Attachment of mRNA to the cytoskeletal framework and translational control of gene expression in rat L6 muscle cells

Abstract

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 α-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 α-actin mRNA was associated with polysomes. Furthermore, most of the small amount of α-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. Translationally repressed mRNA which fails to bind to ribosomes may, therefore, be preferentially released from the cytoskeleton framework.