this report. The sclerotia contain about 50% of the poly(A)-containing RNA [poly(A)+RNA] complement of microplasmodia as determined by [³H]-poly(U) hybridization. The sclerotial poly(A)+RNA sequences are associated with proteins in a ribonucleoprotein complex [poly(A)+mRNP] which sediments more slowly than the polysomes. Sclerotial poly(A)+RNP sediments more rapidly than poly(A)+RNP derived from the polysomes of microplasmodia despite the occurrence of poly(A)+RNA molecules of a similar size in both particles suggesting the existence of differences in protein composition. Isolation of poly(A)+RNP by oligo (dT)-cellulose chromatography and the analysis of its associated proteins by polyacrylamide gel electrophoresis show that sclerotial poly(A)+RNP contains at least 14 major polypeptides, 11 of which are different in electrophoretic mobility from the polypeptides found in polysomal poly(A)+RNP. Three of the sclerotial poly(A)+RNP polypeptides are associated with the poly(A) sequence (18, 46, and 52 × 10³ mol. wt. components), while the remaining eight are presumably bound to non-poly(A) portions of the poly(A)+RNA. Although distinct from polysomal poly(A)+RNP, the sclerotial poly(A)+RNP is similar in sedimentation behavior and protein composition (with two exceptions) to the microplasmodial free cytoplasmic poly(A)+RNP. The results suggest that dormant sclerotia store mRNA sequences in association with a distinct set of proteins and that these proteins are similar to those associated with the free cytoplasmic poly(A)+RNP of vegetative plasmodia.     

Complex formation between metarhodopsin II and GTP-binding protein in bovine photoreceptor membranes leads to a shift of the photoproduct equilibrium

Cap binding protein (CBP)-related polypeptides were identified in different cytoplasmic RNP particles of embryonic chick muscles using monoclonal antibody to purified CBP. A single immunoreactive peptide (M_r 78000) was present in preparations of both free mRNP particles and a novel 10 S translation inhibitory RNP particle. In contrast, proteins isolated from these particles showed two new low-M_r immunoreactive peptides (M_r 43000 and M_r 29000). No CBP related protein could be detected in polysomal mRNP, although an immunoreactive M_r 43000 CBP-related protein was present in polysomes. The relevance of the association of different CBP-related polypeptides with cytoplasmic RNP particles and polysomes are discussed.     

Translational control of protein synthesis after infection by vesicular stomatitis virus.

Four hours after infection of BHK cells by vesicular stomatitis virus (VSV), the rate of total protein synthesis was about 65% that of uninfected cells and synthesis of the 12 to 15 predominant cellular polypeptides was reduced to a level about 25% that of control cells. As determined by in vitro translation of isolated RNA and both one- and two-dimensional gel analyses of the products, all predominant cellular mRNA's remained intact and translatable after infection. The total amount of translatable mRNA per cell increased about threefold after infection; this additional mRNA directed synthesis of the five VSV structural proteins. To determine the subcellular localization of cellular and viral mRNA before and after infection, RNA from various sizes of polysomes and nonpolysomal ribonucleoproteins (RNPs) was isolated from infected and noninfected cells and translated in vitro. Over 80% of most predominant species of cellular mRNA was bound to polysomes in control cells, and over 60% was bound in infected cells. Only 2 of the 12 predominant species of translatable cellular mRNA's were localized to the RNP fraction, both in infected and in uninfected cells. The average size of polysomes translating individual cellular mRNA's was reduced about two- to threefold after infection. For example, in uninfected cells, actin (molecular weight 42,000) mRNA was found predominantly on polysomes with 12 ribosomes; after infection it was found on polysomes with five ribosomes, the same size of polysomes that were translating VSV N (molecular weight 52,000) and M (molecular weight 35,000) mRNA. We conclude that the inhibition of cellular protein synthesis after VSV infection is due, in large measure, to competition for ribosomes by a large excess of viral mRNA. The efficiency of initiation of translation on cellular and viral mRNA's is about the same in infected cells; cellular ribosomes are simply distributed among more mRNA's than are present in growing cells. About 20 to 30% of each of the predominant cellular and viral mRNA's were present in RNP particles in infected cells and were presumably inactive in protein synthesis. There was no preferential sequestration of cellular or viral mRNA's in RNPs after infection.     

'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.     

Developmental Changes in Levels of Translatable mRNAs for Enolase Isozymes in Chicken Brain

Using chicken brain mRNAs, alpha and gamma enolase precursors were synthesized in the rabbit reticulocyte cell-free translation system. The product proteins showed molecular weights almost identical to those of the mature subunits. The levels of translatable mRNAs for alpha and gamma subunits were determined by the cell-free translation system and immunoprecipitation with specific antisera, during development of chicken brain. The level of alpha mRNA was high at any developmental stage of the brain. On the other hand, the gamma mRNA level was very low at the early embryonic stage, and increased rapidly during development of the brain. These changes were closely correlated with those of the corresponding enzyme activities, indicating that the levels of enolase activities in developing brain were controlled primarily by the level of the translatable alpha and gamma mRNAs.     

Polysomal and non-polysomal messenger RNA in neuroblastoma cells: Lack of correlation between polyadenylation or initiation efficiency and messenger RNA location

Neuroblastoma cytoplasm was fractionated on sucrose gradients into polysomes (>90 S) and non-polysomal particles (<90 S). Purified RNA from these fractions was translated using a wheat germ lysate and translation products were compared by two-dimensional gel electrophoresis. Non-polysomal messenger RNA directed the synthesis of a specific subset of polysomal mRNA translation products. Careful comparison of individual translation products demonstrated that specific mRNAs were not randomly distributed between polysomes and the non-polysomal fraction.Fractionation of both RNA populations into polyadenylated (poly(A)⁺) and non-adenylated (poly(A)^?) species indicated that specific, abundant non-polysomal mRNAs were not less adenylated than their polysomal counterparts. Furthermore, comparison of translation products from assays of subsaturating and supersaturating RNA concentrations demonstrated that no simple correlation could be made between the relative initiation efficiency of a specific mRNA and its distribution between polysomes and non-polysomal particles.     

L-bodies are RNA–protein condensates driving RNA localization in Xenopus oocytes

Ribonucleoprotein (RNP) granules are membraneless compartments within cells, formed by phase separation, that function as regulatory hubs for diverse biological processes. However, the mechanisms by which RNAs and proteins interact to promote RNP granule structure and function in vivo remain unclear. In Xenopus laevis oocytes, maternal mRNAs are localized as large RNPs to the vegetal hemisphere of the developing oocyte, where local translation is critical for proper embryonic patterning. Here we demonstrate that RNPs containing vegetally localized RNAs represent a new class of cytoplasmic RNP granule, termed localization-bodies (L-bodies). We show that L-bodies contain a dynamic protein-containing phase surrounding a nondynamic RNA-containing phase. Our results support a role for RNA as a critical component within these RNP granules and suggest that cis-elements within localized mRNAs may drive subcellular RNA localization through control over phase behavior.