Stabilization of tubulin mRNA by inhibition of protein synthesis in sea urchin embryos.

Gong, Zhiyuan; Brandhorst, Bruce P.

doi:10.1128/mcb.8.8.3518

Cited by 21 publications

(11 citation statements)

References 30 publications

(52 reference statements)

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“…5). This confirms earlier observations that subunit-dependent changes in ox-and f-tubulin mRNA levels are prevented when polysomes are disrupted by protein synthesis inhibitors (15,25), whereas slowing ribosome translocation accelerates autoregulated degradation of both a-and ,B-tubulin mRNAs.…”

Section: Discussionsupporting

confidence: 80%

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

Bachurski¹,

Theodorakis²,

Coulson³

et al. 1994

Mol. Cell. Biol.

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The steady-state level of a-and ,I-tubulin synthesis is autoregulated by a posttranscriptional mechanism that selectively alters a-and j-tubulin mRNA levels in response to changes in the unassembled tubulin subunit concentration. For (1-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 jI-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 ,-tubulin autoregulation is the amino-terminal tetrapeptide MR(E/D)I. Although tubulin-dependent changes in ox-tubulin mRNA levels are shown to result from changes in cytoplasmic mRNA stability, transfection of wild-type and mutated a-tubulin genes reveals that a-and I8-tubulin mRNA degradation is not mediated through a common pathway. Not only does the amino-terminal a-tubulin tetrapeptide MREC fail to confer regulated mRNA degradation, neither wild-type oa-tubulin transgenes nor an a-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 a-and I-tubulin mRNAs, degradation of a-tubulin transgene mRNAs is not enhanced, and in one case, the mRNA is actually stabilized. We conclude that, despite similarities, a-and ,-tubulin mRNA destabilization pathways utilize divergent determinants to link RNA instability to tubulin subunit concentrations.The two major microtubule subunits, a(-and ,B-tubulin, are encoded in higher eukaryotes by multigene families whose expression is regulated at both the transcriptional and posttranscriptional levels (reviewed in references 3, 4, and 34). Although transcriptional activation determines the repertoire of tubulin isotypes expressed in each cell type (reviewed in reference 31), a posttranscriptional autoregulatory mechanism controls the rate of tubulin subunit synthesis relative to the unassembled tubulin subunit concentration. Elevation of tubulin subunit concentration by colchicine-or nocodazole-induced microtubule disassembly (1, 6) or by direct microinjection of unassembled tubulin into cells (7) causes a concomitant loss of both a-and ,B-tubulin mRNAs. Conversely, taxol, a drug which depletes the unassembled tubulin subunit pool by inducing assembly of microtubules, causes tubulin mRNA levels to rise (6).Several lines of evidence suggest that the autoregulation of both ao-and ,B-tubulin mRNA abundance is posttranscriptional. In nuclear run-on transcription assays using probes that cross-hybridized with all isotypes, no differences in the overall transcription rates of ax-and 3-tubulin genes were seen between nuclei isolated from colchicine-treated and untreated cells (5). Studies with enucleated cells confirmed the cytoplasmic location of the ,-tubulin autoregulatory machinery: cytoplasts were able to respond to colchicine by decreasing the synthesis of ,B-t...

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Section: Discussionsupporting

confidence: 80%

“…The simplest view is that only ,B-tubulin mRNAs attached to elongating ribosomes are substrates for regulated degradation. As with ,B-tubulin mRNA, autoregulation of ot-tubulin mRNA is blocked by complete inhibition of translation (15,25).…”

mentioning

confidence: 99%

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

Bachurski¹,

Theodorakis²,

Coulson³

et al. 1994

Mol. Cell. Biol.

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“…Examples of mRNAs that do need to be translated in order to be degraded include those for the cell cycleregulated histones (18,40) and 3-tubulin (16,17). In the case of histone mRNAs, ribosomes must translate to the vicinity of the destabilizing hairpin structure in order for rapid degradation to take place.…”

Section: Resultsmentioning

confidence: 99%

“…For some mRNAs, abundance is increased if the coding region is not translated in full. Examples of this type include mRNAs for the replication-dependent human histones (10,18,26); human c-fos (19,41,42); murine c-myc (50); yeast MATel (35); and hamster, chicken, murine, and sea urchin P-tubulin (16,17,34,51,52). In each case, translation to at least a certain point within the coding region of the mRNA appears to be required for mRNA degradation, and nonsense codons that reside upstream of that point are associated with an increase in mRNA abundance.…”

mentioning

confidence: 99%

Nonsense codons in human beta-globin mRNA result in the production of mRNA degradation products.

Lim¹,

Sigmund²,

Gross³

et al. 1992

Mol. Cell. Biol.

110

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Each transgene produces properly processed albeit abnormally unstable mRNA as well as several smaller RNAs in erythroid cells. These smaller RNAs are detected only in the cytoplasm and, relative to mRNA, are longer-lived and are missing sequences from either exon I or exons I and H. In this communication, we show by using genetics and Si nuclease transcript mapping that the premature termination of 1-globin mRNA translation is mechanistically required for the abnormal RNA metabolism. We also provide evidence that generation of the smaller RNAs is a cytoplasmic process: the 5' ends of intron 1-containing pre-mRNAs were normal, the rates of removal of introns 1 and 2 were normal, and studies inhibiting RNA synthesis with actinomycin D demonstrated a precursor-product relationship between full-length mRNA and the smaller RNAs. In vivo, about 50% of the full-length species that undergo decay are degraded to the smaller RNAs and the rest are degraded to undetectable products. Exposure of erythroid cells that expressed a normal human 13-globin transgene to either cycloheximide or puromycin did not result in the generation of the smaller RNAs.Therefore, a drug-induced reduction in cellular protein synthesis does not reproduce this aspect of cytoplasmic mRNA metabolism. These data suggest that the premature termination of 13-globin mRNA translation in either exon I or exon II results in the cytoplasmic generation of discrete mRNA degradation products that are missing sequences from exon I or exons I and II. Since these degradation products appear to be the same for all nonsense codons tested, there is no correlation between the position of translation termination and the sites of nucleolytic cleavage. mRNA exists to be translated into protein. However, evidence suggests that in the process of being translated, the abundance of an mRNA can be altered. For some mRNAs, abundance is increased if the coding region is not translated in full. Examples of this type include mRNAs for the replication-dependent human histones (10,18,26); human c-fos (19,41,42); murine c-myc (50); yeast MATel (35); and hamster, chicken, murine, and sea urchin P-tubulin (16,17,34,51,52). In each case, translation to at least a certain point within the coding region of the mRNA appears to be required for mRNA degradation, and nonsense codons that reside upstream of that point are associated with an increase in mRNA abundance. While similar in this regard, however, each of these mRNAs is not degraded as a function of the same features of translation. Histone mRNA degradation at the end of S phase requires that ribosomes translate to within a specific distance of a hairpin structure that resides in the 3' untranslated region. In contrast, c-fos, c-myc, and MATod mRNA degradation seems to require ribosome translation across a specific region of the coding sequence. And, as another variation, ,B-tubulin mRNA decay depends upon an excess of free tubulin that presumably interacts with the first 4 amino acids of an actively elongating, nascent peptide. (2,...

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“…However, this experiment did not eliminate the possibility that translation elongation was indeed required for the mRNA destabilization if the residual (albeit reduced) translation rate was still sufficient for efficient signal transduction. This possibility became all the more likely in view of similar protein synthesis inhibition experiments done in developing sea urchin embryos (11), which found tubulin mRNA autoregulation to be disrupted by emetine, a drug that principally inhibits translation elongation.…”

mentioning

confidence: 93%

Autoregulatory control of beta-tubulin mRNA stability is linked to translation elongation.

Sisodia¹,

Cleveland²

1989

Proc. Natl. Acad. Sci. U.S.A.

152

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Tubulin synthesis in animal cells is controlled in part by an autoregulatory mechanism that modulates the stability of ribosome-bound tubulin mRNAs. For f3 tubulin, the initial recognition event for this selective RNA instability has previously been shown to be a cotranslational binding (presumably by tubulin itself) to the nascent amino-terminal 13-tubulin tetrapeptide just after it emerges from the ribosome. Although this "autoregulation" of tubulin expression is thus obligatorily linked to the translation process, the mechanism of how a cotranslational protein-protein binding event ultimately triggers RNA degradation is unknown. Using protein synthesis inhibitors to slow and ultimately to block translation elongation, we now show that the mRNA destabilization pathway requires ongoing ribosome translocation.In most animal cells, the appropriate quantitative level of tubulin expression is established in part through an autoregulatory pathway in which the apparent intracellular concentration of tubulin heterodimers (comprising one a-and one P-tubulin polypeptide) modulates the stability of tubulin mRNAs (1-7). Evidence for such a self-regulated pathway emerged initially from experiments with inhibitors that induced microtubule depolymerization: the corresponding rise in the intracellular concentration of unassembled subunits leads to a rapid and specific depression of synthesis of both a-and f3-tubulin polypeptides. This decrease in synthesis [induced either by treatment with microtubule-destabilizing drugs or by direct microinjection of tubulin subunits (8)] was next shown to be the result of a posttranscriptional mechanism that alters the stability of cytoplasmic tubulin mRNAs after changes in the concentration of unpolymerized tubulin subunits (3-5, 9). The sequences that are necessary and sufficient to specify f3-tubulin mRNAs as substrates for this autoregulated instability have been shown by DNA transfection experiments (6) to lie within the first 13 translated nucleotides [which encode the first four ,3-tubulin amino acids (Met-Arg-Glu-Ile)]. Further, by using inhibitors of protein synthesis and transfection ofgenes bearing premature translation termination codons, it was determined that only tubulin mRNAs that are attached to polyribosomes are destabilized by increased subunit concentrations (6, 10). Most recently, introduction of 25 different nucleotide base substitutions into this 13-base regulatory element has documented that 13-tubulin RNAs are selectively targeted as substrates for destabilization not through recognition of specific RNA sequences but rather through cotranslational recognition of the amino-terminal P-tubulin tetrapeptide after its emergence from the ribosome (7).Many important details of this mRNA instability pathway remain to be determined. For example, it is not known how the putative interaction involving the nascent tubulin polypeptide and a cellular factor(s) (presumably the tubulin heterodimer) can be transduced through the ribosome to yield enhanced degradation of the ...

show abstract

Stabilization of tubulin mRNA by inhibition of protein synthesis in sea urchin embryos.

Cited by 21 publications

References 30 publications

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

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

Nonsense codons in human beta-globin mRNA result in the production of mRNA degradation products.

Autoregulatory control of beta-tubulin mRNA stability is linked to translation elongation.

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