Vol. 12, No. 2MOLECULAR AND CELLULAR BIOLOGY, Feb. 1992, p. 791-7990270-7306/92/020791-09$02.00/0Copyright C 1992, American Society for Microbiology

Physical Evidence for Cotranslational Regulation of3-Tubulin mRNA Degradation

NICHOLAS G. THEODORAKIS AND DON W. CLEVELAND*Department of Biological Chemistry, Johns Hopkins University School of Medicine,

725 North Wolfe Street, Baltimore, Maryland 21205

Received 4 September 1991/Accepted 25 November 1991

Tubulin synthesis is controlled by an autoregulatory mechanism through which an increase in theintracellular concentration of tubulin subunits leads to specific degradation of tubulin mRNAs. The sequencenecessary and sufficient for the selective degradation of a "-tubulin mRNA in response to changes in the levelof free tubulin subunits resides within the first 13 translated nucleotides that encode the amino-terminalsequence of 1-tubulin, Met-Arg-Glu-Ile (MREI). Previous results have suggested that the sequence responsiblefor autoregulation resides in the nascent peptide rather than in the mRNA per se, raising the possibility thatthe regulation of the stability of tubulin mRNA is mediated through binding of tubulin or some other cellularfactor to the nascent amino-terminal tubulin peptide. We now show that this putative cotranslationalinteraction is not mediated by tubulin alone, as no meaningful binding is detectable between tubulin subunitsand the amino-terminal 1-tubulin polypeptide. However, microinjection of a monoclonal antibody that bindsto the 13-tubulin nascent peptide selectively disrupts the regulation of 13-tubulin, but not a-tubulin, synthesis.This finding provides direct evidence for cotranslational degradation of "-tubulin mRNA mediated throughbinding of one or more cellular factors to the 13-tubulin nascent peptide.

Microtubules are dynamic filamentous structures that par-ticipate in a wide variety of cellular processes, includingmitosis, vesicle transport, and cellular motility. The princi-ple component of microtubules is tubulin, a heterodimer of aand i subunits that exist in a state of dynamic equilibriumwith the microtubule polymer. The synthesis of tubulin istightly coupled to the state of polymerization of the micro-tubule. Increasing the cytoplasmic concentration of tubulinsubunits, for example by depolymerizing microtubules withdrugs (1, 8) or by microinjection of unassembled tubulinsubunits (9), leads to a rapid and specific arrest of thesynthesis of a- and ,B-tubulin, caused by a decrease in thelevel of tubulin mRNAs (8).A number of observations have led to the conclusion that

the modulation of tubulin synthesis in response to changes intubulin subunit concentration is a posttranscriptional cyto-plasmic event. First, run-on transcription in isolated nucleireveals no changes in the transcription rates of tubulin geneswhen microtubules are depolymerized by colchicine (7).Second, the colchicine-induced decrease in tubulin synthesisrates occurs even in enucleated cells (5, 26). Finally, hybridgenes composed of tubulin-coding sequences and heterolo-gous promoters still respond to changes in tubulin subunitlevels in transfected cells (13, 32, 33).

Previous efforts have shown that the sequence necessaryand sufficient for the selective degradation of a ,B-tubulinmRNA in response to changes in the level of free tubulinsubunits resides within the first 13 translated nucleotides thatencode the amino-terminal sequence of 3-tubulin, MREI(32). Several lines of evidence indicate that P-tubulin mRNAstability is regulated cotranslationally. First, only translatedmRNAs are substrates for regulation (24). Second, prema-ture translation termination results in an mRNA that is nolonger a substrate for regulation (24, 32). Third, all mutationsthat alter the mRNA sequence at the second or third codon

* Corresponding author.

but still encode identical polypeptides result in an mRNAthat is still a substrate for regulation (33). Fourth, in order toconfer regulated instability, the minimal mRNA domain thatspecifies instability must be translated in the correct readingframe (33). These data, taken together with the observationthat changes in unassembled tubulin concentration appar-ently trigger the degradation of 1-tubulin mRNA, suggest amodel in which unassembled tubulin subunits bind cotrans-lationally to nascent tubulin peptides, somehow triggeringdegradation of the polysome-bound mRNA. However, so farthere is no direct physical evidence that tubulin subunits, orany other cellular factor, can bind to the P-tubulin nascentpeptide.

In this study, we tested two predictions of this autoregu-latory model: whether tubulin can bind to a peptide thatcorresponds to the amino terminus of P-tubulin, and whetherthe binding of a cellular component to the ,-tubulin nascentpeptide is an obligatory event for the regulation of tubulinsynthesis.

MATERIALS AND METHODS

Labelling of synthetic peptide and binding assays. A syn-thetic peptide corresponding to the amino terminus of ,B-tu-bulin (MREIVHIQAGQCY) was prepared by the JohnsHopkins University Peptide Facility and sequenced to con-firm its identity. (Note that the C-terminal tyrosine of thepeptide is not encoded in 3-tubulin, but was added for theconvenience of radiolabelling and for monitoring absorbanceat 280 nm.) High-pressure liquid chromatography (HPLC)analysis confirmed that the peptide was greater than 95%pure. Peptide was radiolabelled with Na125I (Amersham,Arlington Heights, Ill.) by using Iodobeads (Pierce, Rock-ford, Ill.) according to the manufacturer's directions. Thepeptide was separated from the free label by chromatogra-phy on a Bio-Rad P-10 column or on a disposable Sep-PakC18 column (Waters). Before labelling, the reactive sulfhy-dryl group on the peptide was blocked by treatment with

791

792 THEODORAKIS AND CLEVELAND

iodoacetic acid (11) to prevent its reaction with the iodina-tion reagent. Briefly, the peptide was dissolved in degassed0.1 M sodium phosphate buffer (pH 7.4) and equilibratedwith nitrogen gas. lodoacetic acid was added to a fivefoldmolar excess from a 1 M stock, and the solution was titratedto pH 7.4 with sodium hydroxide. After incubation in thedark at room temperature for 2 h, the reaction was termi-nated by the addition of excess 2-mercaptoethanol and thepeptide was purified on a disposable Sep-Pak C18 column(Waters). The extent of carboxymethylation was confirmedby reverse-phase HPLC; the carboxymethylated form has aslightly earlier retention time and was typically greater than75% of the reaction products.For measurement of binding by equilibrium gel filtration

(17), a 2-ml Sephadex G-75 Superfine colui n (0.4 by 16 cm)was equilibrated in buffer [30 mM KCI, 2( iM piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES; A 6.8), 1 mMethylene glycol-bis(P-aminoethyl ether)-N,N,N',N'-tetraace-tic acid (EGTA), 1 mM MgCl2] alone, buffer containing 10 ,uMpeptide at 2,000 cpm/,ul, or buffer containing 2.0 mg ofphosphocellulose-purified bovine brain tubulin per ml. Pep-tide (25,000 cpm, or "100 ng) was diluted to 20 ,li with bufferor buffer containing 60 ,g of antipeptide antibody and appliedto the column. Fractions of 3 drops (-60 ,ul) were collectedand assayed for radioactivity, using a gamma counter.

Equilibrium dialysis assays were performed in chambersmade from 0.5-ml plastic snap-cap centrifuge tubes. Tubulin(100 ,ug in 100 pl of buffer) and peptide were added to the capend of the centrifuge tube, which was covered with dialysistubing (molecular weight cutoff of 50,000; Spectrapor). Pep-tide was added in 100 p,l to the tube end, which was invertedover the cap. The liquid in the tube end was then flickeddown over the membrane and allowed to equilibrate with thecap end for 24 h at 4°C with gentle shaking. Dialysis wasterminated by briefly centrifuging the tubes to separate theliquid in the tube end from the membrane; the tube was thencut in half with dog toenail clippers, and the radioactivity inthe two chambers was assayed by gamma counting.

Production of monoclonal antibodies (MAbs) and immuno-logical methods. A synthetic peptide (MREIVHIQAGQCY)was coupled to keyhole limpet hemocyanin (KLH), using theheterobifunctional cross-linking reagent m-maleimidoben-zoyl-N-hydroxysuccinimide ester (MBS; Pierce). Briefly,KLH was incubated with MBS in 0.1 M potassium phos-phate (pH 7.2) at room temperature for 2 h. Unreacted MBSwas removed by gel filtration. The reacted KLH was thenincubated with peptide for 2 h at room temperature and thenincubated in 2-mercaptoethanol. The unreacted productswere removed by gel filtration.Female BALB/c mice were immunized five times intraper-

itoneally with peptide-conjugated KLH (20 p,g in Freund'sadjuvant) at 2-week intervals. Splenocytes were fused toP3-X63 mouse myeloma cells, and hybridomas (18) wereselected as described previously (10, 15). Cells secretingantitubulin antibodies were screened first by enzyme-linkedimmunosorbent assay (ELISA) against purified chickenbrain tubulin; ELISA-positive cells were expanded andrescreened by immunoblot against whole-cell extracts, andanti-,-tubulin-secreting cells were cloned twice by limitingdilution. Antibody classes and isotypes were determined byusing an isotyping kit (Amersham).Immunofluorescence was performed as described previ-

ously (20) under conditions that maintain microtubule integ-rity (28). Cells were washed in microtubule stabilizing buffer(MTSB; 0.1 M PIPES [pH 6.8], 4 M glycerol, 1 mM MgCl2,2 mM EGTA, 0.1 mM EDTA) at room temperature, ex-

tracted with 0.5% Triton X-100 in MTSB, washed again inMTSB, and fixed in methanol at -20°C. Alternatively, cellswere fixed directly in methanol after washing in MTSBwithout prior extraction. The cells were then rehydrated inphosphate-buffered saline containing 3% bovine serum albu-min (PBS/BSA) and incubated in PBS/BSA containing as-cites fluid diluted 1,000-fold or rabbit antiserum diluted30-fold for 1 to 2 h in a humidified atmosphere. The cellswere then washed five times in PBS and incubated inPBS/BSA containing fluorescein- or Texas red-conjugatedhorse anti-mouse immunoglobulin G (IgG) or fluoresceinatedgoat anti-rabbit IgG (Vector Laboratories, Burlingame, Cal-if.), washed as described above, and mounted with Gel/Mount (Biomeda Corp., Foster City, Calif.). Microscopywas performed with an Olympus BH-2 microscope equippedfor epifluorescence.

Immunoblotting was performed essentially as describedpreviously (20). Protein was separated by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis as de-scribed previously (19) except that the separating gels con-tained Tris at pH 9.1 and were formulated with lowerconcentrations of bisacrylamide (2). Protein was electro-phoretically transferred to nitrocellulose (Schleicher &Schuell); transfer efficiency was checked by staining with0.5% Ponceau S in 1% acetic acid. The blots were incubatedin PTX (10 mM sodium phosphate buffer [pH 7.5], 150 mMNaCl, 1 mM EGTA, 0.2% Triton X-100) containing 4% BSA.Ascites fluid was added at 1,000-fold dilution, and the blotswere incubated for 1 to 2 h with gentle shaking. The blotswere then washed vigorously five times in GB (50 mMtriethanolamine [pH 7.5], 100 mM NaCl, 2 mM EDTA, 0.5%Triton X-100, 0.1% SDS). Secondary antibody ('25I-sheepanti-mouse; Amersham) was incubated at 1,000-fold dilutionin PTX/BSA, and incubation and washing were continued asdescribed above.

Microinjection of MAbs. Mouse ascites fluid was prepared(Bioproducts for Science, Indianapolis, Ind.), and IgG waspurified by ammonium sulfate fractionation and DEAE-cellulose chromatography as described previously (15).Briefly, ascites fluid was adjusted to 50% saturation ofammonium sulfate. The precipitate was collected by centrif-ugation, resuspended in 10 mM Tris (pH 8.6), and dialyzed.The protein was applied to a column of DEAE-cellulose(DE52; Whatman) in 10 mM Tris and eluted with a lineargradient of 5 column volumes of 0 to 150 mM NaCl.Fractions containing IgG were identified by gel electropho-resis, pooled, and concentrated by ammonium sulfate pre-cipitation. Purified IgG was resuspended at 15 mg/ml ininjection buffer (0.1 M KCl, 10 mM potassium phosphate[pH 6.8]) and dialyzed. Gel electrophoresis showed that thepurified antibody was greater than 95% IgG.CHO-Kl cells (American Type Culture Collection; CCL

61) were grown in Ham's F-12 medium containing 10% fetalbovine serum at 37°C in a humidified atmosphere of5% CO2.Microinjection of CHO cells was performed under phasemicroscopy as described previously (9). For immunofluores-cence experiments, cells were grown on etched griddedcoverslips (Bellco). After injection, the cells were allowed torecover for 3 to 6 h before fixation and staining. Formetabolic labelling experiments, CHO cells were grown onl1-mm2 glass chips. Chips that contained 50 to 100 cellswere selected for injection. After injection, cells were al-lowed to recover for 30 to 60 min, incubated in fresh mediumin the absence or presence of colchicine (0.5 pug/ml) for 4 h,washed in methionine-free medium, and incubated under a5-pul drop of methionine-free medium containing 25 ,uCi of

MOL. CELL. BIOL.

REGULATION OF ,-TUBULIN mRNA DEGRADATION 793

A

i10

a

B

0

0-

CO4 '0

X

92

0 10 20 30 40

Fracton No.

FIG. 1. Evidence that tubulin does not bind to a peptide corre-sponding to the amino terminus of P-tubulin. (A) A peptide corre-

sponding to the amino terminus of P-tubulin was radiolabelled with125I and chromatographed on a 2-ml Sephadex G-75 Superfinecolumn (0.4 by 16 cm) equilibrated in buffer (30 mM KCI, 20 mMPIPES [pH 6.8], 1 mM EGTA, 1 mM MgCl2) in the absence ofprotein (circles) or after the addition of 60 ,ug of antipeptide antibody(squares). Fractions of 3 drops (=60 ,u) were collected and assayedfor radioactivity, using a gamma counter. (B) Equilibrium binding oftubulin to peptide. A Sephadex G-75 column was equilibrated in 10,uM radiolabelled peptide; 200 p.g of phosphocellulose-purified bo-vine brain tubulin was added, chromatography was continued, andradioactivity was assayed (open circles). Alternatively, the columnwas equilibrated in 20 ,uM tubulin; radioactive peptide was added,and chromatography was continued in the presence of 20 pLM tubulin(filled circles).

[35S]methionine (800 Ci/mmol; Amersham) for 1 h. Labelledproteins were analyzed by two-dimensional gel electropho-resis (23), using isoelectric focusing over a pH gradient of 5to 7 in the first dimension and SDS-gel electrophoresis on10% polyacrylamide gels in the second dimension as de-scribed previously (9). Isofocusing gels contained 9 M urea,3.8% acrylamide, 0.21% bisacrylamide, 2% Nonidet P-40,1.6% ampholines in the pH range of 5 to 7 (Bio-Rad), and0.4% ampholines in the pH range of 3 to 10 (Serva).

RESULTS

Purified tubulin subunits do not bind to a l-tubulin amino-terminal peptide. One prediction of the autoregulatory modelof 1-tubulin synthesis is that unassembled tubulin itselfmight bind to the ,-tubulin nascent peptide. However, sincethe intracellular concentration of free tubulin is high (=10p.M) even in cells with normal microtubules, a low affinity oftubulin subunits for the nascent peptide might be expected.

TABLE 1. Binding of amino-terminal ,-tubulin peptide totubulin, using equilibrium dialysisa

Peptide (,ug) cpm in: nmol of peptide:added toeach Upper chamber Lower chamber Freeb Bound

chamber (=free) free)

160.0 65,940 66,760 113.58 1.4180.0 32,657 32,293 57.46 (-0.64)40.0 16,456 16,393 28.63 (-0.11)16.0 59,447 60,082 11.37 0.128.0 29,765 29,754 5.72 (0)4.0 14,676 16,213 2.71 0.281.6 58,478 59,329 1.13 0.020.8 26,294 30,091 0.53 0.080.4 13,248 16,195 0.26 0.06

a Tubulin (100 FLg = 1 nmol) was added in 100 j.l to the lower chamber.Equal amounts of peptide were added to the upper (100 ,ul) and lowerchambers in 50 mM KCI-20 mM PIPES (pH 6.9)-i mM EGTA-1 mMMgCI2-1 mM GTP. Dialysis was allowed to proceed for 24 h at 4°C.

b Calculated from the fraction of the total counts per minute in the upperchamber, assuming the molecular weight of the peptide to be 1,400.

1 Calculated from the counts per minute in the lower chamber subtractedfrom the counts per minute in the upper chamber.

Otherwise, tubulin binding to the nascent peptide would besaturated at all times. Accordingly, we used equilibrium gelfiltration (17) to test the ability of tubulin to bind to aradiolabelled synthetic peptide corresponding to the amino-terminal 12 residues of P-tubulin (MREIVHIQAGQCY).Chromatography of the peptide alone on a Sephadex G-75column results in its elution in the included volume (Fig. 1A,fractions 27 to 35). In an initial experiment, an antibody thatbinds to the peptide was added before chromatography, andthis resulted in the expected shift of a significant proportionof the peptide into the void volume (fractions 11 to 16). Totest the binding of tubulin to peptide under equilibriumconditions (17), we equilibrated the column with 10 ,uMpeptide. Tubulin (200 ,ug in 50 ,ul of buffer containing 10 ,uMpeptide) was added, and chromatography continued in thepresence of 10 ,uM peptide. If under these conditions tubulinbound a significant proportion of the peptide, then an in-creased concentration of the peptide would elute with tubu-lin in the void volume of the column. However, no suchchanges in the level of peptide were detected (Fig. 1B, opencircles).

In these experiments, the concentration of tubulin appliedto the column (4 mg/ml = 40 ,uM) is close to the solubility ofpurified tubulin, and this limits the concentration of tubulinthat can be applied to the column. If the Kd of tubulin for thepeptide is much greater than the 10 ,uM peptide concentrationin the column, it would be difficult to detect binding above thebackground noise of the assay. To further increase thesensitivity of the binding assay, we reversed the normalprocedure and equilibrated the column in tubulin at 2 mg/ml(=20 ,uM) before adding a small amount of radiolabelledpeptide. This would allow us to detect small changes in theelution profile of the peptide; however, when the sample waschromatographed in the presence of 20 FM tubulin, no changein the elution profile of the peptide was detectable (Fig. 1B,closed circles). Since a shift of as little as 10% of the peptideout of the included fraction could easily have been detected(in fact, less than 1% of the radiolabel was shifted bychromatography in the presence of tubulin), the dissociationconstant for peptide binding to tubulin must be greater than180 ,uM. {If the total tubulin concentration is 20 ,uM and is

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794 THEODORAKIS AND CLEVELAND

Bco < C

C - T-

115 -

97

68

45

1 8D6+ Peptidem I

DM1 A 18D6

1 2 3 4 5FIG. 2. Recognition of P-tubulin by MAb 18D6. (A) Immunoblot analysis of antitubulin antibodies. CHO cell extract was fractionated on

SDS-containing polyacrylamide gels, blotted to nitrocellulose, and probed with the MAb 18D6 (lanes 1, 4, and 5), DM1A (lane 2), or DM1B(lane 3) in the presence of no peptide (lanes 1 to 3) or in the presence of MREIVHIQAGQCY (lane 4) or MREIVY (lane 5). (B)Immunofluorescence. CHO cells were extracted, fixed, and stained for microtubules, using MAb DM1A or 18D6. The insets show the stainingpatterns in mitotic cells.

much greater than the peptide concentration, then the freetubulin concentration is also 20 ,uM. Assuming that we couldeasily detect 10% of the peptide bound, then (free peptide)/(total peptide) = 0.9; thus, (free peptide)/([free peptide] +[bound peptide]) = 0.9, and therefore (free peptide) =

9(bound peptide). If we assume one potential binding site ontubulin for the peptide, then Kd = (free peptide)(free tubulin)/(bound peptide), which means that Kd = 9(bound peptide)20,uM/(bound peptide) = 180 ,uM.} With an intracellular concen-tration of total tubulin of about 20 ,uM (16), no physiologicallyrelevant binding of pure tubulin to isolated peptide is possible.We confirmed the low affinity of tubulin for the amino-

terminal 0-tubulin peptide by a series of equilibrium dialysisexperiments. If peptide is allowed to diffuse across a dialysismembrane that retains tubulin on one side of the membrane,then if tubulin bound to the peptide, the concentration of thepeptide would be higher in that side. Preliminary experi-ments in which radiolabelled peptide was added to onechamber and allowed to diffuse to the other chamber in theabsence of added protein indicated that equilibration pro-ceeded to 90% within 24 h. In the actual experiment, whentubulin was added to one side of the membrane, no bindingto the peptide was detectable over a wide range of peptideconcentrations (Table 1). We conclude that the affinity oftubulin for the 1-tubulin amino-terminal peptide is, at best,very low.MAbs to the I8-tubulin nascent peptide disrupt autoregula-

tion in microinjected cells. The proposal that unassembled

tubulin levels are an intrinsic component in regulating therate of new tubulin synthesis is supported by two directexperiments. First, drugs that cause changes in unassembledtubulin levels (by interfering with microtubule assembly)also cause concomitant changes in tubulin synthetic rates (1,8) and tubulin mRNA levels (8). Second, microinjection ofunassembled tubulin subunits into cells suppresses tubulinsynthetic levels (9). That a cotranslational interaction of thenascent P-tubulin peptide with a cellular factor(s) is a crucialstep in regulating tubulin RNA stability has been inferredfrom transfection experiments with mutant genes (32, 33).Our inability here to detect binding of tubulin to its amino-terminal peptide raised the question as to whether tubulin (orany other component) truly interacted with the nascentpeptide in vivo. We reasoned that we could test for thepresence of factors that interact with the P-tubulin nascentpeptide by introducing into cells an antibody to the nascentpeptide; the presence of this antibody might prevent suchputative cellular factors from recognizing the nascent pep-tide, thereby blocking the normal regulation of P-tubulinsynthesis. Alternatively, we considered the possibility thatintroduction of the peptide into cells might disrupt theregulation of tubulin synthesis; however, our inability todetect binding of tubulin to the synthetic peptide made usreluctant to pursue this approach.

Accordingly, we raised MAbs to a synthetic peptidecorresponding to the amino-terminal 12 residues of P-tubulin(MREIVHIQAGQCY). Hybridomas (prepared by fusion of

A

.Is.p

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REGULATION OF p-TUBULIN mRNA DEGRADATION 795

FIG. 3. Evidence that microinjection of antibody does not perturb microtubule dynamics. Uninjected CHO cells (A and C) or cells injectedwith MAb 18D6 (B and D) were incubated in the presence (C and D) or absence (A and B) of colchicine (0.5 ,ug/ml) for 4 h. The distributionof tubulin was revealed by immunofluorescence microscopy, using a rabbit antiserum that recognizes tubulin.

splenocytes to mouse myeloma cells) were assayed for theproduction of antitubulin antibodies by ELISA and byimmunoblot. One of the antitubulin IgG-secreting lines(18D6) was cloned by limiting dilution, and immunoblotanalysis revealed that its antibody (MAb 18D6) recognizes aprotein of approximately 55 kDa that is present in CHO cellextracts (Fig. 2A, lane 1) as well as in phosphocellulose-purified chicken brain tubulin (not shown). The antigenrecognized by MAb 18D6 has the same apparent molecularweight as that recognized by the anti-fi-tubulin antibodyDM1B (3) (lane 3) and is slightly smaller than that recognizedby the anti-a-tubulin antibody DM1A (3) (lane 2). Peptidecompetition experiments confirmed the specificity of MAb18D6. Incubation of the antibody in the presence of excesspeptide (MREIVHIQAGQCY) abolished the binding to tu-bulin on immunoblots (lane 4). However, since a shorterpeptide corresponding to the extreme amino terminus ofP-tubulin (MREIVY) failed to abolish binding (lane 5), theepitope for MAb 18D6 must reside within the first 12amino-terminal amino acids of P-tubulin but probably not atthe extreme amino terminus. The specificity of MAb 18D6was also tested by indirect immunofluorescence (Fig. 2B);after conventional fixation, MAb 18D6 revealed interphaseand mitotic patterns of microtubules similar to those re-vealed by the anti-a-tubulin antibody DM1A.As we wished to microinject this antibody and determine

its effects on regulation of tubulin mRNA stability, an initial

concern was whether the antibody would affect microtubulearrays in vivo. The antibody was microinjected into CHOcells, and the cells were then stained for the presence ofmicrotubules by using a rabbit polyclonal antiserum thatrecognizes tubulin. Immunofluorescence microscopy re-vealed that the microtubule array in injected cells (Fig. 3B)was similar to that seen in uninjected cells (Fig. 3A).Therefore, microinjection of the antibody did not disrupt thearray of microtubules. Incubation of either uninjected cells(Fig. 3C) or injected cells (Fig. 3D) in the presence ofcolchicine resulted in the disassembly of microtubules, indi-cating that the presence of the antibody did not prevent thecolchicine-induced depolymerization of microtubules. Todetermine the subcellular distribution of microinjected anti-body, injected cells were fixed and stained for the presenceof the antibody by using a fluorescent anti-mouse antibody(Fig. 4). When cells were fixed without prior extraction, theantibody decorated structures that resemble microtubules;indeed, in injected cells that were undergoing mitosis, amitotic spindle could clearly be seen (Fig. 4, inset). How-ever, if cells were extracted with nonionic detergent beforefixation (Fig. 4B), no specific staining was observed. Conse-quently, injected antibody is not tightly bound to microtu-bules in living cells, nor does it impair the ability of tubulinto reorganize during mitosis or during colchicine-induceddepolymerization.We next determined whether injection of the antibody

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796 THEODORAKIS AND CLEVELAND

0'-.z......

FIG. 4. Intracellular distribution of microinjected antibody. CHO cells were injected with MAb 18D6, allowed to recover for 3 h, andstained for the presence of MAb 18D6, using fluoresceinated anti-mouse antibody. Panel A reveals the distribution of the injected antibodyin cells fixed directly in methanol without prior extraction. In panel B, cells were extracted with Triton X-100 before fixing and staining withanti-mouse IgG. The inset shows the localization of the antibody in an injected cell that is undergoing mitosis.

could perturb the regulation of tubulin synthesis in CHOcells. If the binding of a cellular factor to the ,B-tubulinnascent peptide could cause the degradation of the poly-some-bound P-tubulin mRNA, then either of two outcomesmight be predicted after antibody injection. One possibilityis that the antibody itself mimics the binding of a normalcellular factor and stimulates the degradation of tubulinmRNA. To test this, cells were microinjected with MAb18D6, allowed to recover for 4 h, and metabolically labelledwith [35S]methionine, and the labelled proteins were ana-lyzed by two-dimensional gel electrophoresis and fluorog-raphy. Cells injected with MAb 18D6 displayed no changesin the general pattern of protein synthesis or in the levels oftubulin synthesis (Fig. 5A). Therefore, we conclude thatinjection of the antibody does not prevent the normal trans-lation of P-tubulin, nor does it repress or induce the synthe-sis of other cellular proteins.The alternative possibility is that the binding of the anti-

body to the P-tubulin nascent peptide prevents the binding ofthe putative regulatory factor. In this case, the colchicine-induced repression of ,-tubulin synthesis should be blockedby the presence of the antibody. We tested this possibility byincubating antibody-injected cells with colchicine and usingtwo-dimensional gel electrophoresis to monitor tubulin syn-thetic rates. In uninjected cells, colchicine-induced microtu-bule disassembly resulted in the inhibition of a- and ,-tubu-lin synthesis, as expected (Fig. 5B). However, when cells

were injected with the antibody and then treated withcolchicine, we observed little or no change in the level of3-tubulin synthesis (Fig. 5B). Therefore, injection of MAb18D6 prevents the normal colchicine-induced decrease inP-tubulin synthesis. This result has been observed six timesin five separate experiments using two different preparationsof antibody. Although the antibody prevented the decreaseof P-tubulin synthesis in injected cells, a-tubulin synthesiswas repressed to the same degree as it was in uninjectedcells. This is an important internal control that demonstratesthat the effect of antibody injection is specific for P-tubulinand cannot be the result of depleting the pool ofunassembledtubulin subunits. We conclude that antibody-dependent dis-ruption of 1-tubulin synthesis must reflect interference ofbinding by some cellular factor to the P-tubulin nascentpeptide. These results provide direct physical evidence thatthe binding of a cellular factor to P-tubulin nascent peptide isa critical step in the degradation of 1-tubulin mRNA.

DISCUSSION

Previous results using mutant gene constructs have sup-ported a model in which the nascent peptide of P-tubulin isthe regulatory target for a cellular factor that causes degra-dation of polysome-bound tubulin mRNA. We now haveobtained direct physical evidence that the cotranslationalbinding of a cellular factor to the ,-tubulin nascent peptide is

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REGULATION OF 3-TUBULIN mRNA DEGRADATION 797

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4~-4PM..+ o13 Uninj.+ col.

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FIG. 5. Evidence that microinjection of MAb 18D6 disrupts the down-regulation of P-tubulin synthesis. (A) CHO cells growing on glasschips were injected with antibody (Inj.) or left uninjected (Uninj.) and allowed to recover for 4 h. The cells were then metabolically labelledwith [35S]methionine; labelled proteins were analyzed by two-dimensional gel electrophoresis and fluorography. The positions of a- andP-tubulin are indicated by arrows. Another spot that is not a tubulin but that migrates near the tubulins (presumably vimentin) is indicatedby the arrowhead. (B) Cells that were untreated (Uninj. - col.), treated with colchicine for 4 h (Uninj. + col.), or microinjected with antibodyand treated with colchicine (0.5 ,ug/ml) for 4 h (Inj. + col.) were analyzed as described above.

an obligatory step in the regulation of 1-tubulin mRNAstability. Although the simplest interpretation of the auto-regulatory model is that unassembled tubulin subunits mightbe the factor that interacts with the P-tubulin nascent pep-tide, we have been not been able to detect any binding oftubulin to a synthetic peptide that corresponds to the aminoterminus of P-tubulin. Possibly the effector molecule is someother nontubulin protein that binds to f-tubulin nascentpeptide and whose availability is elevated by an increase inthe concentration of unassembled tubulin. Perhaps a morelikely interpretation is that tubulin requires a cellular cofac-tor for binding to the nascent peptide. Inasmuch as 1-tubulinmRNA is cotranslationally degraded, and the process iscoupled to translational elongation (12), a reasonable possi-bility is that the cofactor is the ribosome itself. In this regard,it is interesting to note that ribosomes can associate with seaurchin microtubules in vitro (29), although the significance ofthis finding to tubulin autoregulation is obscure.With regard to the physiological significance of tubulin

autoregulation, although we have no direct evidence, it isreasonable to propose that it is always in place as a fine-tuning mechanism for establishing tubulin subunit levels. Aspecific example of its operation may be at mitosis: tubulin isone of only a few proteins whose synthesis is known toincrease during mitosis (4). Since the many additional micro-tubules assembled at mitosis are thought to lead to a fall inthe unassembled subunit concentration (22), the mitotic

increase in synthesis is as predicted if the autoregulatorymachinery is at work. Yet if this fine-tuning mechanism isalso continually working during interphase (i.e., in thecytoplasm of the cells that we have examined here bymicroinjection), it might be expected that binding of injectedantibody to the nascent P-tubulin peptide would stabilizeP-tubulin mRNAs even without drug-induced microtubuledisassembly. This predicts an increase in P-tubulin synthe-sis, but we did not observe it (Fig. 5). However, thisprobably reflects that in the rapidly growing cells examinedhere, tubulin mRNA stability (and protein synthesis) isalready maximal; in such cells, decreases in the subunit pool(e.g., following taxol-stimulated assembly of all cell tubulin)are not accompanied by increases in new synthesis (J. S.Pachter and D. W. Cleveland, unpublished data), althoughsuch increases are easily seen in cells with slower growthrates (6).

Cotranslational regulation of mRNA degradation has beenimplicated in several other systems. These include the cellcycle regulation of histone mRNA degradation, which re-quires translation to within 300 nucleotides of a stem-loopstructure at the 3' end of histone mRNA (14). In the yeastMATal gene, translation of a 42-nucleotide segment contain-ing rare codons is required for its rapid degradation (25). Athird example may be found in the mRNA encoding c-fos;one of the regulatory elements conferring instability is lo-cated within the protein-coding domain (27). Although the

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specifics of the various RNA degradation pathways differ, acommon thread is linkage to translation.

In the case of P-tubulin, we have now proven that cotrans-lational regulation involves the binding of a cellular factor tothe 1-tubulin nascent peptide, although the identity of thatfactor remains unproven. Interactions involving the nascentpeptide have intriguing parallels in at least two other cases ofgene regulation. During the synthesis of proteins destined fortransport to the endoplasmic reticulum, signal recognitionparticle binding to the nascent peptide can induce a transientarrest of translational elongation until the ribosome isdocked appropriately to the membrane (30). Another exam-ple of involvement of the nascent peptide in the modulationof gene expression is in bacteriophage T4 gene 60, in which50 bases of the RNA are translationally skipped, an eventinferred to require an interaction with the nascent peptide(31). Although in both of these latter examples cotransla-tional events do not expedite mRNA degradation, theynevertheless provide examples of regulation of gene expres-sion mediated by recognition of a nascent peptide.For tubulin autoregulation, the major unresolved ques-

tion, second to the identity of the peptide-binding factor, ishow binding to the nascent peptide is then transduced as asignal for degradation of the mRNA. Clearly, nascent pep-tide binding is not a sufficient stimulus for mRNA degrada-tion, as injection of the antibody inhibits degradation ratherthan accelerates it. Moreover, binding of the signal recogni-tion particle to the nascent peptides of microsome-boundpolysomes does not cause mRNA degradation and mayactually stabilize those mRNAs (21). Further dissection ofthe mechanism of cotranslational degradation of tubulinmRNA thus seems likely to uncover common features of thisgeneral category of cotranslational gene regulation.

ACKNOWLEDGMENTS

We thank Mark Pittenger for advice regarding microinjection,Vicki Saylor and Tim Yen for advice on raising monoclonal anti-bodies, Margaret Lopata for the rabbit polyclonal antitubulin serum,and Doug Murphy for the gift of tubulin. We are also grateful to P.Shenbagamurthi for synthesis of the peptide and advice on peptidechemistry and to the Peterson laboratory for advice on the equilib-rium dialysis assays.These studies were supported by a grant from the National

Institutes of Health to D.W.C. N.G.T. was supported by a postdoc-toral fellowship from the National Institutes of Health.

REFERENCES1. Ben Ze'ev, A., S. R. Farmer, and S. Penman. 1979. Mechanisms

of regulating tubulin synthesis in cultured mammalian cells. Cell17:319-325.

2. Blattler, D. P., F. Garner, K. Van Slyke, and A. Bradley. 1972.Quantitative electrophoresis in polyacrylamide gels of 2-40%. J.Chromatogr. 64:147-155.

3. Blose, S. H., D. I. Meltzer, and J. R. Feramisco. 1984. 10-nmfilaments are induced to collapse in living cells microinjectedwith monoclonal and polyclonal antibodies against tubulin. J.Cell Biol. 98:847-858.

4. Bravo, R., and J. E. Celis. 1980. A search for differentialpolypeptide synthesis throughout the cell cycle of HeLa cells. J.Cell Biol. 84:795-802.

5. Caron, J. M., A. L. Jones, L. B. Ball, and M. W. Kirschner.1985. Autoregulation of tubulin synthesis in enucleated cells.Nature (London) 317:648-651.

6. Cleveland, D. W. 1988. Autoregulated instability of tubulinmRNAs: a novel eukaryotic regulatory mechanism. TrendsBiochem. Sci. 13:339-343.

7. Cleveland, D. W., and J. C. Havercroft. 1983. Is apparent

autoregulatory control of tubulin synthesis nontranscriptionallycontrolled? J. Cell Biol. 97:919-924.

8. Cleveland, D. W., M. A. Lopata, P. Sherline, and M. W.Kirschner. 1981. Unpolymerized tubulin modulates the level oftubulin mRNAs. Cell 25:537-546.

9. Cleveland, D. W., M. F. Pittenger, and J. R. Feramisco. 1983.Elevation of tubulin levels by microinjection suppresses newtubulin synthesis. Nature (London) 305:738-740.

10. Compton, D. A., T. J. Yen, and D. W. Cleveland. 1991.Identification of novel centromere/kinetochore-associated pro-teins using monoclonal antibodies generated against humanmitotic chromosome scaffolds. J. Cell Biol. 112:1083-1097.

11. Crestfield, A. M., S. Moore, and W. H. Stein. 1%3. Thepreparation and enzymatic hydrolysis of reduced and S-car-boxymethylated proteins. J. Biol. Chem. 238:622-627.

12. Gay, D. A., S. S. Sisodia, and D. W. Cleveland. 1989. Autoreg-ulatory control of ,B-tubulin mRNA stability is linked to trans-lational elongation. Proc. Natl. Acad. Sci. USA 86:5763-5767.

13. Gay, D. A., T. J. Yen, J. T. Y. Lau, and D. W. Cleveland. 1987.Sequences that confer 3-tubulin autoregulation through modu-lated mRNA stability reside within exon 1 of a 13-tubulin mRNA.Cell 50:671-679.

14. Graves, R. A., N. B. Pandey, N. Chodchoy, and W. F. Marzluff.1987. Translation is required for regulation of histone mRNAdegradation. Cell 48:615-626.

15. Harlow, E., and D. Lane. 1988. Antibodies: a laboratory man-ual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

16. Hiller, G., and K. Weber. 1978. Radioimmunoassay for tubulin:a quantitative comparison of the tubulin content of differentestablished tissue culture cells and tissues. Cell 14:795-804.

17. Hummel, J. P., and W. J. Dryer. 1962. Measurement of protein-binding phenomena by gel filtration. Biochim. Biophys. Acta63:530-532.

18. Kohler, G., and C. Milstein. 1975. Continuous culture of fusedcells secreting antibody of predefined specificity. Nature (Lon-don) 256:495-497.

19. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

20. Lopata, M. A., and D. W. Cleveland. 1987. In vivo microtubulesare copolymers of available p-tubulin isotypes: localization ofeach of six vertebrate P-tubulin isotypes using polyclonal anti-bodies elicited by synthetic peptide antigens. J. Cell Biol.105:1707-1719.

21. Mason, J. O., G. T. Williams, and M. S. Neuberger. 1988. Thehalf-life of immunoglobulin mRNA increases during B-cell dif-ferentiation: a possible role for targeting to membrane-boundpolysomes. Genes Dev. 2:1003-1011.

22. Mitchison, T. J., and M. W. Kirschner. 1987. Some thoughts onthe partitioning of tubulin between monomer and polymer underconditions of dynamic instability. Cell Biophys. 11:35-55.

23. O'Farrell, P. 1975. High resolution two-dimensional electropho-resis of proteins. J. Biol. Chem. 250:4007-4021.

24. Pachter, J. S., T. J. Yen, and D. W. Cleveland. 1987. Autoreg-ulation of tubulin expression is achieved through specific deg-radation of polysomal tubulin mRNAs. Cell 51:283-292.

25. Parker, R., and A. Jacobsen. 1990. Translation and a 42-nucleotide segment within the mRNA encoded by the yeastMATal gene are involved in promoting rapid mRNA decay.Proc. Natl. Acad. Sci. USA 87:2780-2784.

26. Pittenger, M. F., and D. W. Cleveland. 1985. Retention ofautoregulatory control of tubulin synthesis in cytoplasts: dem-onstration of a cytoplasmic mechanism that regulates the levelof tubulin expression. J. Cell Biol. 101:1941-1952.

27. Shyu, A.-B., M. E. Greenberg, and J. G. Belasco. 1989. Thec-fos transcript is targeted for rapid mRNA decay by twodistinct mRNA degradation pathways. Genes Dev. 3:60-72.

28. Solomon, F., M. Magendantz, and A. Salzman. 1979. Identifica-tion with microtubules of one of the co-assembling microtubule-associated proteins. Cell 18:431-435.

29. Suprenant, K. A., L. B. Tempero, and L. E. Hammer. 1989.Association of ribosomes with in vitro assembled microtubules.

MOL. CELL. BIOL.

REGULATION OF ,B-TUBULIN mRNA DEGRADATION 799

Cell Motil. Cytoskel. 14:401-415.30. Walter, P., and G. Blobel. 1981. Translocation of proteins across

the endoplasmic reticulum. III. Signal recognition particlecauses signal sequence-dependent and site-specific arrest ofchain elongation that is released by microsomal membranes. J.Cell Biol. 91:557-561.

31. Weiss, R. B., W. M. Huang, and D. M. Dunn. 1990. A nascentpeptide is required for ribosomal bypass of the coding gap inbacteriophage T4 gene 60. Cell 62:117-126.

32. Yen, T. J., D. A. Gay, J. S. Pachter, and D. W. Cleveland. 1988.Autoregulated changes in stability of polyribosome-bound 1-tu-bulin mRNAs are specified by the first thirteen translatednucleotides. Mol. Cell. Biol. 8:1224-1235.

33. Yen, T. J., P. S. Machlin, and D. W. Cleveland. 1988. Autoreg-ulated instability of P-tubulin mRNAs by recognition of thenascent amino terminus of 1-tubulin. Nature (London) 334:580-585.

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