Autoregulated control of tubulin synthesis in animal cells

D.W. Cleveland

Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

Current Opinion in Cell Biology 1989, 1:10-14

Introduction

Expression of ~- and [3-tubulin, the two major compo- nents of microtubules, is regulated in animal ceils by events that operate on two levels. The first of these is the transcriptional activation of one or more members of the small multigene families of about six to seven functional genes that encode either subunit. In addition to this, the appropriate quantitative level of tubulin expression is es- tablished through an apparently autoregulated pathway in which the stability of tubulin messenger RNA (mRNA) is governed by the concentration of its own end products, the tubulin subunits themselves.

The initial observation, now nearly 10 years old (Ben Ze'ev et al., Cell 1979, 17:317-325), that suggested the existence of such a novel regulatory pathway emerged from analyzing the level of tubulin synthesis in cells treated with drugs that interfere with normal microtubule assembly. For example, following colchicine-induced mi- crotubule depolymerization (and a corresponding eleva- tion in the unassembled subunit concentration), the only observable change in the pattern of newly synthesized

proteins was a 5- to 10-fold lower level of synthesis of 0t- and ]3-tubulins. Treatment of cells with a second micro- tubule inhibitor, vinblastine, led to a very different result: tubulin synthesis was specifically elevated. Since vinblas- tine not only induces microtubule disassembly but also precipitation of the disassembled subunits, it was pro- posed that regulation of tubulin expression was autoreg- ulated by the level of unassembled subunits.

This initial work was quickly confirmed by additional investigators using pulse-labeling approaches and ad- ditional microtubule inhibitors to alter the level of unassembled tubulin subunits. Overall, it was shown that inhibitors that increase the concentration of unassem- bled subunits (e.g. colchicine and nocodazole) induced suppression of new synthesis, whereas those that lower intracellular subunit levels (e.g. vinblastine and taxol) stimulated the level of new synthesis. Figure I gives an ex- ample of the alteration in tubulin synthesis rates in cells with a colchicine-induced elevation (Fig. lb) or taxol-in- duced depletion (Fig. lc) of the unassembled subunit concentration. This apparent autoregulation of synthesis has been demonstrated in almost all animal cells tested

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Fig. 1. Specific alteration in synthesis of ¢¢- and ~-tubulin polypeptides following an alteration in the unpolymerized tubulin subunit con- centration. Newly synthesized proteins in cultured hamster cells were pulse-labeled and analyzed by two-dimensional gel electrophoresis. Proteins are synthesized in (a) control cells with a normal level of unassembled tubulin subunit, (b) cells whose tubulin subunit con- centration was depleted by taxol-stimulated microtubule polymerization or (c) cells with a colchicine-induced elevation in unassembled tubulin concentration. Data from Pachter and Cleveland (private communications).

Abbreviations mRNA--messenger RNA; tk--thymidine kinase.

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Autoregulation of tubulin synthesis in animals Cleveland 11

(Cleveland et al., Cell 1981, 20:95-105), including those from species as divergent as mosquito and human (Cleve- land and Havercroft, J Cell Bio11983, 97:919-924). Fur- ther, autoregulation appears to control tubulin synthesis during at least one portion of early sea urchin develop- ment [1,2].

The obvious conclusion from these efforts was that tubulin synthesis is somehow specified by the level of unassembled subunits, although a nagging doubt con- cerning the absolute specificities and mechanisms of ac- tion of each of the drugs employed could not initially be discounted. These difficulties were overcome by directly elevating tubulin subunits inside cells, using microinjec- tion. With this approach, a rapid and specific suppression of new tubulin synthesis was achieved by injection of as little as 25--50% of additional cell tubulin (Cleveland et al., Na ture 1983, 305:738-740).

The level of unassembled subunits affects stability of cytoplasmic tubulin rnRNA

In determining the molecular source for the changes in the rate of translation of new tubulin polypeptides, after alterations to the intracellular concentration of unassem- bled subunits, the original report (Ben Ze'ev' etal., 1979) demonstrated that the changes in tubulin synthesis were reflected in corresponding changes in translatable tubu- lin mRNA levels. Once sequences for ~- and [3-tubulin were cloned, it was relatively simple, using RNA blot- ting, to show that the primary effect was on tubulin mRNA levels (Cleveland et al., Cell 1981, 21:530-537). Cloned sequences also allowed the measurement of tubulin transcription rates, by using 'run-off transcrip- tion' in isolated nuclei. These experiments revealed that no transcriptional changes accompanied the altered cy- toplasmic tubulin RNA levels (Cleveland and Havercroft, 1983). Indeed, two groups eliminated the involvement of all nuclear effects (e.g. transcription and RNA pro- cessing/transport) by demonstrating that enucleated cells quantitatively retained the mechanism responsible for changing [3-tubulin synthetic rates (Pittenger and Cleve- land, J Cell Biol 1985, 101:1941-1952; Caron et al., Na- ture 1985, 317:648~551). It was concluded that autoregu- lation must be specified by selective changes in cytoplas- mic tubulin mRNA stability.

Identifying the nucleotides that specify tubulin RNA for autoregulated instability

Recent work [3-5] has identified the sequences carried by [3-tubulin RNA that label it as a substrate for selec- tive degradation when the tubulin subunit concentration is elevated. These authors used transient DNA transfec- tion of hybrid gene constructs that carry portions of tubu- l ingene segments inserted into reporter genes. Initially,

they found that insertion into a thymidine kinase (tk) gene of as few as 106 nucleotides (57 bases of 5' un- translated region and the first 49 translated nucleotides) from a [3-tubulin gene were sufficient to regulate the ex- pression of the resultant chimeric mRNA as efficiently as authentic [3-tubulin mRNA [3]. Further, [3-tubulin RNA in which the entire [3-tubulin 5' untranslated region was substituted with the corresponding domain from metal- lothionein also remained a substrate for autoregulated instability. It was thus clear that instability was speci- fied through sequences lying within the first 16 translated codons of [3-tubulinmRNA,

Next, the minimal domain that could confer autoregu- lated instability was identified as a 13-nucleotide segment that encoded the amino-terminal four amino acids of j3- tubulin [4]. This was determined by constructing a se- ries of genes carrying progressively reduced amounts of amino-temlinal [3-tubulin sequences linked in the correct translational reading frame to a tk gene body. Additional gene constructs demonstrated that the amino-terminal [3- tubulin domain has been functionally conserved through- out evolution, since sequences isolated from the anal- ogous region of [3-tubulin genes from species as diver- gent as yeast and human also conferred regulated RNA instability. Finally, this 13-nucleotide domain proved not only sufficient for autoregulation but also necessary, since deletion of codon 2 or codons 3 and 4 from an otherwise authentic [3-tubulin gene disrupted autoregulated instabil- ity of its RNA transcripts [4].

Destabilization of tubulin RNA requires translation

Although transfection experiments identified the nu- cleotide sequences carried by autoregulated RNAs, they shed little light on the exact cytoplasmic site of degrada- tion. Several experiments have now shown that it is the polyribosome-bound RNA that is selectively destabilized, while RNA not attached to ribosomes remains stable. The first of these experiments used drugs that block protein synthesis to show that inhibitors which remove RNA from polysomes (e.g. pactamycin and puromycin) completely disrupt the linkage of tubulin RNA content with the le- vel of unassembled subunits [6]. However, efficient pro- tein synthesis p e r se is not required, since blockage of translation elongation with the inhibitor cyclohexit~de enhanced tubulin RNA instability in the presence of el- evated subunit concentrations. Further, by introducing premature translation termination codons into an authen- tic [3-tubulin gene [6] or into tubulin-tk chimeras [4], it was detem~ined that RNA transcribed from these genes was not autoregulated if the translation was terminated prior to codon 42. From these collective findings, it is clear that for RNA to be a substrate for autoregulated in- stability, not only must it carry the 13-nucleotide coding sequence, but it must also be ribosome bound and its translation must proceed 3' to codon 41.

12 Cytoplasm and cell motility

The nascent, amino-terminal ]3-tubulin polypeptide is the true signal for autoregulated mRNA instability

The Preceding data were consistent with two possible models for the mechanism that establishes autoregulated instability of polyribosome bound ~-mbulin RNA, In the first model, RNA degradation is facilitated by unpolymer- ized tubulin subunits binding (either directly or indi- rectly) to the 13-nucleotide recognition sequence present in 13-tubulin mRNA. The fact that only ribosome-bound RNA is a substrate for degradation can be explained ff translation is required to make the recognition domain accessible for binding. The second model arises from the realization that the sequence conferring regulated RNA instability actually encodes the first four amino-ter- minal [~-tubulin amino acids, thereby suggesting that the true recognition evenf is a protein-protein interaction involving unassembled subunits interacting (directly or indirectly) with the nascent mbulin polypeptide imme- diately after it emerges from the large ribosomal sub- unit. This putative binding event must somehow be trans- duced through the ribosome to signal the degradation of the RNA being translated.

To distinguish between these two models (recognition of the nucleotide sequence or recognition of the encoded polypeptide), site-directed mutagenesis was used system- atically to introduce 25 different nucleotide base substi- tutions into the 13-base regulatory element of a human ]3-mbulin gene (Fig. 2) [5]. From 16 mutants introduced into codon 2 (which normally encodes arginine), it was clear that the nucleotide sequence itself was not impor- tant; instead, the only five mutants whose RNA was still a substrate for autoregulation were those that carried the other five arginine codons. A similar situation emerged from mutagenesis of the wild type glutamic acid codon found at position 3. In this case, only the RNA carrying glutamic or aspartic acid codons was still regulatable.

An even more powerful test was performed by chang- ing the translational reading frame of the 13-nucleotide identifier sequence so that while its position within the mRNA remained virtually unchanged it was translated in an inappropriate reading frame (producing a polypeptide with a different amino acid sequence). RNA derived from these frame-shifted genes was not a substrate for autoreg- ulation [5]. The obvious conclusion from these experi- ments is that the recognition element is the amino-ter- minal four amino acids of [3-mbtflin (Met-Arg-Glu-Ile).

Fig. 2. Only [3-tubulin messenger RNA (mRNA) that encodes arginine at codon 2 is autoregulated. (a) Schematic dia- gram of a human ~-tubulin gene, for which site-directed rnutagenesis was used to substitute nucleotides at codon 2. , pUC sequences;- , ~ tubulin 5' and 3' or intron sequences; [ ~ , exon sequences corresponding to cod~ ing regions: [ ~ , exon sequences corresponding to 5' and 3' untrans- lated regions. (b) Quantitative analy- sis of levels of cytoplasmic RNA from cells transfected with various mutant [3- tubulin genes. The wild type sequence of the first three codons is shown, fol- lowed by the various nucleotide sub- stitutions (capital letters) introduced at codon 2. Encoded amino acids are indicated below the nucleic acid se- quences. Equal amounts of cytoplasmic RNA from cells,with normal ( - ) or el- evated (+) unpolymerized tubulin sub- units were analysed for endogenous ~- tubulin mRNA or for RNA from each transfected tubulin gene. Data are from Yen et aL [5].

Autoregulation of tubulin synthesis in animals Cleveland 13

Finally since transposition of the tubulin amino-terminal tetrapeptide to an internal region of a translation unit pre- vented its recognition by the autoregulatory factor(s), it seems quite possible that an amino-terminal localization of the four amino acid 'identifier peptide' may be neces- sary for autoregulation.

Similar, but not identical conclusions were reached by Gong and Brandhorst in their investigation of tubulin regulation in early sea urchin embryogenesis [1]. Like Pachter et al. [6] these investigators found that tubulin RNA instability was linked to continuing translation. How- ever, they also found that when protein synthesis was inhibited by the drug emetine, which principally blocks translation elongation, tubulin RNA remains stable to in- creses in the subunit concentration. This suggests that the RNA that is selectively degraded is the RNA unattached to the polyribosomes. In view of the site-directed muta- genesis experiments of Yen e ta l . [5], it is difficult to rec- oncile the two sets of data, although it seems unlikely that different mechanisms are used to establish RNA stability in the two animal cell systems.

A model for autoregulated tubulin mRNA instability

The model that best incorporates the available results is presented in Fig. 3. As the unpolymerized tubulin sub- unit pool is elevated, the initial event that leads to regu- lated degradation of fl-tubulin RNA is the interaction be-

tween the.autoregulatory factor(s), possibly tubulin itself (as shown in the figure) and the nascent amino-terminal tubulin tetrapeptide just after it emerges from the ribo- some. How this protein interaction is transduced through the ribosome to yield degradation of the corresponding RNA is not known, although there are two general pos- sibilities. First, the binding event could activate a cellu- lar RNase (which might itself be a peripheral ribosomal component). Alternatively, binding could induce trans- ient stalling of the ribosome, leaving the RNA in an ex- posed conformation that would be a better substrate for a non-specific RNase. No data are yet available to distin- guish between these two possibilities. Further, although Fig. 3 shows the binding event as occurring immediately after the amino-terminus of ~-tubulin emerges from the ribosome, the actual position within a polysome profile where this interaction occurs has not been determined experimentally.

It may seem surprising that so short a sequence as a te- trapeptide can be specifically recognized. However, in- spection of the predicted amino-terminal four amino acids, among all the [3-tubulin genes sequenced to date, reveals that the tetrapeptide Met-Arg--Glu-Ile is abso- lutely conserved at the amino-terminus. Furthermore, this tetrapeptide sequence has been found only in [3- tubulins. Since cz-tubulin carries a common tripeptide (Met-Arg-CGlu) at its amino-terminus it is tempting to speculate that this domain may also confer regulated RNA instability on ~x-tubulins.

The model easily explains why it is that the RNA that yields only short translation products is not autoregu-

Ribosomes Tubulin AUG / k mRNA

,'l¢~pp A444/t4

Microtubule M E R M

Fig. 3. Proposed model for autoregulated instability of [3-tubulin messenger RNA (mRNA). Unpolymerized tubulin subunits bind directly [or activate a factor(s) which binds] to the nascent amino terminal tetrapeptide (Met-Arg-Glu-Ile) of ~-tubulin. This binding is transduced through the adjacent ribosome to activate an RNase which degrades the ribosome-bound mRNA. The RNase has been drawn ( © ) as ribosome-associated, although this has not yet been demonstrated. MREI, the amino terminal J3-tubulin polypeptide. Published by permission [5].

14 Cytoplasm and cell motility

lared, even though it is efficiently associated with ribo- somes [4]. The answer is that during translation the large ribosomal subunit covers the 30-40 most recently added amino acids; therefore at least this number of amino acids must be translated before the nascent peptide can be ac- cessible for binding by additional cellular factors,

In vivo autoregulation

The identification of the sequences that specify mbu- lin RNA as a substrate for autoregulated instability fur- ther highlights the as yet unanswered question of the functional end that autoregulation serves. At first glance, autoregulation appears to be largely redundant, since the polymerization reaction is sell-buffering in vitro, subunits continue to assemble until the level of free subunits is de- pleted to the 'critical concentration', the level at which the subunits are at steady state with the polymer. However, Mitchison and Kirschner [7] have recently demonstrated that the concept of a unique critical concentration for a microtubule array in a closed system (a cell with a fixed amount of tubulin, in which microtubules are nucleated from specific centrosomal sires) is not meaningful. Micro- tubules can polymerize at almost any subunit concentra- tion provided nucleation is sufficiently rapid. In this type of system, the number and stability of nucleated micro- tubules is dependent on two factors: nucleation capacity and unassembled subunit concentration. Thus, autoreg- ulation of tubulin synthesis may represent a portion of a cellular mechanism that establishes a level of unpoly- merized subunits necessary to ensure a nucleated array of dynamic microtubules [4].

The challenge that lies ahead is to test experimentally the consequence of disruption of the normal regulatory path- way. An experiment that sheds some light on this issue has been provided by Boggs and Cabral [8], who used somatic cell genetics to obtain a hamster cell line that accumulates only about 2/3 of the normal level of cellular tubulin. This was achieved by inactivating one or more of the handful of genes that are normally expressed in this line. The simplest prediction was that the remaining gene(s) would be expressed at elevated levels in order to maintain a constant level of cellular tubulin. In fact, this was not observed, although it is not clear whether the complex interaction between the regulatory factors and microtubule dynamics was actually altered. It may well be that in this example the stringent selection process for the inactivation of a specific gene led to concurrent changes both in tubulin synthetic capacity and in nucle- ation ability. If this were the case, then the dynamic as- sembly properties would remain largely unchanged, as would the unpolymerized tubulin concentration. Future DNA transfection experiments designed to force elevated levels of synthesis will probably be required to establish

unambiguously the in vivo role of autoregulated tubulin RNA instability.

Annotated references and recommended reading

• Of interest 0 • Of outstanding interest

1. GONG Z, BRANDHORST B: Autogenous regulation of tubulin • synthesis via RNA stability during sea urch in embryogen-

esis. Development 1988, 102:31-43. This study is the first demonstration of the use of autoregulated tubulin RNA instability during a true in vivo situation, in this instance early sea urchin developmem.

2. G O N G Z, BRANDHORST B: Multiple levels of regulation of • tubulin gene express ion during sea urch in embryogenesis~

Dev Biol 1988, 130:144-153. During sea urchin embryogenesis there are multiple levels of regula- tion of tubulln gene expression. First there is the translation activation of maternal mRNA, followed by a translational elevation; later, autoreg ulated RNA instability becomes operative.

3. GAY DA~ YEN TJ, LAU JTY, CLEVELAND DW: Sequences that • confer ~-tubulin autoregulation th rough modulated mRNA

stability reside wi th in Exon 1 of a ~-tubulin mRNA. Cell 1987, 50:671q579.

This study was the first to determine which sequences specify tubulin RNA as substrate for autoregulated instability.

4. YEN TJ, GaY DA, PACHTER JS, CLEVELAND DW: Autoregu- • • lated changes in stability of polyribosome-bound ~3-tubulin

mRNAs are specified by the first 13 translated nucleotides. Mol Cell Biol 1988, 8:1224-1235.

Following [3], the authors demonstrate unambiguously, using DNA transfection, that the stability of [~-tubulin RNA is specified through the first 13 translated nucleotides.

5- YEN TJ, MACHLIN PS, CLEVELAND DW: Autoregulated instability • • of [~-tubulin mRNAs by recognit ion of the nascent amino

te rminus of ~-tubulin. Nature 1988, 334:580-584. An extensive series of site-directed mutants was used to demonstrate that the signal [3-tubulin RNA for autoregulated instability is, in fact, its encoded amino-terminal [3-tubulin potypeptide.

6. PACHTEU JS, YEN TJ, CtEVEtAND DW: Autoregulation of tubu- • lin express ion is achieved th rough specific degradation of

polysomal tubulin mRNAs, Cell 1987, 51:283-292. Protein synthesis inhibitors were used to suggest that tubulin mRNA instability takes place while the RNA is attached to polysomes. This no- tion was further confirmed by the use of transfection of genes bearing premature translation termination codons.

7. MITCHISON TJ, KIRSCHNER IV[X~: Some thoughts on the par- o * titioning of tubulin be tween m o n o m e r and polymer un-

der condit ions of dynamic instability. Cell Biophys 1987, 11:35-56.

A very thoughtful study that describes the interplay between tubulin assembly and dynamic instability not in a test-tube, but in the closed system of an intact cell.

8. BOGGS B, CABRAL F: Mutations affecting assembly and sta- • bility of tubulin: evidence for a nonessential ~-tubulin in

CHO cells. Mol Cell Biol 1987, 7:270(~2707. This study examines the consequences of selecting for inactivation of an individual tubulin gene. Among those SUlviving cells that do not express a specific gene, the total level of accumulated tubulin declines. This finding is somewhat surprising in view of the apparent autoregulatory pathway.