Inhibition of tubulin assembly by RNA and other polyanions: evidence for a required protein.

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Abstract
Nonneural cell extracts contain a heat-stable, nondialyzable activity that will inhibit the spontaneous assembly in vitro of partially purified [beef] brain tubulin. The sensitivity of this inhibitory activity to ribonucleases but not to a variety of other hydrolytic enzymes indicated that the inhibitor was an RNA. This conclusion was supported by the observation that purified RNAs from sea urchins, chinese hamster ovary cells and brain all inhibited spontaneous microtubule assembly in vitro. The snythetic polynucleotides [poly(A), (C), (G), and (U) were also inhibitory. This inhibition appeared to be nonspecific since the RNA base composition was unimportant and a variety of other nonnucleic acid polyanions also functioned as inhibitors. The treatment of assembly competent tubulin preparations with an insoluble RNA in the form of poly(A) covalently linked to agarose beads produced a stripped tubulin which did not assemble microtubules unless a heat-stable, trypsin-sensitive fraction eluted with increased ionic strength was mixed with the stripped tubulin. Similar results were obtained with other cation exchangers, including phosphocellulose and carboxymethylcellulose. The heat-stable protein sequestered by poly(A)-agarose appeared to be identical to the .tau. factor recently described by Kirschner and coworkers. Reconstitution experiment indicated that there was a stoichiometric requirement for these factors. Spontaneous assembly of microtubules in nonneural cell extracts may be blocked because the endogenous factors are complexed with RNA. The ratio of tubulin to RNA is low in cultured cell extracts but very high in neural tissue extracts.