α-Tubulin limits its own synthesis: Evidence for a mechanism involving translational repression

Abstract

A Chinese hamster α-tubulin cDNA was modified to encode an 11-amino acid carboxyl-terminal extension containing the immunodominant epitope from influenza hemagglutinin antigen (to create HAα1-tubulin) and was cloned into a vector for expression in mammalian cells. 12 stable CHO cell lines expressing this HAαl-tubulin were isolated and characterized. HAα1-tubulin incorporated into all classes of microtubules, assembled to the same extent as the endogenous tubulin, and did not perturb the growth of the cells in which it was expressed. However, overexpression of HAα1-tubulin strongly repressed the synthesis of endogenous α-tubulin while having little or no effect on the synthesis of β-tubulin. Treatment of transfected cells with sodium butyrate to induce even greater expression of HAα1-tubulin led to a further decrease in synthesis of endogenous α-tubulin that was fully reversible upon removal of the inducer. Decreased synthesis of α-tubulin in transfected cells did not result from decreased levels of α-tubulin mRNA, as demonstrated by ribonuclease protection assays. On the other hand, colchicine, a drug previously shown to destabilize the tubulin message, caused a clear reduction in both protein synthesis and mRNA levels for transfected HAα1-tubulin and endogenous α-tubulin, thus indicating that the decreased α-tubulin synthesis observed as a result of HAα1-tubulin overexpression is distinct from the previously described autoregulation of tubulin. The results are consistent with a mechanism in which free α- tubulin inhibits the translation of its own message as a way of ensuring stoichiometric synthesis of α- and β-tubulin.