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Synthesis of poly(A)-binding protein is regulated at the translational level. We have investigated the binding of proteins to this mRNA on the premise that the protein(s) of the mRNP complex may be involved in regulating the expression of the mRNA. We found the first 243 nucleotides of the 5' untranslated region to contain sequences essential for RNP formation. A large, single-stranded bulge structure encompassing stretches rich in adenine nucleotides and a potential stem-loop domain appear to be the primary sites for protein binding. Removal of the 243-nucleotide segment results in a drastic reduction in protein binding and a concomitant increase in translational efficiency in vitro. We suggest that proteins binding to this region, including poly(A)-binding protein itself, may be essential for regulating translation of this mRNA. [ABSTRACT FROM AUTHOR]

Translation of the mRNA for a housekeeping polypeptide of 40 kDa (P40) was found to be regulated under a variety of conditions in rat L6 cells. This mRNA was translated in the proliferating myoblasts but not in the non-proliferating myotubes. A number of chemicals, such as dimethyl sulfoxide, sodium butyrate and aphidicolin, were used to prevent expression of muscle-specific genes in mitogen-poor differentiation medium. In the absence of any detectable accumulation of muscle-specific α-actin mRNA, the P40 mRNA remained in the translated state. A fourth chemical, EGTA, a known inhibitor of fusion of muscle cells, blocked translation of muscle-specific actin and tropomyosin mRNAs. On the other hand, it showed no effect on the translation of P40 mRNA. Addition of Ca2+ to the EGTA-treated cultures, however, almost completely reversed the block of translation of actin tropomyosin mRNAs within four days. Concomitant to Ca2+ reversal of the translational block of muscle mRNAs, P40 mRNA entered the non-translated state. An inverse relationship, therefore, was observed between the translation of housekeeping P40 mRNA and muscle-specific mRNAs. The ability to mimic in vivo regulation of P40 mRNA translation was examined in mRNA-dependent micrococcal-nuclease-treated homologous cell-free extracts. The extracts from myoblasts and myotubes were able to translate P40 mRNA. Furthermore, ribosomes of both myoblast and myotube extracts containing endogenous mRNAs were also able to bind to P40 and actin mRNAs. Myotube extract, however, showed a lower binding ability to P40 mRNA than to the actin mRNA. The ability of ribosomes of myotube extract to bind P40 mRNA was somewhat enhanced by addition of proteins derived from washing these ribosomes with a high-ionic-strength buffer. In order to elucidate the role of interaction between mRNA and proteins in translational control of P40 mRNA, the polypeptide complements of polysomal and free P40 mRNA-protein (mRNP) complexes were also examined. Hybrid selection of polysomal and free P40 mRNP complexes followed the covalent joining of the RNA and protein moieties of mRNP complexes by ultraviolet irradiation of rat L6 cells. Analysis of buoyant densities of these complexes showed that free P40 mRNP had slightly less protein than polysomal P40 mRNP. Furthermore, analysis of the polypeptide complements of both free and polysomal P40 mRNP complexes showed that they were composed of identical polypeptides. The only detectable difference between the polypeptide complements of these complexes was that two polypeptides of 72 kDa and 55 kDa were more abundant in the polysomal P40 mRNP than free P40 mRNP. [ABSTRACT FROM AUTHOR]

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 α and β 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 post- mitotic 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 1)12 with rat genornic 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. [ABSTRACT FROM AUTHOR]

Cytoplasmic messenger RNAs of eukaryotic cells are distributed between polysomal and post-polysomal fractions (free) as protein-bound complexes. These studies were designed to determine whether a specific mRNA isolated from different subcellular compartments is complexed with the same family of polypeptides. As a first approach we have examined the proteins associated with mRNA which codes for histone H4. To perform these experiments HeLa cells were exposed to ultraviolet light to cross-link in vivo polypeptides which are closely associated with nucleic acid. To identify the polypeptides associated with mRNA specific for histones a genomic probe for histone H4 mRNA was immobilized on epoxy-cellulose. By hybrid selection specific mRNPs containing histone mRNA were isolated. Our results reveal the existence of a number of polypeptides associated with both polysomal and post-polysomal historic mRNAs. In polysomal histone mRNA two polypeptides of Mr=49000 and 52500 were the major components. In contrast polypeptides of Mr=43000 and 57000 were the major polypeptide components of post-polysomal (or free) histone mRNA. Furthermore, these results also suggest that the polypeptides associated with either polysomal or free H4 histone mRNA represent a subset of proteins found in poly(A)-free fractions or poly(A)-rich mRNA fractions. [ABSTRACT FROM AUTHOR]

Irradiation of chicken muscle cells with ultraviolet light (254 nm) to cross-link RNA and protein moieties was used to examine the polypeptide complements of cytoplasmic mRNA-protein complexes (mRNP). The polypeptides of translationally active mRNP complexes released from polysomes were compared to the repressed nonpolysomal cytoplasmic (free) mRNP complexes. In general, all of the polypeptides present in free mRNPs were also found in the polysomal mRNPs. In contrast to polysomal mRNPS, polypeptides of Mr 28000, 32000, 46000, 65000 and 150000 were either absent or present in relatively smaller quantities in free mRNP complexes. On the other hand, and relative proportion of polypeptides of Mr 130000 and 43000 was higher in free mRNPs than in polysomal mRNP complexes. To examine the role of cytoplasmic mRNP complexes in protein synthesis or mRNA metabolism, the changes in these complexes were studied following (a) inhibition of mRNA synthesis and (b) heat-shock treatment to alter the pattern of protein synthesis. Actinomycin D was used to inhibit mRNA synthesis in chick myotubes. The possibility of newly synthesized polypeptides of cytoplasmic mRNP complexes being assembled into these complexes in the absence of mRNA synthesis was examined. These studies showed that the polypeptides of both free and polysomal mRNP complexes can bind to pre-existing mRNAs, therefore suggesting that polypeptides of mRNP complexes can be exchanged with a pool of RNA-binding proteins. In free mRNP complexes, this exchange of polypeptides in significantly slower than in the polysomal mRNP complexes. Heat-shock treatment of chicken myotubes induces the synthesis of three polypeptides of Mr = 81000, 65000 and 25000 (heat-shock polypeptides). Whether this altered pattern of protein synthesis following heat-shock treatment could affect the polypeptide composition of translationally active polysomal mRNPs was examined. The results of these studies show that, compared to normal cells, more newly synthesized polypeptides were assembled into polysomal mRNPs following heat-shock treatment. A [35S]methionine-labeled polypeptide of Mr = 80000 was detected in mRNPs of heat-shocked cells, but not of normal cells. This polypeptide was, however, detected by AgNO3 staining of the unlabeled polypeptide of mRNP complexes of normal cells. These results, therefore, suggest thai the assembly of newly synthesized 80000-Mr polypeptide to polysomal mRNPs was enhanced following induction of new heat-shock mRNAs. The results of these studies reported here have been discussed in relation to the concept that free mRNP complexes are inefficiently translated in vivo. Failure to bind to regulatory protein(s) due to competition among mRNAs may be responsible for their inability to participate in protein synthesis. [ABSTRACT FROM AUTHOR]

Exposure of chick myotube cultures to a temperature (45°C) higher than their normal growing temperature (37°C) caused extensive synthesis of three major polypeptides of Mr = 25000, 65000 and 81000 referred to as ‘heat-shock polypeptides’ (hsps). When these cells were allowed to recover from heat-shock treatment at 37°C for 6–8 h, the rate of accumulation of isotope into the 65000-Mr and 81000-Mr hsps declined to levels comparable to those in control cultures maintained at 37°C. However, incorporation of isotope in the 25000-Mr hsp continued at an elevated rate for a longer period than the 65000-Mr and 81000-Mr hsps. When heat-shocked cells were allowed to recover at 37°C in the presence of actinomycin D to block new mRNA synthesis, the hsp synthesis as measured by the incorporation of radioactive isotope in these polypeptides continued at levels comparable to those in heat-shocked cells prior to recovery. The block of recovery by actinomycin D was due to the presence of a greater amount of functional hsp mRNAs in the polysomes as compared to untreated controls. The role of competition between the mRNAs for hsps and normal cellular proteins for the translation machinery in regulating protein synthesis during the recovery from heat shock has been discussed. [ABSTRACT FROM AUTHOR]

When primary cultures of chicken myoblasts were subjected to incubation at a temperature higher than their normal growing temperature of 36–37°C, the pattern of protein synthesis was altered. This condition of heat shock induced a vigorous production of a number of proteins collectively known as 'heat-shock proteins'. The synthesis of heat-shock proteins was achieved without a significant decrease in the production of a broad spectrum of proteins by muscle cells. The synthesis of three major heat-shock polypeptides with Mr values of 81000, 65000 and 25000 was observed in both mononucleated dividing myoblast cells and terminally differentiated myotubes. Two-dimensional electrophoretic separation of the heat-induced polypeptides synthesized by myogenetic cultures further established that same set of polypeptides with Mr of 65000 (pI 6.0 and 5.5), 81000 (pI 6.2) and 25000 (pI 5.6 and 5.3) were produced in myoblasts and myotubes. The effect of the changes in pattern of protein synthesis on the mRNA and protein moieties of non-polysomal cytoplasmic mRNA-protein complexes (free mRNP) was examined. Free mRNP complexes sedimenting at 20–35 S were isolated from the post-ribosomal supernatant of both normal and heat-shocked myotube cultures by centrifugation in a sucrose gradient. A 10–20S RNA fraction isolated from these complexes stimulated protein synthesis in a cell-free system. The RNA fraction obtained from heat-shocked cells appeared to direct the synthesis of all three major heat-shock proteins. In contrast, synthesis of these polypeptides was not detected when RNA from free mRNP complexes of normal cells was used for translation. The free mRNP complexes of both normal and heat-shocked cells showed a buoyant density of 1.195 g/cm3 in metrizamide gradients. A large number of polypeptides of Mr = 35000–105000 were present in the highly purified free mRNP complexes isolated from the metrizamide gradient. Similar sets of polypeptides were found in these complexes from both normal and heat-shocked myotube culture. However, the relative proportion of a 65000-Mr polypeptide was dramatically increased in the free tuRN P complexes of heat-shocked cells. Two-dimensional gel electrophoretic analysis revealed that this polypeptide and the 65000-Mr heat-shock polypeptide exhibit similar electrophoretic migration properties. These observations suggest that, following heat-shock treatment of chicken myotube cultures, the changes in the pattern of protein synthesis is accompanied by alteration of the mRNA and protein composition of free mRNP complexes. [ABSTRACT FROM AUTHOR]

A class of protein(s) with high affinity for RNA was isolated by affinity chromatography on poly(U)-Sepharose from an extract of embryonic chicken muscle which had been freed of ribo- somes and cytoplasmic messenger ribonucleoprotein complexes. The RNA-binding protein comprised about 1% of the total protein of the post-ribosomal extract. Analysis of the polypeptide com- position of the RNA-binding proteins by two-dimensional gel electrophoresis, using separation according to charge in the first dimension and molecular weight in the second, showed the presence of at least 37 polypeptides. These polypeptides were non-ribosomal in nature. The major class of polypeptides falls into three molecular weight groups with Mr of 34000-41000, 46000- 54000 and 68000-78000. Nine of these polypeptides showed similar electrophoretic migration properties to that of polypeptides from nonpolysomal cytoplasmic messenger ribonucleoprotein. Furthermore, four polypeptides, including one acidic 38000-Mr polypeptide, displayed electrophoretic migration properties similar to those of polypeptides present in crude initiation factor preparation. The RNA-binding proteins can form a ribonucleoprotein complex(es) when incubated with either synthetic or natural RNAs. Furthermore, in competition binding studies, mRNAs were found to be more efficient in complexing with the RNA-binding proteins than were non-mRNA molecules. A cyclic-AMP-independent protein kinase activity was detected in the RNA-binding proteins. This protein kinase phosphorylates several polypeptides of the RNA-binding proteins. Among these, the 38000-Mr acidic polypeptide was the best acceptor of phosphate group(s). In contrast, ribosomal proteins were found to be poor substrates for the protein kinase. The acidic 38000-Mr polypeptide of RNA-binding protein, which is most intensely phosphorylated by the endogenous protein kinase, also appeared to be the common polypeptide found in the crude initiation factor preparation. The similarity of this protein kinase with the cyclic-AMP-independent protein kinase activity, previously reported in the nonpolysomal cytoplasmic messenger ribonucleoprotein complexes of embryonic chicken muscle [Bag, J. and Sells, B. H. (1979) J. Biol. Chem. 254, 3137-3140] has been discussed. These RNA-binding proteins interfered with translation of both homologous and heterologous mRNAs in the rabbit reticulocyte cell-free system. The effects obtained, however, were concentration-dependent in the range 7-30 μg/ml: the proteins inhibited polypeptide formation while at higher concentrations (70 μg/ml) they stimulated translation. The presence of both inhibitor and activator of mRNA translation in RNA-binding protein has been discussed. [ABSTRACT FROM AUTHOR]

The distribution of pulse-labeled poly(A)-containing RNAs between the polysomal and non-polysomal (free) cytoplasmic fractions of embryonic chicken muscle was examined by affinity chromatography on oligo(dT)-cellulose. Approximately 60% of the total cytoplasmic poly(A)-containing RNAs were present in the polysome; the remainder were present outside the polysome, the 3H-labeled ones being present in the form of ribonucleoprotein complexes. These free mRNA · protein complexes can be divided into two populations: one can bind to an oligo(dT)-cellulose column as a ribonucleoprotein complex, suggesting that the 3′-poly(A) region was available for interaction with the oligo(dT) residues; in the other, this region was presumably covered with proteins and can only bind to the oligo(dT)-cellulose column following their removal. The mRNA · protein bound to oligo(dT)-cellulose was subfractionated into three populations. The first fraction was eluted from the column by using low-ionic-strength buffer at 2°C, the major portion was eluted by a combination of low-ionic-strength buffer and a temperature of 37°C, while the remainder was eluted with 50% formamide. Each fraction displayed a distinctive buoyant density in a CsCl gradient, suggesting a difference in their protein content. The nature of polypeptides associated with these fractions was analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Polypeptides of Mr 36500, 41000, 45000, 47000, 51000 and 75000 were common to all three mRNA protein fractions. The third population (eluted with 50% formamide) contains five additional polypeptides of Mr 54000, 66000, 68000, 73000 and 105000. The direct interaction of free proteins with oligo(dT)-cellulose was studied by modification of the binding of mRNA · protein to oligo(dT) by saturation of oligo(dT) sequences with poly(A) or the poly(A) segment of mRNA · protein with poly(U). Two polypeptides of Mr 41000 and 54000 showed independent binding to these columns; they were eluted with 50% formamide but not at 37 °C in low-salt buffer. Therefore, the source of the 41000-Mr and 54000-Mr polypeptides was considered as other than mRNA · protein. The translation of these mRNA · protein fractions showed that all three can be translated either as protein-free RNA or complexed with proteins. [ABSTRACT FROM AUTHOR]