Membrane-bound ribosomes of myeloma cells. VI. Initiation of immunoglobulin mRNA translation occurs on free ribosomes

Immunoglobulin heavy (Ig H) and light (Ig L) chain mRNA molecules have been released from the endoplasmic reticulum (ER) membranes as free (F) mRNP particles when MOPC 21 (P3K) mouse myeloma cells are exposed to a hypertonic initiation block (HIB). The subsequent fate of these mRNA sequences has been examined when the cells are returned to normal growth medium. Upon return to isotonicity, all previously translated mRNA molecules reassociate with ribosomes and form functional polysomes. Ig H mRNA is found incorporated first into F polysomes and then into membrane-bound (MB) polysomes. Kinetic studies indicate that the time of passage of Ig H mRNA in F polysomes is approximately 30 s, during which a nascent polypeptide chain of approximately 80 amino acids would have been completed. When the rate of polypeptide elongation is depressed with emetine during the recovery from HIB, both Ig H and L mRNA molecules accumulate in small F polysomes. These results indicate that the formation of Ig-synthesizing polysomes proceeds in the sequence: mRNA leads to F polysomes leads to MB polysomes. With the additional observation that during HIB recovery puromycin completely prevents the reassociation of Ig mRNA with the ER, these findings support a model of MB polysome formation in which the specificity of membrane attachment is determined by the nature of the N- terminal amino acid sequence of the nascent polypeptide chain.     

Characterization and Complexity of Wheat Developing Endosperm mRNAs

Free and membrane-bound (MB) polysomes and the corresponding polyadenylated RNAs (polyA⁺ RNAs) have been isolated from developing wheat endosperm (Triticum aestivum L.) Free and MB poly(A)⁺ RNAs, analyzed on isokinetic sucrose gradient with [³H]polyuridylic acid [poly(U)] hybridization detection, appear to be 11S to 12S in size with a 7% poly(A) tail for MB RNAs. cDNAs synthesized using both of these mRNA populations in presence of a potent RNase inhibitor (RNasin), have been used for hybridization kinetics experiments. The mean square fitting analysis of the hybridization kinetics between MB cDNA and its template reveals the presence of two abundance classes representing roughly ⅔ and ⅓ of the MB poly(A)⁺ RNAs and containing the information for approximately 75 superabundant species (21,000 copies per cell) and 750 intermediate species (530 copies per cell), respectively. The mRNA population extracted from free polysomes is divided into three abundance classes. The first one is composed of superabundant sequences which would correspond to the MB superabundant mRNAs. The free mRNAs consist of about 11,000 diverse sequences, most of them being rare sequences. Heterologous hybridizations of MB cDNAs to free mRNAs have shown that some mRNAs are common to both populations. This could be explained either by a partial contamination or by free polysomes en route to their membrane destination. Contrary to the low number of diverse mRNAs corresponding to the legume seed storage proteins, the wheat endosperm superabundant mRNAs consist of about 75 different sequences which would encode most of the seed storage proteins, especially gliadins.     

Isolation of separate mRNAs for α- and β-Tubulin and characterization of the corresponding in vitro translation products

The messenger RNAs coding for α- and β-tubulin have been isolated from embryonic chick brain. Although the mRNAs for the two tubulin subunits have been resolved on native gels, they are very similar in molecular weight (650,000 daltons) as judged by mobility on denaturing gels containing methyl mercury. The mRNAs for β- and γ-actin have also been resolved on native gels, but migrate as an unresolved peak (molecular weight 650,000–700,000 daltons) under denaturing conditions. Since the nonmuscle actins are substantially smaller proteins than α- and β-tubulin, the large size of chick nonmuscle actin mRNAs suggests an unusually long untranslated region.Since tubulin and actin polypeptides are internal structural proteins, one would expect them to be synthesized only on free polysomes. Translation of mRNA derived directly from a purified membrane fraction or by puromycin release from that fraction, however, showed the synthesis of a small proportion of these proteins on polysomes that are membrane-associated. Peptide mapping has in all cases confirmed the identity of the products of cell-free synthesis with authentic α-tubulin, β-tubulin and actin. Approximately 67% of the α- and 13% of the β-tubulin chains produced by in vitro translation are competent for co-assembly into microtubules with added carrier microtubule protein.     

Distribution of messenger ribonucleic acid in polysomes and nonpolysomal particles of sea urchin embryos: translational control of actin synthesis

We have used cell-free translation and two-dimensional gel electrophoresis to examine the complexities of the polysomal and cytoplasmic nonpolysomal [ribonucleo-protein (free RNP)] messenger ribonucleic acid (mRNA) populations of sea urchin eggs and embryos. We show that all species of mRNA detected by this method are represented in both the polysomes and free RNPs; essentially all messages present in polysomes are also in the free RNP fraction. However, the cytoplasmic distribution is clearly nonrandom since some templates are relatively concentrated in the free RNPs and others are predominantly in the polysomes. The polypeptides synthesized under the direction of unfertilized egg mRNA are qualitatively indistinguishable from those made by using embryonic mRNA, indicating that the complexity of the abundant class mRNA remains unchanged from egg through early development. However large changes in the abundancies of specific mRNAs occur, and changes are detected in the polysomal/free RNP distribution of some mRNAs through development. The differences in the realtive abundancies of specific mRNAs between polysomes and free RNPs and the developmental changes that take place indicate significant cytoplasmic selection of mRNA for translation. Three different forms of actin (termed alpha, beta, and gamma) were identified among the translation products. Messages for all three are present in the unfertilized egg and early cleavage embryo, yet the gamma form is preferentially located in the polysomes and the alpha and beta in the free RNPs. The relative concentrations of the three change greatly during development as do their relative distributions into polysomes and free RNPs. Examinations of in vivo labeled proteins largely support the in vitro findings. The results indicate that the synthesis of actin mRNAs increases greatly during development and that the expression of the actin mRNAs is partly controlled at the translation level during early development.     

Subcellular localization of histone messenger RNAs on cytoskeleton-associated free polysomes in HeLa S3 cells

We have examined the subcellular distribution of histone mRNA-containing polysomes in HeLa S3 cells to assess the possible relationship between localization of histone mRNAs and the regulation of cellular histone mRNA levels. The distribution of histone mRNAs on free and membrane bound polysomes was examined as well as the association of histone mRNA-containing polysomes with the cytoskeleton. The subcellular localization of histone mRNAs was compared with that of HLA-B7 mRNAs which encode a cell surface antigen. Histone mRNAs were localized predominantly on the free polysomes, whereas the HLA-B7 mRNA was found almost exclusively on membrane bound polysomes. However, both species of mRNA were found associated with the cytoskeleton. Interruption of DNA synthesis by hydroxyurea treatment resulted in a rapid and selective destabilization of histone mRNAs in each subcellular fraction; in contrast, the stability of HLA-B7 mRNA appeared unaffected. The results presented confirm that histone mRNAs are predominantly located on non-membrane bound polysomes and suggest that these polysomes are associated with the cytoskeletal framework.     

In vitro synthesis of superoxide dismutases of rat liver

The syntheses of copper, zinc-superoxide dismutase (Cu,Zn-SOD) and manganese-superoxide dismutase (Mn-SOD) in vitro were studied. Both Cu,Zn-SOD and Mn-SOD were preferentially synthesized by free polysomes. Mn-SOD was synthesized as a large precursor (26,000 daltons), which was processed to the mature size (22,500 daltons) by in vitro incubation with a rat liver mitochondrial fraction. On the other hand, Cu,Zn-SOD was synthesized as the mature size product. It was shown that Cu,Zn-SOD and Mn-SOD synthesized in vitro represented 0.018% and 0.016% of the total translation products of free polysomes, respectively.     

In yeast, the 3' untranslated region or the presequence of ATM1 is required for the exclusive localization of its mRNA to the vicinity of mitochondria

We isolated mitochondria from Saccharomyces cerevisiae to selectively study polysomes bound to the mitochondrial surface. The distribution of several mRNAs coding for mitochondrial proteins was examined in free and mitochondrion-bound polysomes. Some mRNAs exclusively localize to mitochondrion-bound polysomes, such as the ones coding for Atm1p, Cox10p, Tim44p, Atp2p, and Cot1p. In contrast, mRNAs encoding Cox6p, Cox5a, Aac1p, and Mir1p are found enriched in free cytoplasmic polysome fractions. Aac1p and Mir1p are transporters that lack cleavable presequences. Sequences required for mRNA asymmetric subcellular distribution were determined by analyzing the localization of reporter mRNAs containing the presequence coding region and/or the 3'-untranslated region (3'UTR) of ATM1, a gene encoding an ABC transporter of the mitochondrial inner membrane. Biochemical analyses of mitochondrion-bound polysomes and direct visualization of RNA localization in living yeast cells allowed us to demonstrate that either the presequence coding region or the 3'UTR of ATM1 is sufficient to allow the reporter mRNA to localize to the vicinity of the mitochondrion, independently of its translation. These data demonstrate that mRNA localization is one of the mechanisms used, in yeast, for segregating mitochondrial proteins.     

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.     

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.     

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.     

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.     

Contribution of polyadenylate sequences to the translational efficiency of globin messenger RNAs.

mRNAs from reticulocyte polysomes were fractionated by chromatography on poly(U)-Sepharose and thermal elution. The molar ratio of alpha- to beta-globin mRNA was found to be 2:1 and 1:1 respectively in short- and long-poly(A) size classes. Translational analyses indicated that the globin mRNAs containing long poly(A) tracts (with a mean length of about 70 nucleotides) directed protein synthesis with higher rates than did mRNA containing short poly(A) tracts (15-35 nucleotides). Experiments performed with sub-saturating mRNA concentrations showed that the digestion with RNAase H induced a decrease in the translational capacity of both globin mRNAs and an increase in the alpha- to beta-globin synthesis ratio. No correlation was observed between the size of the poly(A) tail in mRNA and the optimal K+ requirement for translation.     

P bodies, stress granules, and viral life cycles

Eukaryotic mRNAs are in a dynamic equilibrium between different subcellular locations. Translating mRNAs can be found in polysomes, mRNAs stalled in translation initiation accumulate in stress granules and mRNAs targeted for degradation or translation repression can accumulate in P bodies. Partitioning of mRNAs between polysomes, stress granules, and P bodies affects rates of translation and mRNA degradation. Host proteins within P bodies and stress granules can enhance or limit viral infection, and some viral RNAs and proteins accumulate in P bodies and/or stress granules. Thus, an important interplay among P bodies, stress granules, and viral life cycles is beginning to emerge.     

Isolation and partial purification of ceruloplasmin messenger RNA from rat liver

Partially purified ceruloplasmin mRNA was isolated using indirect immunoprecipitation of rat liver polysomes and poly(U)-Sepharose chromatography of polysomal RNA. This RNA programmed the synthesis of ceruloplasmin polypeptides in a cell-free system from mitochondria. Immunochemical analysis of the translation products revealed a 40-fold enrichment of the ceruloplasmin mRNA activity. The purified ceruloplasmin mRNA migrated as a major homogeneous component with an apparent molecular weight about 1×10⁶ daltons in polyacrylamide gels containing sodium dodecyl sulfate. The immunoprecipitated products of the cell-free translation had molecular weights in the range 4.5–5.4×10⁴ daltons as estimated by gel-electrophoresis under denaturating conditions. These values approach the weight of the half-molecule of native ceruloplasmin.     

Biosynthesis of carnitine octanoyltransferase and carnitine palmitoyltransferase

Male Wistar rats were fed a diet with or without di(2-ethylhexyl)phthalate (DEHP) for 2 weeks. Carnitine octanoyltransferase (COT) in the liver was increased 23.5-fold in rats given DEHP. It was found by in vivo experiments using L-[4,5-3H]leucine and the immunoprecipitation technique that the rate of synthesis of COT was 14.1-fold higher and that of its degradation was 1.5-fold lower in the DEHP group. COT was translated much more effectively in free polysomes than in membrane-bound polysomes. The molecular size of the in vitro product was the same as that of the mature enzyme. The translation activity of mRNA coding for COT measured with total hepatic RNA was 16.6-fold higher in the DEHP group. Carnitine palmitoyltransferase (CPT) was increased 5.9-fold after administration of DEHP. The rate of synthesis of CPT measured in the in vivo experiment was 5.0-fold higher in the DEHP group. The rate of its degradation was the same in the two groups. CPT was also translated much more effectively in free polysomes. The size of the preenzyme was larger than that of the subunit of the mature enzyme by about 2,400 daltons. In contrast to COT, the increase in the translation activity of mRNA for CPT by administration of DEHP was markedly higher than the increase in the rate of its synthesis measured in the in vivo experiment.