scholarly journals An amino-terminal tetrapeptide specifies cotranslational degradation of beta-tubulin but not alpha-tubulin mRNAs.

1994 ◽  
Vol 14 (6) ◽  
pp. 4076-4086 ◽  
Author(s):  
C J Bachurski ◽  
N G Theodorakis ◽  
R M Coulson ◽  
D W Cleveland
Keyword(s):  
Degradation Mechanism ◽  
Full Range ◽  
Mrna Degradation ◽  
Mrna Levels ◽  
Wild Type ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Amino Terminal ◽  
Tubulin Subunit ◽  
Tubulin Mrna

The steady-state level of alpha- and beta-tubulin synthesis is autoregulated by a posttranscriptional mechanism that selectively alters alpha- and beta-tubulin mRNA levels in response to changes in the unassembled tubulin subunit concentration. For beta-tubulin mRNAs, previous efforts have shown that this is the result of a selective mRNA degradation mechanism which involves cotranslational recognition of the nascent amino-terminal beta-tubulin tetrapeptide as it emerges from the ribosome. Site-directed mutagenesis is now used to determine that the minimal sequence requirement for conferring the full range of beta-tubulin autoregulation is the amino-terminal tetrapeptide MR(E/D)I. Although tubulin-dependent changes in alpha-tubulin mRNA levels are shown to result from changes in cytoplasmic mRNA stability, transfection of wild-type and mutated alpha-tubulin genes reveals that alpha- and beta-tubulin mRNA degradation is not mediated through a common pathway. Not only does the amino-terminal alpha-tubulin tetrapeptide MREC fail to confer regulated mRNA degradation, neither wild-type alpha-tubulin transgenes nor an alpha-tubulin gene mutated to encode an amino-terminal MREI yields mRNAs that are autoregulated. Further, although slowing ribosome transit accelerates the autoregulated degradation of endogenous alpha- and beta-tubulin mRNAs, degradation of alpha-tubulin transgene mRNAs is not enhanced, and in one case, the mRNA is actually stabilized. We conclude that, despite similarities, alpha- and beta-tubulin mRNA destabilization pathways utilize divergent determinants to link RNA instability to tubulin subunit concentrations.

An amino-terminal tetrapeptide specifies cotranslational degradation of beta-tubulin but not alpha-tubulin mRNAs

1994 ◽  
Vol 14 (6) ◽  
pp. 4076-4086
Author(s):  
C J Bachurski ◽  
N G Theodorakis ◽  
R M Coulson ◽  
D W Cleveland
Keyword(s):  
Degradation Mechanism ◽  
Full Range ◽  
Mrna Degradation ◽  
Mrna Levels ◽  
Wild Type ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Amino Terminal ◽  
Tubulin Subunit ◽  
Tubulin Mrna

The steady-state level of alpha- and beta-tubulin synthesis is autoregulated by a posttranscriptional mechanism that selectively alters alpha- and beta-tubulin mRNA levels in response to changes in the unassembled tubulin subunit concentration. For beta-tubulin mRNAs, previous efforts have shown that this is the result of a selective mRNA degradation mechanism which involves cotranslational recognition of the nascent amino-terminal beta-tubulin tetrapeptide as it emerges from the ribosome. Site-directed mutagenesis is now used to determine that the minimal sequence requirement for conferring the full range of beta-tubulin autoregulation is the amino-terminal tetrapeptide MR(E/D)I. Although tubulin-dependent changes in alpha-tubulin mRNA levels are shown to result from changes in cytoplasmic mRNA stability, transfection of wild-type and mutated alpha-tubulin genes reveals that alpha- and beta-tubulin mRNA degradation is not mediated through a common pathway. Not only does the amino-terminal alpha-tubulin tetrapeptide MREC fail to confer regulated mRNA degradation, neither wild-type alpha-tubulin transgenes nor an alpha-tubulin gene mutated to encode an amino-terminal MREI yields mRNAs that are autoregulated. Further, although slowing ribosome transit accelerates the autoregulated degradation of endogenous alpha- and beta-tubulin mRNAs, degradation of alpha-tubulin transgene mRNAs is not enhanced, and in one case, the mRNA is actually stabilized. We conclude that, despite similarities, alpha- and beta-tubulin mRNA destabilization pathways utilize divergent determinants to link RNA instability to tubulin subunit concentrations.

Physical evidence for cotranslational regulation of beta-tubulin mRNA degradation

1992 ◽  
Vol 12 (2) ◽  
pp. 791-799
Author(s):  
N G Theodorakis ◽  
D W Cleveland
Keyword(s):  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Amino Terminal ◽  
Selective Degradation ◽  
Cellular Factors ◽  
Nascent Peptide ◽  
Tubulin Mrna ◽  
The Stability ◽  
Necessary And Sufficient ◽  
Specific Degradation

Tubulin synthesis is controlled by an autoregulatory mechanism through which an increase in the intracellular concentration of tubulin subunits leads to specific degradation of tubulin mRNAs. The sequence necessary and sufficient for the selective degradation of a beta-tubulin mRNA in response to changes in the level of free tubulin subunits resides within the first 13 translated nucleotides that encode the amino-terminal sequence of beta-tubulin, Met-Arg-Glu-Ile (MREI). Previous results have suggested that the sequence responsible for autoregulation resides in the nascent peptide rather than in the mRNA per se, raising the possibility that the regulation of the stability of tubulin mRNA is mediated through binding of tubulin or some other cellular factor to the nascent amino-terminal tubulin peptide. We now show that this putative cotranslational interaction is not mediated by tubulin alone, as no meaningful binding is detectable between tubulin subunits and the amino-terminal beta-tubulin polypeptide. However, microinjection of a monoclonal antibody that binds to the beta-tubulin nascent peptide selectively disrupts the regulation of beta-tubulin, but not alpha-tubulin, synthesis. This finding provides direct evidence for cotranslational degradation of beta-tubulin mRNA mediated through binding of one or more cellular factors to the beta-tubulin nascent peptide.

Transfer and amplification of a mutant beta-tubulin gene results in colcemid dependence: use of the transformant to demonstrate regulation of beta-tubulin subunit levels by protein degradation

1986 ◽  
Vol 6 (5) ◽  
pp. 1422-1429
Author(s):  
C Whitfield ◽  
I Abraham ◽  
D Ascherman ◽  
M M Gottesman
Keyword(s):  
Cho Cells ◽  
Single Gene ◽  
Chinese Hamster ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Tubulin Gene ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Tubulin Subunit ◽  
Steady State Levels

Total genomic DNA from a temperature-sensitive, colcemid-resistant Chinese hamster ovary (CHO) cell mutant expressing an electrophoretic variant beta-tubulin was used to transform wild-type CHO cells to colcemid-resistant cells at 37 degrees C. Southern blot analysis of the transformant demonstrated the three- to fivefold amplification of one of many beta-tubulin sequences compared with that of the wild type or mutant, thereby identifying a functional tubulin gene in CHO cells. This amplification of one tubulin-coding sequence resulted in a threefold increase in two beta-tubulin mRNA species, suggesting that both species may be encoded by a single gene. Pulse-chase experiments showed that in the transformant, total beta-tubulin was synthesized and degraded faster than in the revertant or wild-type cells, so that the steady-state levels of beta-tubulin and alpha-tubulin were unchanged in the transformant compared with those of wild-type, mutant, or revertant cells. Increased ratios of mutant to wild-type beta-tubulin made the transformant dependent on microtubule-depolymerizing drugs for growth at 37 but not 34 degrees C and supersensitive to the microtubule-stabilizing drug taxol at 34 degrees C.

scholarly journals Physical evidence for cotranslational regulation of beta-tubulin mRNA degradation.

1992 ◽  
Vol 12 (2) ◽  
pp. 791-799 ◽  
Author(s):  
N G Theodorakis ◽  
D W Cleveland
Keyword(s):  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Amino Terminal ◽  
Selective Degradation ◽  
Cellular Factors ◽  
Nascent Peptide ◽  
Tubulin Mrna ◽  
The Stability ◽  
Necessary And Sufficient ◽  
Specific Degradation

Tubulin synthesis is controlled by an autoregulatory mechanism through which an increase in the intracellular concentration of tubulin subunits leads to specific degradation of tubulin mRNAs. The sequence necessary and sufficient for the selective degradation of a beta-tubulin mRNA in response to changes in the level of free tubulin subunits resides within the first 13 translated nucleotides that encode the amino-terminal sequence of beta-tubulin, Met-Arg-Glu-Ile (MREI). Previous results have suggested that the sequence responsible for autoregulation resides in the nascent peptide rather than in the mRNA per se, raising the possibility that the regulation of the stability of tubulin mRNA is mediated through binding of tubulin or some other cellular factor to the nascent amino-terminal tubulin peptide. We now show that this putative cotranslational interaction is not mediated by tubulin alone, as no meaningful binding is detectable between tubulin subunits and the amino-terminal beta-tubulin polypeptide. However, microinjection of a monoclonal antibody that binds to the beta-tubulin nascent peptide selectively disrupts the regulation of beta-tubulin, but not alpha-tubulin, synthesis. This finding provides direct evidence for cotranslational degradation of beta-tubulin mRNA mediated through binding of one or more cellular factors to the beta-tubulin nascent peptide.

scholarly journals Transfer and amplification of a mutant beta-tubulin gene results in colcemid dependence: use of the transformant to demonstrate regulation of beta-tubulin subunit levels by protein degradation.

1986 ◽  
Vol 6 (5) ◽  
pp. 1422-1429 ◽  
Author(s):  
C Whitfield ◽  
I Abraham ◽  
D Ascherman ◽  
M M Gottesman
Keyword(s):  
Cho Cells ◽  
Single Gene ◽  
Chinese Hamster ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Tubulin Gene ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Tubulin Subunit ◽  
Steady State Levels

Total genomic DNA from a temperature-sensitive, colcemid-resistant Chinese hamster ovary (CHO) cell mutant expressing an electrophoretic variant beta-tubulin was used to transform wild-type CHO cells to colcemid-resistant cells at 37 degrees C. Southern blot analysis of the transformant demonstrated the three- to fivefold amplification of one of many beta-tubulin sequences compared with that of the wild type or mutant, thereby identifying a functional tubulin gene in CHO cells. This amplification of one tubulin-coding sequence resulted in a threefold increase in two beta-tubulin mRNA species, suggesting that both species may be encoded by a single gene. Pulse-chase experiments showed that in the transformant, total beta-tubulin was synthesized and degraded faster than in the revertant or wild-type cells, so that the steady-state levels of beta-tubulin and alpha-tubulin were unchanged in the transformant compared with those of wild-type, mutant, or revertant cells. Increased ratios of mutant to wild-type beta-tubulin made the transformant dependent on microtubule-depolymerizing drugs for growth at 37 but not 34 degrees C and supersensitive to the microtubule-stabilizing drug taxol at 34 degrees C.

scholarly journals alpha-Tubulin limits its own synthesis: evidence for a mechanism involving translational repression.

1996 ◽  
Vol 135 (6) ◽  
pp. 1525-1534 ◽  
Author(s):  
M L Gonzalez-Garay ◽  
F Cabral
Keyword(s):  
Mammalian Cells ◽  
Chinese Hamster ◽  
Mrna Levels ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Transfected Cells ◽  
Influenza Hemagglutinin ◽  
Tubulin Mrna ◽  
Ribonuclease Protection ◽  
Expression In Mammalian Cells

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.

Regulation of tubulin and actin mRNA production in rat brain: expression of a new beta-tubulin mRNA with development

1983 ◽  
Vol 3 (8) ◽  
pp. 1333-1342
Author(s):  
J F Bond ◽  
S R Farmer
Keyword(s):  
Rat Brain ◽  
Morphological Changes ◽  
In Vitro Translation ◽  
Brain Maturation ◽  
Cdna Clones ◽  
Mrna Levels ◽  
Beta Tubulin ◽  
Mrna Species ◽  
Tubulin Mrna ◽  
Actin Mrna

The expression of alpha-tubulin, beta-tubulin, and actin mRNA during rat brain development has been examined by using specific cDNA clones and in vitro translation techniques. During brain maturation (0 to 80 days postnatal), these mRNA species undergo a significant decrease in abundance. The kinetics of this decrease varies between the cerebrum and the cerebellum. These mRNAs are most abundant in both tissues during week 1 postnatal, each representing 10 to 15% of total mRNA activity. Both alpha- and beta-tubulin mRNA content decreases by 90 to 95% in the cerebrum after day 11 postnatal, and 70 to 80% decreases in the cerebellum after day 16. Actin sequences also decrease but to a lesser extent in both tissues (i.e., 50%). These decreases coincide with the major developmental morphological changes (i.e., neurite extension) occurring during this postnatal period. These studies have also identified the appearance of a new 2.5-kilobase beta-tubulin mRNA species, which is more predominant in the cerebellar cytoplasm. The appearance of this form occurs at a time when the major 1.8-kilobase beta-tubulin mRNA levels are declining. The possibility that the tubulin multigene family is phenotypically expressed and then this expression responds to the morphological state of the nerve cells is discussed.

Sto1p, a fission yeast protein similar to tubulin folding cofactor E, plays an essential role in mitotic microtubule assembly

1999 ◽  
Vol 112 (12) ◽  
pp. 1979-1988 ◽  
Author(s):  
E.L. Grishchuk ◽  
J.R. McIntosh
Keyword(s):  
Fission Yeast ◽  
Microtubule Assembly ◽  
Wild Type ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Cold Sensitive ◽  
Alpha Beta ◽  
Sensitive Allele ◽  
Delta Cells

The proper functioning of microtubules depends crucially on the availability of polymerizable alpha/beta tubulin dimers. Their production occurs concomitant with the folding of the tubulin polypeptides and is accomplished in part by proteins known as Cofactors A through E. In the fission yeast, Schizosaccharomyces pombe, this tubulin folding pathway is essential. We have taken advantage of the excellent cytology available in S. pombe to examine the phenotypic consequences of a deletion of sto1(+), a gene that encodes a protein similar to Cofactor E, which is required for the folding of alpha-tubulin. The interphase microtubule cytoskeleton in sto1-delta cells is severely disrupted, and as cells enter mitosis their spindles fail to form. After a transient arrest with condensed chromosomes, the cells exit mitosis and resume DNA synthesis, whereupon they septate abnormally and die. Overexpression of Spo1p is toxic to cells carrying a cold-sensitive allele of the alpha- but not the beta-tubulin gene, consistent with the suggestion that this protein plays a role like that of Cofactor E. Unlike its presumptive partner Cofactor D (Alp1p), however, Sto1p does not localize to microtubules but is found throughout the cell. Overexpression of Sto1p has no toxic effects in wild-type cells, suggesting that it is unable to disrupt alpha/beta tubulin dimers in vivo.

scholarly journals Regulation of tubulin and actin mRNA production in rat brain: expression of a new beta-tubulin mRNA with development.

1983 ◽  
Vol 3 (8) ◽  
pp. 1333-1342 ◽  
Author(s):  
J F Bond ◽  
S R Farmer
Keyword(s):  
Rat Brain ◽  
Morphological Changes ◽  
In Vitro Translation ◽  
Brain Maturation ◽  
Cdna Clones ◽  
Mrna Levels ◽  
Beta Tubulin ◽  
Mrna Species ◽  
Tubulin Mrna ◽  
Actin Mrna

The expression of alpha-tubulin, beta-tubulin, and actin mRNA during rat brain development has been examined by using specific cDNA clones and in vitro translation techniques. During brain maturation (0 to 80 days postnatal), these mRNA species undergo a significant decrease in abundance. The kinetics of this decrease varies between the cerebrum and the cerebellum. These mRNAs are most abundant in both tissues during week 1 postnatal, each representing 10 to 15% of total mRNA activity. Both alpha- and beta-tubulin mRNA content decreases by 90 to 95% in the cerebrum after day 11 postnatal, and 70 to 80% decreases in the cerebellum after day 16. Actin sequences also decrease but to a lesser extent in both tissues (i.e., 50%). These decreases coincide with the major developmental morphological changes (i.e., neurite extension) occurring during this postnatal period. These studies have also identified the appearance of a new 2.5-kilobase beta-tubulin mRNA species, which is more predominant in the cerebellar cytoplasm. The appearance of this form occurs at a time when the major 1.8-kilobase beta-tubulin mRNA levels are declining. The possibility that the tubulin multigene family is phenotypically expressed and then this expression responds to the morphological state of the nerve cells is discussed.

Assembly properties of altered beta-tubulin polypeptides containing disrupted autoregulatory domains

1989 ◽  
Vol 9 (8) ◽  
pp. 3418-3428
Author(s):  
W Gu ◽  
N J Cowan
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Tubulin Isotype ◽  
The Stability ◽  
Carboxy Terminal ◽  
Amino Terminal Domain

beta-Tubulin synthesis in eucaryotic cells is subject to control by an autoregulatory posttranscriptional mechanism in which the first four amino acids of the beta-tubulin polypeptide act either directly or indirectly to control the stability of beta-tubulin mRNA. To investigate the contribution of this amino-terminal domain to microtubule assembly and dynamics, we introduced a series of deletions encompassing amino acids 2 to 5 of a single mammalian beta-tubulin isotype, M beta 1. Constructs carrying such deletions were inserted into an expression vector, and the ability of the altered polypeptide to coassemble into microtubules was tested by using an anti-M beta 1-specific antibody. We show that the M beta 1 beta-tubulin polypeptide was competent for coassembly into microtubules in transient transfection experiments and in stably transfected cell lines when it lacked either amino acid 2 or amino acids 2 and 3. The capacity of these mutant beta-tubulins to coassemble into polymerized microtubules was only slightly diminished relative to that of unaltered beta-tubulin, and their expression did not influence the viability or growth properties of cell lines carrying these deletions. However, more extensive amino-terminal deletions either severely compromised or abolished the capacity for coassembly. In analogous experiments in which alterations were introduced into the amino-terminal domain of a mammalian alpha-tubulin isotype, M alpha 4, deletion of amino acid 2 did not affect the ability of the altered polypeptide to coassemble, although removal of additional amino-terminal residues essentially abolished the capacity for competent coassembly. The stability of the altered assembly-competent alpha- and beta-tubulin polypeptides was measured in pulse-chase experiments and found to be indistinguishable from the stability of the corresponding unaltered polypeptides. An assembly-competent M alpha 4 polypeptide carrying a deletion encompassing the 12 carboxy-terminal amino acids also had a half-life indistinguishable from that of the wild-type alpha-tubulin molecule. These data suggest that the universally conserved amino terminus of beta-tubulin acts largely in a regulatory role and that the carboxy-terminal domain of alpha-tubulin is not essential for coassembly in mammalian cells in vivo.

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