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Translation of an mRNA in rat L6 muscle cells is regulated within the cell cycle
1Department of Molecular Biology and Genetics, University of Guelph, Ontario, Canada.
Abstract:
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.
Insights
Researchers identified a specific 1.3-kilobase messenger RNA (mRNA) that is repressed in non-dividing cells, such as myotubes. This repressed mRNA can be translated in vitro, suggesting cell cycle regulation of its protein synthesis.
Area of Science:
- Molecular Biology
- Cell Biology
- Gene Regulation
Background:
- Cytoplasmic messenger RNA (mRNA) exists in two populations: actively translating mRNA bound to ribosomes and a smaller, repressed population found as free mRNA-protein complexes.
- Understanding the regulation of these mRNA populations is crucial for comprehending cellular differentiation and function.
Purpose of the Study:
- To investigate the nature and regulation of repressed mRNA populations in differentiating muscle cells.
- To identify and characterize specific repressed mRNAs and their potential roles in cell cycle control.
Main Methods:
- Construction of a complementary DNA (cDNA) library from repressed mRNA of rat L6 myoblast cells.
- Differential hybridization of radioactively labeled cDNA probes to polysomal and free mRNA populations.
- Hybrid selection and in vitro translation of specific mRNA identified by recombinant plasmid D12.
- Analysis of mRNA distribution in different cellular fractions and cell types.
Main Results:
- A 1.3-kilobase (kb) poly(A)-rich mRNA was identified, preferentially associated with the repressed fraction in non-dividing myotubes, unlike actin mRNAs.
- This 1.3-kb mRNA's translational control appears linked to withdrawal from the cell cycle, not cell fusion, and is present in other rat cell types (liver, kidney).
- In vitro translation of hybrid-selected 1.3-kb mRNA produced a 40-kDa polypeptide, indicating its translatability from a repressed state.
Conclusions:
- A specific 1.3-kb mRNA is translationally regulated during the cell cycle, becoming repressed in terminally differentiated, non-dividing cells.
- The regulation of this mRNA's translation is associated with cell cycle exit rather than differentiation status alone.
- The identified mRNA is derived from moderately repetitive rat DNA and can be translated in vitro from its repressed form.