MESSENGER-RNA ASSOCIATION WITH THE CYTOSKELETAL FRAMEWORK LIKELY REPRESENTS A PHYSIOLOGICAL BINDING EVENT

A multitude of studies has indicated that the vast majority of mRNA and polyribosomes is associated with the detergent-resistant cytoskeletal framework (CSK). However, the nature and purpose of this association remain unclear. To begin unraveling the factors which may mediate this phenomenon, we examined the extent of association of four mRNAs (tubulin, vimentin, actin, and histone mRNA) with the CSKs of NIH 3T3 cells over a wide range of salt concentrations. Results indicate that the vast majority (> 90%) of each of these mRNAs remains associated with the CSK after detergent extraction of cells in low ionic strength buffer (25 mM NaCl). This association is manifest under conditions that cause the complete depolymerization of microtubules but that leave microfilaments and intermediate filaments intact. Even after extensive washing in buffer of approximately physiological ionic strength (150 mM NaCl), 75-85% of these mRNAs still remain associated with the CSK. However, at least 50% of each of these mRNAs can be eluted from the CSK by washing with buffer containing 250 mM NaCl. Not all the mRNAs, though, display the same elution profile. This suggest that difference binding sites and/or different binding affinities may exist for different mRNAs. Surprisingly, close to 50% of the polyribosome population remains bound to the CSK despite washing in as much as 1.0 M NaCl. These adherent polyribosomes appear to be of the same size as those that are eluted, allaying the possibility that they are retained by the CSK simply due to size exclusion. Collectively, these data strongly imply that mRNAs are neither weakly adsorbed to the CSK nor physically trapped within the meshwork of cytoskeletal filaments. Instead, mRNA appears to be bound to the CSK via interactions that are, at least partially, ionic in nature and strong enough to exist intracellularly. Moreover, the binding sites for these mRNAs on the CSK are not associated with microtubules, but may be associated with either the microfilament or intermediate filament network.