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There is undetectable transcription of 11S storage protein (medicagin) mRNA by nuclei isolated from pre-cotyledonary-stage somatic embryos of alfalfa (Medicago sativa L). However, this message exists at steady-state levels in the embryos at this stage of development without concomitant synthesis of the storage protein. At the pre-cotyledonary stage, therefore, the transcriptional rate for 11S mRNA is low; what message is transcribed is sequestered in the form of mRNP complexes and is not recruited into polysomes in vivo (33). Both transcription (in vivo and in isolated nuclei) and translation of the 11S mRNA are evident at the onset of cotyledon development in somatic and zygotic embryos, reaching a maximum during expansion of the cotyledons and then declining as the embryos mature. Pre-cotyledonary-stage somatic embryos which do not utilize the 11S-mRNA in polysomes lack certain mRNA-binding proteins (32, 36 and 38 kD) which are present at later stages of development. These mRNA-binding proteins may be responsible for the initiation of large polysome formation since they were exclusively present in the translational extracts of cotyledonary somatic and zygotic embryos in which there was no repression of storage protein synthesis. In contrast, the pre-cotyledonary somatic embryos contained a different set of 11S-mRNA-binding proteins (28, 50, 55, and 62 kD) whose presence in the cotyledonary stage embryos was very rare or non-existent; these could be responsible for preventing translation.

The relationship between attachment of mRNA to the cytoskeletal framework and its translation was examined using the mRNA for a polypeptide of 40 kDa (P-40) which is translated in rat L6 myoblasts but not in the myotubes. In both myoblasts and myotubes this mRNA was found to be associated with the cytoskeletal framework. Furthermore, the stability of the association between P-40 mRNA and the cytoskeletal framework in absence of RNA and protein synthesis was examined by using actinomycin D and NaF to block RNA and protein synthesis, respectively. In absence of RNA synthesis portions of both nontranslated P-40 mRNA and translated actin mRNA of myotubes were released into the soluble fraction. In myoblasts, however, both mRNAs remained associated with the cytoskeletal framework following inhibition of RNA synthesis. Inhibition of protein synthesis, on the other hand, had a more dramatic effect on the association between the cytoskeletal framework and P-40 mRNA in myoblasts but not in myotubes. In contrast, the association between actin mRNA and cytoskeletal framework was unaffected by inhibition of protein synthesis in both myoblasts and myotubes. The results of these studies show that the molecular nature of association between cytoskeletal framework and mRNA may differ among mRNAs and may also depend on whether the cells are dividing or are terminally differentiated. Furthermore, no direct relationship between the translation of mRNA and its attachment to the cytoskeletal framework was observed.

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.

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 alpha-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.

The distribution of mRNA between the detergent-soluble and insoluble (cytoskeleton) fractions in rat L6 myoblast and myotube cells was examined. Approximately 85% of cytoplasmic mRNA in both myoblasts and myotubes was found associated with the cytoskeletal framework. The cytoskeleton-bound mRNA was present as polysomes. In contrast, the mRNA of the detergent-soluble fraction was not associated with ribosomes and was thus considered to be the repressed population. The association of mRNA with the cytoskeletal framework was not affected by treatments leading to dissociation of polysomes. Differential distribution of mRNA between the soluble and cytoskeleton-bound fractions was analyzed by in vitro translation. The mRNAs coding for polypeptides of molecular masses 40 kDa and 60 kDa were preferentially enriched in the soluble fraction. The nature of binding between mRNA and the cytoskeletal framework was examined following in vivo cross-linking of RNA and protein by irradiating muscle cells with ultraviolet light. It was observed that this treatment covalently linked RNA and the neighbouring protein moieties without any detectable damage to the cytoskeletal framework, as measured by the distribution of RNAs and proteins between the cytoskeleton and soluble fractions. Analysis of the polypeptide moieties cross-linked to the mRNA have shown that a large number of polypeptides of molecular masses between 15-220 kDa were associated with both cytoskeleton-bound and soluble mRNAs. The polypeptide moieties of these mRNA-protein complexes were not only similar in the cytoskeleton and soluble mRNA-protein complexes but also were similar between myoblasts and myotubes. However, one polypeptide of 165 kDa was preferentially associated with the cytoskeleton-bound mRNA-protein complexes. Interestingly this 165-kDa polypeptide was also preferentially enriched in the total proteins from the cytoskeleton fraction. This result suggests a possible role of the 165-kDa polypeptide in association between mRNA and the cytoskeletal framework. To examine the mechanism of interaction between mRNA and the cytoskeletal framework we have reported here a ghost monolayer transcription system from myotubes. This transcription system was able to synthesize rRNA and mRNA. The mRNA transcribed in vitro was preferentially associated with the cytoskeleton structure present in the ghost monolayer system.

The mRNA coding for a 40-kDa polypeptide (P-40) was previously cloned and sub-cellular distribution of this mRNA was examined in rat L6 myoblast cells under different conditions [Pramanik, S. & Bag, J. (1987) Eur. J. Biochem. 170, 59-67]. The translation of this mRNA was found to be regulated during differentiation of myoblasts. This mRNA was translated in proliferating myoblasts but not in the non-dividing differentiated myotubes. We have further examined whether the mRNA present in the polysomal fraction of myoblasts and that in the free non-polysomal fraction in myotubes was identical by nuclease S1 mapping. The coding strand of the 600-base-pair PstI fragment of the recombinant clone was 3'-end-labeled with cordycepin 5'-[alpha-32P]triphosphate and hybridized with RNA from either myoblasts or myotubes. The results of these studies have shown that RNA from both preparations was fully able to hybridize with the probe DNA and, therefore, protected the 600-nucleotide-long fragment from nuclease S1 digestion, thus suggesting that the sequence of 600 nucleotides at 3' ends of both translationally active polysomal mRNA of myoblasts and repressed free mRNA of myotubes are identical. These results also confirmed the results of our earlier studies on the subcellular distribution of this mRNA by Northern blot analysis. Further studies were also performed to determine whether withdrawal of muscle cells from the cell cycle during differentiation to form myotubes alone was responsible for regulating translation of P-40 mRNA. The results of the subcellular distribution of this mRNA in proliferating myoblasts following inhibition of DNA synthesis by cytosine arabinoside have shown that translation of P-40 mRNA continued in absence of DNA synthesis. This observation suggests that an additional signal is necessary to block the translation of P-40 mRNA in myotubes. The relationship between the translation of P-40 mRNA and its stability was examined. Two different methods were used to determine the stability of mRNAs. The first approach was by determining the steady-state levels of this mRNA following inhibition of RNA synthesis by actinomycin D. In the second method, we have determined the amount of 3H-labeled P-40 mRNA during pulse and chase experiments. Both methods produced similar results. It was found that the stability of P-40 mRNA was not altered during differentiation of rat L6 cells. The results of pulse and chase studies have also shown that P-40 mRNA was synthesized in both myoblasts and myotubes.(ABSTRACT TRUNCATED AT 400 WORDS)

The mRNA of rat L6 muscle cells was distributed between a detergent-insoluble fraction containing the cytoskeletal framework and a detergent-soluble fraction. The majority of cytoskeleton-bound mRNA was translationally active and present as polysomes. The mRNA of the detergent-soluble fraction was not associated with the ribosomes and, thus, considered to be the repressed free population. The binding of mRNA was not mediated through ribosomes or the poly(A) region of mRNA. Cross-linking of RNA and proteins was used to examine whether proteins of the cytoskeletal framework were involved in binding mRNA to this structure. Analysis of the mRNA-protein complexes has shown that a large number of polypeptides of molecular masses between 15 and 220 kilodaltons (kDa) were associated with both cytoskeleton-bound and soluble mRNAs. However, a 165-kDa polypeptide was preferentially associated with cytoskeleton-bound mRNA-protein complexes. This polypeptide was also enriched in the total proteins of the cytoskeleton fraction. We have suggested a receptor-like role for the 165-kDa polypeptide in binding mRNA to the cytoskeletal framework. The mechanism of interaction between the cytoskeleton and mRNA was further examined by using a ghost-monolayer transcription system. The mRNA synthesized by this transcription system was preferentially retained in the detergent-insoluble cytoskeleton component of the ghost-monolayer preparation. To understand the physiological significance of the distribution of mRNA between the translationally active cytoskeleton-bound and repressed soluble fractions we have isolated a cDNA clone for a 1.3-kilobase (kb) mRNA. This mRNA was preferentially repressed in myotubes. Distribution of this mRNA was determined by Northern blot analysis using the recombinant plasmid. This analysis indicates that nearly 90% of this mRNA was not associated with ribosomes. In contrast, only 3% of alpha-actin mRNA was found in the repressed population. However, approximately 25% of the 1.3-kb mRNA was present as repressed free messenger ribonucleoprotein. This behaviour is again different from that of actin. All of the cytoskeleton-bound alpha-actin mRNA was associated with polysomes. Furthermore, most of the small amount of alpha-actin mRNA which was present in the soluble fraction was also associated with polysomes. We have, therefore, concluded from these observations that binding of mRNA to the cytoskeleton framework and translation of mRNA are two separate events. We have suggested that mRNA is transported to the cytoplasm as a cytoskeleton-associated complex and further interaction with ribosomes may stabilize this complex.(ABSTRACT TRUNCATED AT 400 WORDS)