alpha-Tubulin limits its own synthesis: evidence for a mechanism involving translational repression.
A Chinese hamster alpha-tubulin cDNA was modified to encode an 11-amino acid carboxyl-terminal extension containing the immunodominant epitope from influenza hemagglutinin antigen (to create HA alpha 1-tubulin) and was cloned into a vector for expression in mammalian cells. 12 stable CHO cell lines expressing this HA alpha 1-tubulin were isolated and characterized. HA alpha 1-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 HA alpha 1-tubulin strongly repressed the synthesis of endogenous alpha-tubulin while having little or no effect on the synthesis of beta-tubulin. Treatment of transfected cells with sodium butyrate to induce even greater expression of HA alpha 1-tubulin led to a further decrease in synthesis of endogenous alpha-tubulin that was fully reversible upon removal of the inducer. Decreased synthesis of alpha-tubulin in transfected cells did not result from decreased levels of alpha-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 HA alpha 1-tubulin and endogenous alpha-tubulin, thus indicating that the decreased alpha-tubulin synthesis observed as a result of HA alpha 1-tubulin overexpression is distinct from the previously described autoregulation of tubulin. The results are consistent with a mechanism in which free alpha-tubulin inhibits the translation of its own message as a way of ensuring stoichiometric synthesis of alpha- and beta-tubulin.
Gonzalez-Garay
M L
ML
Department of Pharmacology, University of Texas Medical School, Houston 77225, USA.
Cabral
F
F
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D003001:Cloning, Molecular; D003078:Colchicine / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D005786:Gene Expression Regulation; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis / Q000187:drug effects; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014404:Tubulin / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology
D019267:Hemagglutinin Glycoproteins, Influenza Virus; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D003078:Colchicine