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.     

Effect of viral infection on host protein synthesis and mRNA association with the cytoplasmic cytoskeletal structure

We studied the association of several eucaryotic viral and cellular mRNAs with cytoskeletal fractions derived from normal and virus-infected cells. We found that all mRNAs appear to associate with the cytoskeletal structure during protein synthesis, irrespective of their 5' and 3' terminal structures: e.g., poliovirus that lacks a 5' cap structure or reovirus and histone mRNAs that lack a 3' poly A tail associated with the cytoskeletal framework to the same extent as capped, polyadenylated actin mRNA. Cellular (actin) and viral (vesicular stomatitis virus and reovirus) mRNAs were released from the cytoskeletal framework and their translation was inhibited when cells were infected with poliovirus. In contrast, actin mRNA was not released from the cytoskeleton during vesicular stomatitis virus infection although actin synthesis was inhibited. In addition, several other conditions under which protein synthesis is inhibited did not result in the release of mRNAs from the cytoskeletal framework. We conclude that the association of mRNA with the cytoskeletal framework is required but is not sufficient for protein synthesis in eucaryotes. Furthermore, the shut-off of host protein synthesis during poliovirus infection and not vesicular stomatitis virus infection occurs by a unique mechanism that leads to the release of host mRNAs from the cytoskeleton.     

The effect of inhibitors of DNA synthesis on 40-kilodalton polypeptide translation in rat L6 myoblasts

Messenger RNA coding for a polypeptide of 40 kilodaltons (P40) was translated in proliferating rat L6 myoblasts but not in the terminally differentiated myotubes. The relationship between DNA synthesis, differentiation, and P40 mRNA translation was studied. Aphidicolin, a reversible inhibitor of DNA synthesis, was shown to block DNA synthesis in proliferating myoblasts without allowing these cells to differentiate. A second inhibitor, cytosine arabinoside, when added to dividing myoblasts also prevented differentiation. In the absence of biochemical differentiation P40 mRNA remained in the translated state. Translational repression of this mRNA was, therefore, linked to the biochemical differentiation of rat L6 myoblasts.     

Effect of sodium butyrate on gene expression in a rat myogenic cell line

Sodium butyrate, when added in millimolar concentration to a culture of myoblasts of the L6 cell line, inhibits reversibly cell proliferation and differentiation. In the present work, we have studied the effect of Na butyrate on the translational efficiency of the overall poly (A)+ RNA. The mRNA from treated cells was translated in vitro as efficiently as proliferating myoblasts mRNA, while a decrease of translation efficiency was observed with myotubes mRNA. In addition this RNA directs the synthesis of several new polypeptides. on the switch on of alpha actin and myosin heavy chains (MHC), muscle specific genes by the dot blot and Northern blot techniques using cloned probes. Na butyrate prevented the expression of MHC and allowed the switch on of alpha actin gene but at a lesser extent than in normal myotubes. In addition the drug prevented the translocation of alpha actin mRNA into the cytoplasm.     

Cytoskeletal association of muscle-specific mRNAs in differentiating L6 rat myoblasts

The importance of the cytoskeleton in protein synthesis was studied in differentiating L6 rat myoblasts. Soluble and cytoskeletal fractions obtained after gentle, non-ionic detergent lysis of myoblasts and myotubes were analysed for the presence of ribosomes and mRNPs. Polysomal mRNPs were predominantly associated with the cytoskeletal framework and free mRNPs were present in both soluble and cytoskeletal fractions. An examination of the distribution of specific mRNAs in the polysomal and free mRNP populations of both cytoplasmic fractions revealed differences in the pattern of their distribution. It is further demonstrated that in the L6 rat myoblast system, ribosomes and mRNA (or mRNP) are not associated with the microfilaments, unlike in other systems studied.     

Coordinate accumulation of contractile protein mRNAs during myoblast differentiation.

The synthesis of contractile protein mRNAs has been studied during the differentiation of quail skeletal muscle myoblasts in culture. Eight contractile protein mRNAs were identified by translation of total cellular RNA isolated from differentiated myofibers in wheat germ and reticulocyte lysates. Products of the translation systems were fractionated by two-dimensional gel electrophoresis, and incorporation of [³⁵S]methionine into individual contractile proteins was quantitated by computerized densitometry of autoradiograms. These translation assay systems were used to quantitate levels of contractile protein mRNAs in cultures of myoblasts undergoing highly synchronous differentiation. Our results show that dividing myoblasts contain very little, if any, translatable contractile protein mRNA. The mRNAs coding for myosin heavy chain, the musclespecific actin, three myosin light chains, two tropomyosin subunits, and one troponin subunit begin to coordinately accumulate at fusion, when contractile protein synthesis is activated. Their levels increase 50- to 200-fold during the next 30 hr, paralleling increases in the rates of contractile protein synthesis. These results indicate that the contractile protein mRNAs accumulate coordinately during myoblast differentiation and that contractile protein synthesis is regulated by changes in the levels of these mRNAs.     

Changes in distribution of actin mRNA in different polysome fractions following stimulation of MPC-11 cells

Individual mRNA species have been shown to differ both with respect to localization in the cell, and in their distribution upon stimulation of cells with different signals. In this study we have examined the distribution of actin mRNA in the free, cytoskeletal-bound, and membrane-bound RNA fractions, both in starved cells, and in response to stimulation by feeding. These results were then compared with mRNAs for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and histone H4. The results we obtained showed that actin mRNA was located in the free RNA fraction in starved cells, while upon stimulation it was located both in the free, and in the cytoskeletal fraction; no redistribution of GAPDH mRNA occurred between the three RNA fractions, while H4 mRNA showed a different localization upon stimulation. Incubation with the drugs actinomycin-D and cycloheximide showed that an altered localization of actin mRNA from free in starved cells to free and cytoskeletal mRNA fractions following stimulation, was dependent on RNA synthesis, and not on protein synthesis.     

Ultrastructural visualization of cytoskeletal mRNAs and their associated proteins using double-label in situ hybridization

We have been able to visualize cytoskeletal messenger RNA molecules at high resolution using nonisotopic in situ hybridization followed by whole-mount electron microscopy. Biotinated cDNA probes for actin, tubulin, or vimentin mRNAs were hybridized to Triton-extracted chicken embryo fibroblasts and myoblasts. The cells were then exposed to antibodies against biotin followed by colloidal gold-conjugated antibodies and then critical-point dried. Identification of mRNA was possible using a probe fragmented to small sizes such that hybridization of several probe fragments along the mRNA was detected as a string of colloidal gold particles qualitatively and quantitatively distinguishable from nonspecific background. Extensive analysis showed that when eight gold particles were seen in this iterated array, the signal to noise ratio was greater than 30:1. Furthermore, these gold particles were colinear, often spiral, or circular suggesting detection of a single nucleic acid molecule. Antibodies against actin, vimentin, or tubulin proteins were used after in situ hybridization, allowing simultaneous detection of the protein and its cognate message on the same sample. This revealed that cytoskeletal mRNAs are likely to be extremely close to actin protein (5 nm or less) and unlikely to be within 20 nm of vimentin or tubulin filaments. Actin mRNA was found to be more predominant in lamellipodia of motile cells, confirming previous results. These results indicate that this high resolution in situ hybridization approach is a powerful tool by which to investigate the association of mRNA with the cytoskeleton.     

p38 MAPK interacts with actin and modulates filament assembly during skeletal muscle differentiation

Skeletal muscle differentiation is marked by enhanced myotube formation and increased cytoskeletal rearrangement. Actin, a cytoskeletal protein is involved in various cellular functions such as glucose transport, intracellular trafficking, cell shape, and coordinated cell movement in response to various extracellular signals. The present study reveals an association between actin and p38 MAPK only in differentiated myotubes, not in proliferating myoblasts. Actin filament disassembly caused by cytochalasinD can be reversed using the potent activator of p38 MAPK, anisomycin. Pretreatment of myotubes with anisomycin partially resisted the effect of cytochalasinD. However, inhibition of p38 MAPK completely abolished the anisomycin-mediated actin remodeling. Data suggests that p38 MAPK interacts with actin and modulates actin filament rearrangement in differentiated L6E9 skeletal muscle cells.     

Intracellular localization of messenger RNAs for cytoskeletal proteins

We have analyzed intracellular distributions of mRNAs for the cytoskeletal proteins actin, vimentin, and tubulin by in situ hybridization. Although polyadenylated RNA was homogeneously distributed throughout the cell, actin mRNA demonstrated a nonhomogeneous distribution in 95% of randomly selected chicken embryonic myoblasts and fibroblasts, as detected by isotopic and nonisotopic techniques. Actin mRNA concentrations were highest at cell extremities, generally in lamellipodia, where grain densities were up to 16-fold higher than in areas near the nucleus. Vimentin mRNA, unlike actin mRNA, was distributed near the nucleus. Tubulin mRNA appeared most concentrated in the peripheral cytoplasm. These results demonstrate that cytoplasmic mRNAs are localized in specific, nonrandom cellular patterns and that localized concentrations of specific proteins may result from corresponding localization of their respective mRNAs. Hence, actin mRNA distribution may result in increased concentration of actin filaments in lamellipodia of motile cells.     

Stable ribosome binding to the endoplasmic reticulum enables compartment-specific regulation of mRNA translation

In eukaryotic cells, protein synthesis is compartmentalized; mRNAs encoding secretory/membrane proteins are translated on endoplasmic reticulum (ER)-bound ribosomes, whereas mRNAs encoding cytosolic proteins are translated on free ribosomes. mRNA partitioning between the two compartments occurs via positive selection: free ribosomes engaged in the translation of signal sequence-encoding mRNAs are trafficked from the cytosol to the ER. After translation termination, ER-bound ribosomes are thought to dissociate, thereby completing a cycle of mRNA partitioning. At present, the physiological basis for termination-coupled ribosome release is unknown. To gain insight into this process, we examined ribosome and mRNA partitioning during the unfolded protein response, key elements of which include suppression of the initiation stage of protein synthesis and polyribosome breakdown. We report that unfolded protein response (UPR)-elicited polyribosome breakdown resulted in the continued association, rather than release, of ER-bound ribosomes. Under these conditions, mRNA translation in the cytosol was suppressed, whereas mRNA translation on the ER was sustained. Furthermore, mRNAs encoding key soluble stress proteins (XBP-1 and ATF-4) were translated primarily on ER-bound ribosomes. These studies demonstrate that ribosome release from the ER is termination independent and identify new and unexpected roles for the ER compartment in the translational response to induction of the unfolded protein response.     

Uteroglobin mRNA from the lung of the hare (Lepus capensis): cell-free translation and properties

Poly(A)-containing RNA was obtained from the lung of hares (Lepus capensis) and uteroglobin mRNA was characterized by cell-free translation and molecular hybridization to a rabbit uteroglobin cDNA probe. In the cell-free system, hare uteroglobin mRNA was preferentially translated as compared to the whole lung mRNA and it directed the synthesis of a precursor, preuteroglobin, containing about twenty additional amino acids. Hare uteroglobin mRNA was about 40 nucleotides larger than the homologous rabbit one, as analyzed by electrophoresis on agarose gel. Thermal stability of the hybrids formed between rabbit or hare uteroglobin mRNAs and the rabbit cDNA probe indicated differences in the nucleotide sequence of both mRNAs. The levels of uteroglobin mRNA and uteroglobin synthesis in lung are about two-fold higher in hare than in rabbit lung.     

Persistence of chromosomal proteins HMG-14/-17 in myotubes following differentiation-dependent reduction of HMG mRNA.

The expression of chromosomal proteins HMG-14 and HMG-17 during cellular differentiation was studied in cultured mouse myoblasts. During myogenesis the level of both HMG-14 and HMG-17 mRNA decreased to less than 20% of that found in myoblasts. The down-regulation of HMG-14/-17 mRNA occurred simultaneously with activation of muscle-specific actin mRNA and was not linked to DNA synthesis, indicating that it is a differentiation-, rather than a cell cycle-related event. Incorporation of radiolabeled lysine into HMG proteins was similar to that into the major histone fractions in that it was significant in myoblasts and undetectable in myotubes. The decrease in mRNA and protein synthesis did not affect the cellular levels of HMG protein. These results indicate that the regulation of HMG-14/-17 mRNA levels is different from that of the histones and is linked to differentiation rather than to DNA synthesis.     

Cellular localization of muscle and nonmuscle actin mRNAs in chicken primary myogenic cultures: the induction of alpha-skeletal actin mRNA is regulated independently of alpha-cardiac actin gene expression

Specific DNA fragments complementary to the 3' untranslated regions of the beta-, alpha-cardiac, and alpha-skeletal actin mRNAs were used as in situ hybridization probes to examine differential expression and distribution of these mRNAs in primary myogenic cultures. We demonstrated that prefusion bipolar-shaped cells derived from day 3 dissociated embryonic somites were equivalent to myoblasts derived from embryonic day 11-12 pectoral tissue with respect to the expression of the alpha-cardiac actin gene. Fibroblasts present in primary muscle cultures were not labeled by the alpha-cardiac actin gene probe. Since virtually all of the bipolar cells express alpha-cardiac actin mRNA before fusion, we suggest that the bipolar phenotype may distinguish a committed myogenic cell type. In contrast, alpha-skeletal actin mRNA accumulates only in multinucleated myotubes and appears to be regulated independently from the alpha-cardiac actin gene. Accumulation of alpha- skeletal but not alpha-cardiac actin mRNA can be blocked by growth in Ca2+-deficient medium which arrests myoblast fusion. Thus, the sequential appearance of alpha-cardiac and then alpha-skeletal actin mRNA may result from factors that arise during terminal differentiation. Finally, the beta-actin mRNA was located in both fibroblasts and myoblasts but diminished in content during myoblast fusion and was absent from differentiated myotubes. It appears that in primary myogenic cultures, an asynchronous stage-dependent induction of two different alpha-striated actin mRNA species occurs concomitant with the deinduction of the nonmuscle beta-actin gene.     

Cytochalasin releases mRNA from the cytoskeletal framework and inhibits protein synthesis.

Cytochalasin D was shown to be a reversible inhibitor of protein synthesis in HeLa cells. The inhibition was detectable at drug levels typically used to perturb cell structure and increased in a dose-dependent manner. The drug also released mRNA from the cytoskeletal framework in direct proportion to the inhibition of protein synthesis. The released mRNA was unaltered in its translatability as measured in vitro but was no longer translated in the cytochalasin-treated HeLa cells. The residual protein synthesis occurred on polyribosomes that were reduced in amount but displayed a normal sedimentation distribution. The results support the hypothesis that mRNA binding to the cytoskeletal framework is necessary although not sufficient for translation. Analysis of the cytoskeletal framework, which binds the polyribosomes, revealed no alterations in composition or amount of protein as a result of treatment with cytochalasin D. Electron microscopy with embedment-free sections shows the framework in great detail. The micrographs revealed the profound reorganization effected by the drug but did not indicate substantial disaggregation of the cytoskeletal elements.