Purification and characterization of cytoplasmic protamine messenger ribonucleoprotein particles from rainbow trout testis cells

Poly(A)-containing protamine messenger ribonucleoprotein particles [poly(A+) pmRNP particles] have been isolated from the polysomal and free cytoplasmic subcellular fractions of trout testis cells by a two-step isolation procedure. Ethylenediaminetetraacetic acid (EDTA) treated particles from both cytoplasmic fractions were first fractionated by sucrose gradient centrifugation and the putative pmRNP particles localized by utilizing 3H-labeled protamine complementary DNA (pcDNA) probes. In addition, particles present in these fractions were characterized by their translational activity in the heterologous, rabbit reticulocyte cell-free system and the protein components of crude mRNP complexes analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoesis. The final purification step involved affinity chromatography of pooled gradient fractions on oligo(dT)-cellulose from which intact pmRNP could be eluted with distilled water at 40 degrees C. Highly purified particles from both polysomal and free cytoplasmic fractions prepared by this procedure had buoyant densities of 1.35-1.37 g/cm3 in CsCl or a protein content of approximately 82%. Particles isolated from EDTA-dissociated polysomes were actively translated in vitro, while their free cytoplasmic counterparts were not. High salt washed pmRNP particles or the RNA extracted from pmRNP preparations, however, directed the synthesis of trout protamines in this system. A model of the activation of stored pmRNP particles in vitro and in vivo is presented.     

Ribosomal and informational ribonucleoprotein complexes of animal cells. Study on rat liver ribonucleic acids as constituents of ribonucleoprotein complexes by chromatography on nucleoprotein-celite columns.

A novel method of RNA fractionation has been developed. Nuclear and cytoplasmic rat liver RNA species were fractionated as constituents of corresponding ribonucleoprotein particles, which were previously adsorbed on a Celite-column by their protein component. The fractionation is based on a dissociation of the particles (linear concentration gradient of LiCl and urea with subsequent temperature gradient), which results in a gradual release of the RNA molecules from ribonucleoprotein complexes. Thus the fractionation is in accordance with the tightness of the RNA-protein bonds. A gradient elution of RNA from a nucleoprotein-Celite column permitted fractionation of both ribosomal and rapidly labelled non-ribosomal RNA. The latter, both nuclear and cytoplasmic, could be separated by chromatography on nucleoprotein-Celite columns into two main fractions (components I and V). In cytoplasmic RNA components I and V presumably correspond to mlRNA (messenger-like RNA of free cytoplasmic particles) and mRNA (template RNA associated with ribosomes) respectively.     

Labelling kinetics of RNA containing poly(a) in liver subcellular fractions

Kinetics of incorporation of (3H) uridine into cytoplasmic RNA fractions of rat liver is investigated. The fractions include free and membrane bound polysomes, rough membranes sedimenting with mitochondria and free cytoplasmic RNA particles. (1) Poly(A) containing RNA, isolated by oligo-dT cellulose, amounts to 0.4% of the total RNA in the homogenate, 0.5% in bound polysomes, 3.4% in free polysomes and 16% in free cytoplasmic RNA particles. (2) The rate of (3H) uridine incorporation into RNA lacking poly(A) proceeds uniformly in all subcellular fractions except for free cytoplasmic RNA particles, which accumulate negligible amounts of radioactivity. (3) The initial labelling of RNA containing poly(A) is most active in free cytoplasmic RNA particles supporting their identity as mRNA en route to polysomes. The initial specific radioactivities decrease in the following order: homogenate, bound polysomes, rough membranes sedimenting with mitochondria, free polysomes. The data suggest that mRNA is supplied to free and membrane-bound polysomes via different routes. The kinetic analysis indicates that free cytoplasmic RNA particles may be a precursor of mRNA of free polysomes rather than that of bound polysomes. (4) The kinetic differences of free and membrane bound polysomes are also demonstrated by comparing the radioactivity of RNA containing poly(A) to the total radioactivity at various incorporation times. In bound polysomes this decreases from 31% at 1 h to 10% at 25 h, whereas in free polysomes the corresponding ratio increases from 10 to 13%. RNA containing poly(A) of free cytoplasmic RNA particles represents 64% of the total radioactivity throughout the experiment.     

Characterization of poly(A+)RNA in free messenger ribonucleoprotein and polysomes of mouse Taper ascites cells

Cytoplasmic extracts of mouse Taper ascites cells were centrifuged on sucrose gradients to give 0–80 S, monosome, and polysome fractions. CsCl equilibrium density centrifugation of formaldehyde-fixed material from the 0–80 S fraction demonstrated that the messenger RNA in the 0–80 S fraction was in the form of free ribonucleoprotein. The size of the poly(A⁺)RNA and the size of the poly(A) segments of these molecules were shown to be very similar in both the free mRNP² and polysome fractions. The labeling kinetics of the free mRNP poly(A⁺)RNA was similar to that of the polysomal poly(A⁺)RNA.The free mRNP poly(A⁺)RNA efficiently stimulated protein synthesis in the wheat germ cell-free system, supporting the view that it was mRNA. Two-dimensional gel electrophoresis was used to analyze the proteins whose synthesis was directed by free mRNP and polysomal poly(A⁺)RNA. The free mRNP poly(A⁺)RNA directed the synthesis of a simpler set of abundant protein products than did the polysomal poly(A⁺)RNA. Most of the free mRNP abundant protein products were also present in the polysomal products, though obvious quantitative differences were evident, indicating that each individual mRNA had its own characteristic distribution between polysomes and the translationally inactive RNP form.     

The effect of feeding with a tryptophan-free amino acid mixture on rat-liver polysomes and ribosomal ribonucleic acid

1. Starving rats were given complete and tryptophan-deficient amino acid mixtures by stomach tube and were killed from 1 to 7hr. later. The polysome profile in the livers of rats fed with the tryptophan-deficient mixture showed a shift in distribution such that the large aggregates were decreased and the small aggregates were increased, particularly dimers. This polysome shift was reversed when the complete amino acid mixture was given by stomach tube 2hr. after administering the tryptophan-free amino acid mixture. 2. After removal of liver polysomes by centrifugation, some smaller ribosomal aggregates (oligosomes) remaining in suspension were harvested by prolonged centrifugation of the supernatant fluid. A large increase in the dimer population of this fraction was observed in the rats receiving the incomplete mixture. 3. When the polysome and oligosome fractions were incubated with cell sap, an energy-generating system and labelled amino acids dl-[1-(14)C]leucine and l-[Me-(14)C]tryptophan were incorporated into the cell fractions in the ratio 4.5:1. Preparations of polysomes and oligosomes from rats fed with the tryptophan-free amino acid mixture showed a decreased amino acid-incorporating activity compared with particulate preparations made from rats fed with the complete mixture. 4. The yield of free ribosomes prepared from the unfractionated liver microsomes by treatment with iso-octane was 40-50% greater in rats fed with the amino acid mixture deficient in tryptophan. 5. A post-microsomal fraction was prepared from cell sap and was shown to consist of ribosomal sub-units. When the animals were fed with the tryptophan-deficient mixture, there was an increase in content of this post-microsomal fraction and in the ratio 30s RNA/19s RNA. Rats were also given [5-(3)H]orotic acid at the time of feeding with the amino acids. Lack of tryptophan in the mixture caused a decrease in the specific activity of both RNA fractions which affected the 30s RNA more extensively than the 19s RNA. 6. These changes in the distribution and quantity of the cellular components engaged in protein synthesis are discussed in relation to RNA metabolism and amino acid-incorporating activity of the liver cell and their response to feeding with the tryptophan-free amino acid mixture.     

Isolation and characterization of poly(adenylic acid)-containing messenger ribonucleic acid from rat liver polysomes

Undegraded rat liver polysomes were obtained after homogenizing the tissue in a medium containing NH4Cl, heparine, and yeast tRNA. Purification of poly(A)-containing RNA from polysomal RNA was accomplished by affinity chromatography on oligo(dT)-cellulose columns. Poly(A)-containing RNA molecules were monitored by the formation of ribonuclease-resistant hybrids with [3H]poly(U). To improve the separation of messenger RNA and ribosomal RNA by oligo(dT)-cellulose it was found essential to dissociate the aggregates formed between both molecular species by heat treatment in the presence of dimethylsulfoxide (Me2SO) prior to chromatography. Sucrose gradient analysis under denaturing conditions showed that the preparations obtained were virtually free of ribosomal RNA. Poly(A)-containing RNA constituted approx. 2.2% of the total polysomal RNA and the number average size was 1500--1800 nucleotides, as judged by sedimentation analysis on sucrose density gradients containing Me2SO. Approximately 8.2% of the purified preparation obtained was able to anneal with [3H]poly(U); the number average nucleotide length of the poly(A) segment of the RNA population was calculated to be 133 adenylate residues. Based on these values, our preparations appear to be greater than 90% pure. The RNA fractions obtained after oligo(dT)-cellulose chromatography were used to direct the synthesis of liver polypeptides in a heterologous cell-free system derived from wheat-germ. The system was optimized with respect to monovalent and divalent cations, and presence of polyamines (spermine). More than 65% of the translational activity present in the unfractionated polysomal RNA was recovered in the final poly(A)-containing RNA fraction. However, about 25% of the activity was found to be associated with the unbound fraction which was essentially free of poly(A)-containing RNA. Immunoprecipitation analysis with a specific antiserum to rat serum albumin demonstrated that about 6--8% of the labeled synthetic products translated from the poly(A)-containing RNA sample corresponded to serum albumin. Analysis of the translation products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a heterogeneous distribution of molecular sizes ranging from 15 000 to greater than 70 000 daltons. Spermine not only increased the overall yield and extent of protein synthesis, but also resulted in higher yields of large protein products. Under optimal translation conditions a discrete peak representing about 7% of the total radioactivity was observed to migrate with rat serum albumin.     

Direct purification of polyadenylated RNAs from isolated polysome fractions

We report a simplified and improved method for obtaining polyadenylated RNAs (poly(A) RNAs) from polysome fractions. Isolated polysomes were subjected directly to poly(U)-Sephadex column chromatography without conventional purification of polysomal RNAs by phenol extraction followed by ethanol precipitation. The yield of poly(A) RNAs by this direct purification method was about twice that obtained by the conventional method. When the two poly(A) RNA preparations were used in two-dimensional polyacrylamide gel electrophoretic analysis of cell-free translation products and cDNA synthesis, biological activity of the directly purified poly(A) RNA was equal to or even better than that of conventionally purified poly(A) RNA.     

Structure and function of rat liver polysome populations. II. Characterization of polyadenylate-containing mRNA associated with subpopulations of membrane-bound particles

Poly(A)+RNA fractions prepared from free and loosely and tightly membrane-bound polysome populations (poly(A)+RNAfree, poly(A)+RNAloose, and poly(A)+RNAtight) were used to drive cDNA in homologous and heterologous hybridization reactions. A large fraction by mass of sequences was shared among the three poly(A)+RNA populations, but shared sequences exhibited distinct frequency distributions within the different populations. 13-15 in vitro translation products of poly(A)+RNAfree and poly(A)+RNAloose detected by gel electrophoresis were shared. Most of these were produced in different relative quantities by the two RNA populations. Five or six higher mol wt polypeptides were produced by poly(A)+RNAloose that were not detected as products of either poly(A)+free or poly(A)+RNAtight. We suggest that loosely bound polysomes may not be artifactually derived as reflected in their quantitatively distinct poly(A)+RNA population. Two tightly membrane-bound RNP fractions were prepared from rat liver on the basis of their release from or retention on purified rough microsomes or a crude membrane fraction after in vitro disaggregation of polysomes with high-salt and puromycin. Homologous and heterologous hybridizations involving their poly(A)+RNA fractions revealed that a large portion by mass of sequences was shared but that these sequences exhibited distinct frequency distributions in the two fractions. The RNA fractions produced exhibited distinct frequency distributions in the two fractions. The RNA fractions produced an identical set of in vitro translation products but individual polypeptides were produced in different relative quantities. This indicates that the two RNP fractions do not arise by any random artifactual process and suggests that they may represent functionally distinct populations.     

Cellular titers and subcellular distributions of abundant polyadenylate-containing ribonucleic acid species during early development in the frog Xenopus laevis.

The distribution of cytoplasmic messenger ribonucleic acids (RNAs) in translationally active polysomes and inactive ribonucleoprotein particles changes during early development. Cellular levels and subcellular distributions have been determined for most messenger RNAs, but little is known about how individual sequences change. In this study, we used hybridization techniques with cloned sequences to measure the titers of 23 mitochondrial and non-mitochondrial polyadenylate-containing [poly(A)+]RNA species during early development in the frog Xenopus laevis. These RNA species were some of the most abundant cellular poly(A)+ RNA species in early embryos. The concentrations of most of the non-mitochondrial (cytoplasmic) RNAs remained constant in embryos during the first 10 h of development, although the concentrations of a few species increased. During neurulation, we detected several new poly(A)+ RNA sequences in polysomes, and with one possible exception the accumulation of these sequences was largely the result of new synthesis or de novo polyadenylation and not due to the recruitment of nonpolysomal (free ribonucleoprotein) poly(A)+ RNA. We measured the subcellular distributions of these RNA species in polysomes and free ribonucleoproteins during early development. In gastrulae, non-mitochondrial RNAs were distributed differentially between the two cell fractions; some RNA species were represented more in free ribonucleoproteins, and others were represented less. By the neurula stage this differential distribution in polysomes and free ribonucleoproteins was less pronounced, and we found species almost entirely in polysomes. Some poly(A)+ RNA species transcribed from the mitochondrial genome were localized within the mitochondria and were mapped to discrete fragments of the mitochondrial genome. Much of this poly(A)+ RNA was transcribed from the ribosomal locus. Nonribosomal mitochondrial poly(A)+ RNA species became enriched in polysome-like structures after fertilization, with time courses similar to the time course of mobilization of cytoplasmic poly(A)+ RNA.     

Paxillin associates with poly(A)-binding protein 1 at the dense endoplasmic reticulum and the leading edge of migrating cells.

Using mass spectrometry we have identified proteins which co-immunoprecipitate with paxillin, an adaptor protein implicated in the integrin-mediated signaling pathways of cell motility. A major component of paxillin immunoprecipitates was poly(A)-binding protein 1, a 70-kDa mRNA-binding protein. Poly(A)-binding protein 1 associated with both the alpha and beta isoforms of paxillin, and this was unaffected by RNase treatment consistent with a protein-protein interaction. The NH(2)-terminal region of paxillin (residues 54-313) associated directly with poly(A)-binding protein 1 in cell lysates, and with His-poly(A)-binding protein 1 immobilized in microtiter wells. Binding was specific, saturable and of high affinity (K(d) of approximately 10 nm). Cell fractionation studies showed that at steady state, the bulk of paxillin and poly(A)-binding protein 1 was present in the "dense" polyribosome-associated endoplasmic reticulum. However, inhibition of nuclear export with leptomycin B caused paxillin and poly(A)-binding protein 1 to accumulate in the nucleus, indicating that they shuttle between the nuclear and cytoplasmic compartments. When cells migrate, poly(A)-binding protein 1 colocalized with paxillin-beta at the tips of lamellipodia. Our results suggest a new mechanism whereby a paxillin x poly(A)-binding protein 1 complex facilitates transport of mRNA from the nucleus to sites of protein synthesis at the endoplasmic reticulum and the leading lamella during cell migration.     

Major mRNP proteins in the structural organization and function of mRNA in eukaryotic cells

The properties of two universal major proteins of cytoplasmic mRNP, p50 and the poly(A)-binding protein (PABP), are summarized. Their roles in formation of polyribosomal and free inactive mRNP are considered, with the focus on the authors' studies of p50. The parts these mRNP proteins play in translation regulation, stability, and localization of mRNA are described, and the the possible mechanisms of their function are discussed.     

Major mRNP Proteins in the Structural Organization and Function of mRNA in Eukaryotic Cells

The properties of two universal major proteins of cytoplasmic mRNP, p50 and the poly(A)-binding protein (PABP), are summarized. Their roles in formation of polyribosomal and free inactive mRNP are considered, with the focus on the authors' studies of p50. The parts these mRNP proteins play in translation regulation, stability, and localization of mRNA are described, and the possible mechanisms of their function are discussed.     

Yeast mRNA Poly(A) tail length control can be reconstituted in vitro in the absence of Pab1p-dependent Poly(A) nuclease activity

Regulation of poly(A) tail length during mRNA 3'-end formation requires a specific poly(A)-binding protein in addition to the cleavage/polyadenylation machinery. The mechanism that controls polyadenylation in mammals is well understood and involves the nuclear poly(A)-binding protein PABPN1. In contrast, poly(A) tail length regulation is poorly understood in yeast. Previous studies have suggested that the major cytoplasmic poly(A)-binding protein Pab1p acts as a length control factor in conjunction with the Pab1p-dependent poly(A) nuclease PAN, to regulate poly(A) tail length in an mRNA specific manner. In contrast, we recently showed that Nab2p regulates polyadenylation during de novo synthesis, and its nuclear location is more consistent with a role in 3'-end processing than that of cytoplasmic Pab1p. Here, we investigate whether PAN activity is required for de novo poly(A) tail synthesis. Components required for mRNA 3'-end formation were purified from wild-type and pan mutant cells. In both situations, 3'-end formation could be reconstituted whether Nab2p or Pab1p was used as the poly(A) tail length control factor. However, polyadenylation was more efficient and physiologically more relevant in the presence of Nab2p as opposed to Pab1p. Moreover, cell immunofluorescence studies confirmed that PAN subunits are localized in the cytoplasm which suggests that cytoplasmic Pab1p and PAN may act at a later stage in mRNA metabolism. Based on these findings, we propose that Nab2p is necessary and sufficient to regulate poly(A) tail length during de novo synthesis in yeast.     

Direct association of messenger RNA-containing ribonucleoprotein particles with membranes of the endoplasmic reticulum in ethionine-treated rat liver.

The administration of ethionine to female rats causes breakdown of hepatic polysomes. The fate of the mRNA molecules after polysome breakdown was investigated by measuring the amount of poly(A)-containing mRNA in membranous and non-membranous fractions obtained from the cytoplasm of ethionine-treated rat liver. The amount of poly(A)-containing mRNA in the membrane fraction of ethionine-treated liver was found to be the same as that of normal liver. When poly(A)-containing mRNAs from various fractions were translated in a wheat germ system and the products were isolated by immunoprecipitation, the albumin-specific mRNA was found exclusively in the membrane fraction of both normal and ethionine-treated livers. The membrane-bound mRNA in ethionine-treated liver, selectively labeled with [14C]orotate, was banded in CsCl gradient centrifugation at 1.42 g/ml which corresponds to the previously reported mRNA-containing ribonucleoprotein particles. From these results, we concluded that even after the polysome disaggregation by ethionine, most of the mRNA of membrane-bound polysomes remains attached to the endoplasmic reticulum membranes independently of ribosomes and the nascent polypeptide chains.     

Cellular distribution of three mammalian Ca2+-binding proteins related to Torpedo calelectrin.

Addition of Ca2+ to post-microsomal fractions of bovine adrenal or liver produced a sedimentable complex of membrane vesicles and cytoplasmic proteins. Proteins with apparent mol. wts. 70 000, 36 000 and 32 500 were solubilized from this complex by Ca2+ chelation. The 36 000 mol. wt. protein (p36) was immunoprecipitated by an antiserum specific for pp36, a major substrate for Rous sarcoma virus src-gene tyrosine kinase. This protein was present in many mesenchymal cells and associated with membrane cytoskeleton of bovine fibroblasts in a Ca2+-dependent manner. The 70 000 and 32 500 mol. wt. proteins were widely distributed in established cell lines, but were not clearly associated with cell organelles in tissue sections, nor retained in cytoskeleton preparations. On immunoblots p36 reacted strongly with antibodies produced against the electric fish protein Torpedo calelectrin and the similar Ca2+-binding properties and subunit mol. wts. of these proteins suggests that they might be functionally related. Since Torpedo calelectrin, p70, p36 and p32.5 were bound by lipid vesicles or microsomal membranes at micromolar free Ca2+ concentrations, regulated association with intrinsic membrane components may be involved in the functions of these widespread proteins.     

Characterization of a poly(A)+RNA-containing structural component directly associated with cytoplasmic membranes in dormant Artemia cysts

Poly(A)+RNA-containing material was extracted from the purified cytoplasmic membranes of dormant Artemia cysts by treatment with mild detergents. Sedimentation analysis of the extracts showed a predominant poly(A)-containing fraction at 40 S, associated with about 6% of the extracted proteins. Only limited amounts of poly(A)-containing material were found in the heavier fractions. Poly(A)+RNA extracted from the 40-S fraction sedimented around 14 S. The poly(A)-containing 40-S structures could be purified by treatment with non-ionic or zwitterionic detergents followed by resedimentation in sucrose gradients in the presence or absence of detergent. When the 40-S fraction was analyzed by isopycnic centrifugation in Cs2SO4 gradients, the main part of the poly(A)-containing material banded at a density of 1.27 g/ml. Electron-microscopic examination of this fraction revealed circular or slightly bullet-shaped profiles measuring 17-26 nm. When the 40-S fraction had been submitted to mild RNAase treatment prior to density gradient centrifugation, the material was displaced towards lower density and became less distinct. Purified 40-S particles showed a complex protein pattern not very similar to that of polyribosomal poly(A)+RNA-containing particles from developing embryos, but with components in common with unfractionated membranes. The particles also contained some lipids. The experiments indicate that a major part of the membrane-bound, latent poly(A)+RNA in dormant Artemia cysts occurs in the form of relatively uniform, detergent- and Cs2SO4-resistant structures, independent of ribosomes, but intimately associated with membrane components.     

Balbiani ring RNA in the cytoplasm of Chironomus thummi salivary gland cells

In this paper experimental results on the size, transport and stability of cytoplasmic Balbiani ring RNA and on its appearance in polysomes are presented. Cytoplasmic RNA of salivary gland cells from Chironomus thummi contains two large RNA fractions of about 20×10⁶ dalton and 10×10⁶ dalton in size. These RNA fractions correspond both to Balbiani ring BR 1 RNA and BR 2 RNA and are apparently transported from nucleus into cytoplasm without a significant size reduction. Chase experiments illustrate a great stability of giant cytoplasmic Balbiani ring RNA molecules and exclude the possibility of a precursor-product relationship between these and smaller BR RNA molecules also found in cytoplasm. A part of giant cytoplasmic Balbiani ring RNA molecules is bound to poly(U)-sepharose columns and should, therefore, contain poly(A)-sequences. — Polysomes of salivary gland cells extracted by a gentle lysis procedure and centrifuged through sucrose gradients are characterized by a rather broad sedimentation profile. Polysome sizes up to about 800 S have been detected, but in no case a distinct polysome fraction corresponding in size to Balbiani ring RNA has been observed. Hybridization of polysomal RNA with salivary gland chromosomes in situ resulted in labelling of both Balbiani rings BR 1 and BR 2.     

Phosphorylation of proteins in cytoplasmic and nuclear fractions of human cells treated with double-helical RNA and human recombinant interferon type I]

The dynamics of protein kinases activity in nuclear and cytoplasmic fractions of human fibroblasts treated by preparations of natural and synthetic dsRNA (ridostin, rifastin, larifan and poly(I).poly(C), DEAE-dextran and dsRNA complexes with DEAE-dextran), as well as by preparations of recombinant alpha-2 and beta-1 interferons was obtained. The early activation of enzymes in treated cells extracts and their presence in dsRNA-activated and nonactivated forms were found. In cytoplasmic cellular fractions treated by interferons the dsRNA dependent protein kinases (nonactivated forms- were prevalent.r In contrast, in dsRNA treated cells or dsRNA complexes with DEAE-dextran treated ones the dsRNA independent protein kinases (activated forms) were found, while dsRNA dependent forms induced by interferons were found at later periods. Nuclear protein kinases are mainly dsRNA independent making possible the supposition of their intracellular activation by incoming dsRNA or interferon-induced formation of ds-structures in cellular nuclei. In phosphorylated proteins spectre the 90, 69, 45-40 and 30-35 kDa polypeptides were found. At early intervals in nuclear fractions was found a nuclease resistant and partially EDTA resistant high molecular phosphorylated complex (120 kDa). The complex is, probably, capable of dissociation to low mol mass components. DEAE-dextran induces strong activation of protein kinases in cytoplasm and nuclei and increases the content of activated forms of enzyme in larifan treated cells.     

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.