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Association of messenger RNA with the cytoskeletal framework in rat L6 myogenic cells
Abstract:
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.
Insights
Most cytoplasmic messenger RNA (mRNA) in muscle cells binds to the cytoskeleton, forming polysomes. A 165-kDa protein may mediate this mRNA-cytoskeleton association, influencing gene expression.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Cytoplasmic messenger RNA (mRNA) localization is crucial for cellular function.
- The role of the cytoskeleton in mRNA distribution and translation regulation is an area of active research.
Purpose of the Study:
- To investigate the distribution of mRNA between detergent-soluble and insoluble (cytoskeletal) fractions in rat L6 myoblasts and myotubes.
- To identify proteins associated with cytoskeleton-bound and soluble mRNA populations.
- To explore the mechanism of mRNA-cytoskeleton interaction using an in vitro transcription system.
Main Methods:
- Fractionation of cells into detergent-soluble and cytoskeletal components.
- In vitro translation assays to analyze mRNA populations.
- In vivo UV cross-linking to identify RNA-binding proteins.
- Analysis of protein-RNA complexes by SDS-PAGE.
- Development of a ghost monolayer transcription system.
Main Results:
- Approximately 85% of cytoplasmic mRNA is associated with the cytoskeletal framework in both myoblasts and myotubes, existing as polysomes.
- mRNAs encoding 40 kDa and 60 kDa polypeptides are enriched in the soluble fraction.
- UV cross-linking identified numerous polypeptides (15-220 kDa) associated with both soluble and cytoskeleton-bound mRNAs.
- A 165 kDa polypeptide was preferentially associated with cytoskeleton-bound mRNA and enriched in the cytoskeleton fraction, suggesting a role in mRNA-cytoskeleton binding.
- The in vitro transcription system synthesized mRNA that preferentially associated with the cytoskeleton.
Conclusions:
- The majority of cytoplasmic mRNA in muscle cells is bound to the cytoskeleton as active polysomes.
- A specific 165 kDa protein is implicated in mediating the association of mRNA with the cytoskeletal framework.
- The developed ghost monolayer transcription system provides a novel tool for studying mRNA-cytoskeleton interactions in vitro.