analysis of specific mrna destabilization during dictyostelium development · mrna destabilization...

Development 106, 473-481 (1989)Printed in Great Britain © The Company of Biologists Limited 1989

473

Analysis of specific mRNA destabilization during Dictyostelium

development

G. MANGIAROTTI1, S. BULFONE1, R. GIORDA1, P. MORANDINI1, A. CECCARELLI1

and B. D. HAMES2

1Department of Genetics, Biology and Chemistry, University of Turin, Via Santena, 5 bis 10126, Torino, Italy2Department of Biochemistry, University of Leeds, Leeds LS2 9JT, UK

Summary

A number of specific mRNAs are destabilized upondisaggregation of developing Dictyostelium discoideumcells. Analysis of a family of cloned genes indicates thatonly prespore-enriched mRNAs are affected; constitut-ive mRNAs that are expressed throughout developmentand mRNAs that accumulate preferentially in prestalkcells are stable under these conditions. The decay ofsensitive prespore mRNAs can be halted by allowing thecells to reaggregate, indicating that destabilization oc-curs by the progressive selection of individual moleculesrather than on all members of an mRNA subpopulationat the time of disaggregation. Individual molecules of the

sensitive mRNA species remain engaged in proteinsynthesis in the disaggregated cells until selected. De-stabilization of sensitive mRNAs is induced by celldissociation even in the presence of concentrations ofnogalamycin that inhibit RNA synthesis. The reportedprevention of disaggregation-induced mRNA decay byactinomycin D and daunomycin is therefore probably asecondary effect unrelated to the inhibition of transcrip-tion.

Key words: mRNA stability, Dictyostelium, generegulation.

Introduction

Dictyostelium discoideum is an increasingly popularorganism for analysing the regulation of gene ex-pression during cell differentiation. When deprived ofnutrients and deposited on a solid substratum, theamoebae aggregate over a period of 8-10 h to form tightmulticellular complexes. After forming a tip, eachaggregate transforms into a migrating pseudoplas-modium referred to as a grex or slug. At this stage, thepresence of two cell types is already apparent; presporeand prestalk cells. Later in development, during culmi-nation, final differentiation of these cells takes place togive rise to a mass of mature spore cells on top of aslender pillar of vacuolated stalk cells.

Many hundred (Jacquet et al. 1981) and possiblyseveral thousand (Blumberg & Lodish, 1980) newmRNA species are expressed during development,some of which accumulate preferentially in prespore orprestalk cells (Alton & Brenner, 1979; Morrissey et al.1981; Barklis & Lodish, 1983; Borth & Ratner, 1983;Mehdy et al. 1983; Morrissey et al. 1984). Dissociationof cells in developing aggregates leads to the cessationof expression of many late genes and degradation of themRNA already transcribed (Newell et al. 1971, 1972;Alton & Lodish, 1977; Landfear & Lodish, 1980; Chung

etal. 1981; Mangiarotti et al. 1982, 1983; Landfear etal.1982; Mehdy et al. 1983; Bozzaro et al. 1984). Barklis &Lodish (1983) and Chisholm etal. (1984) have suggestedthat the genes sensitive to cell dissociation are thoseexpressed for the first time in the aggregation orpostaggregation stages and can be either prespore orprestalk enriched, while prestalk-enriched genes, in-itially expressed prior to aggregation, and genes ex-pressed constitutively in growth and development(Barklis & Lodish, 1983; Mehdy et al. 1983) areinsensitive to dissociation.

We are interested in elucidating the mechanisms thatcontrol developmental gene expression in Dictyo-stelium. Here we analyse the phenomenon of specificmRNA destabilization and its relationship to the multi-cellular state. The data presented suggest that thestability of mRNAs expressed constitutively throughoutdevelopment or expressed preferentially in prestalkcells is unaffected by cell dissociation. In contrast, allprespore mRNAs examined are rapidly destabilized.We also present evidence concerning the mechanism ofdestabilization. Our studies indicate that disaggregationinduces the instability of sensitive mRNAs in a pro-gressive manner, one by one, rather than affecting theentire mRNA population at the time of cell dis-sociation; that sensitive mRNAs continue to engage in

474 G. Mangiarotti and others

protein synthesis in disaggregated cells until selected forrapid decay; and that, contrary to a recent report(Amara & Lodish, 1987), destabilization is not depen-dent upon RNA synthesis.

Materials and methods

Cell culture conditionsD. discoideum AX2 was grown in HL5 medium as describedelsewhere (Watts & Ashworth, 1970). Cells were harvestedduring exponential growth at a density of 4xlO6 cells ml"1,washed twice with distilled water, once with PDF (Mangiar-otti etal. 1982) and resuspended in PDF at 1-5X108 cells ml"1.Cells were plated for development at a density oflxlO7 cells cm"2 on Whatman 50 filter papers each resting ontwo Whatman 3 filter pads just saturated with PDF. Inexperiments involving radiolabelling with [32P]orthophos-phate, PDF was substituted by MES-PDF (Mangiarotti et al.1982).

Monitoring mRNA decay by dot blot analysisCells plated on filters in MES-PDF were labelled with[32P]orthophosphate (lOmCi per 5X108 cells) between13-17 h of development. After labelling, the cells wereharvested from the filters, washed in PDF by centrifugation(2500 g, 5min), resuspended in 5 ml PDF containing 10 mM-EDTA and dispersed by vortex mixing. The dispersed cellswere then diluted with PDF-EDTA to SxK^cellsml"1 andshaken at 230revsmin~1 at 22°C. At intervals (see Results),cells were harvested by centrifugation. Poly (A)+ RNA wasisolated from each sample (Mangiarotti et al. 1980) andhybridized to DNA dot blots (Mangiarotti et al. 1982). Eachdot contained 2 y.g DNA of selected clones (as indicated in theResults sections) immobilized on nitrocellulose.

Monitoring mRNA decay using nogalamycinFilters bearing cells at the 13 h stage of development weretransferred to pads containing 200 fig ml"1 nogalamycin inPDF. Another batch of cells at the same stage of developmentwas disaggregated as described above but in PDF-EDTAcontaining 200 fig ml"1 nogalamycin and then maintained inshaking suspension (230 revs min"1). At intervals, cells wereharvested from both the filters and the disaggregated cellsuspension and total RNA was isolated as described byWilliams et al. (1987). After electrophoresis on 1-5 % agarosegels in 20 mM-phosphate buffer, (pH7-0) containing 6%formaldehyde and capillary transfer to Hybond N (Amer-sham), the Northern blots were hybridized with DNA pre-pared from clones SC79, D19, EB4 or Per97 previouslylabelled by nick translation (Rigby et al. 1977).

Effect of nogalamycin on RNA synthesis in developingand disaggregated cellsD. discoideum cells were allowed to develop on filters to the15 h stage of development. One batch of filters was thentransferred to pads saturated with 200/igml"1 nogalamycin(Ennis, 1981) in PDF whilst a second (control) batch wastransferred to filters saturated with PDF alone. After 5min,100/xCi [3H]uracil was added by spotting uniformly onto eachfilter using a syringe. Other cells at 15 h of development wereharvested and disaggregated in PDF-EDTA as describedabove. The cell suspension was divided into two halves, toonly one of which was added nogalamycin at 200 ̂ gml"1.After 5 min, 100 /jCi [3H]uracil was added to each suspension.At intervals, cell samples were harvested from the two sets of

filters and the two sets of suspension cultures and total RNAwas isolated (Mangiarotti et al. 1980). For each RNA sample,one sample was treated with 1/ig pancreatic RNAse for30min at room temperature, then precipitated with ice-cold10% TCA and TCA-insoluble radioactivity determined byliquid scintillation counting. Another sample was TCA preci-pitated and counted without RNase digestion. Control RNAsamples treated with RNase contained negligible counts. Thusthe radioactivity of samples not treated with RNase was usedas a direct measure of RNA radioactivity.

Monitoring mRNA decay after disaggregation andreaggregationCells at the 17 h stage of development were dissociated asdescribed above and then maintained in shaking suspension.After 30min incubation, the cell suspension was divided intothree fractions. One fraction was kept shaking without anyfurther addition. To a second fraction of cells was addednogalamycin to 200/igml"1 and then shaking continued. Thethird fraction of cells was collected by centrifugation and thenreplated on filters at 107 cells cm"2 in the presence of200jUgml~1 nogalamycin. At intervals during this protocol asdescribed in Results, cell samples were harvested from allthree fractions and total RNA was extracted (Williams et al.1987). After electrophoresis of 10/ig of each RNA on 1-5%agarose gels and blotting into Hybond N, the Northern blotswere hybridized with selected cloned DNAs previouslylabelled by nick translation (see Results).

The effect of actinomycin D and daunomycin onmRNA decayCells allowed to develop to the 15 h stage of developmentwere dispersed into PDF-EDTA shaking suspension cultureas described above. The suspension culture was then dividedinto two halves. Shaking of one fraction was continuedwithout further addition whilst to the other actinomycin Dand daunomycin were added to 125/igml"1 and 250 jig ml"1

final concentrations respectively and then shaking continued.Total RNA was isolated from cell samples taken at intervalsduring the incubation of each culture and analysed byNorthern blot analysis as described above.

Intracellular localization of mRNA in developing anddisaggregated cellsCells at the 15 h stage of development on filters or disaggre-gated at this time and kept in shaking suspension in PDF-EDTA for 30 min (see above) were lysed and centrifuged on15-30 % sucrose gradients as described by Mangiarotti et al.(1981). After centrifugation, gradients were analysed bycontinuous monitoring at 260 nm. Fractions corresponding to80S ribosomes and lighter material, polyribosomes up to 6-7ribosomes per mRNA and polyribosomes more than 6-7ribosomes per mRNA were separately pooled. Total RNAwas extracted (Williams et al. 1987) from each pooled sampleand analysed by Northern blotting with labelled cloned DNAas described above.

Results

Only prespore-enriched mRNAs are destabilized upondisaggregationEarlier studies have concluded that mRNAs that ac-cumulate preferentially in either prespore or prestalkcells during the aggregation and postaggregation stagesbecome destabilized and are rapidly degraded when the

mRNA destabilization in Dictyostelium 475

cells are disaggregated (Mehdy et al. 1983; Barklis &Lodish, 1983). In contrast, constitutive mRNAs presentin both cell types throughout growth and developmentare unaffected by cell disaggTegation. Since earlierstudies used relatively few clones to reach these con-clusions, we have repeated the analysis using a largebank of clones which are now available. D. discoideumAX2 cells were allowed to develop for 13 h and thenlabelled with 32P for 4h. At 17h of development, thecells were dissociated and shaken fast in PDF-EDTAbuffer to maintain them as single cells. At intervals,samples were taken and used to prepare poly (A)+

RNA which was then hybridized to DNA dot blots

Constitutive

SC79

SC29 « • •*' • •

corresponding to a large panel of clones. Some of theseresults are given in Fig. 1. The data show that severalconstitutive mRNAs, SC79, SC29 and CZ22 (Chung etal. 1981; Barklis & Lodish, 1983; Mangiarotti et al.1983), and a developmental^ regulated non-cell-type-specific mRNA, D15 (Barklis & Lodish, 1983), areindeed stable for the time course of the experiment.Surprisingly, so are five mRNAs that accumulate pref-erentially in prestalk cells; Dll, D14 (Barklis & Lodish,1983), Per79 (E. Barklis; personal communication), 5Dand 5G (Corney et al. 1989). In contrast, the largenumber of prespore-enriched mRNAs examined [D18,D19, EB4 (Barklis & Lodish, 1983); Per97 (E. Barklis,

Prespore

D18 • TP • •

D19 # , ' # %.

CZ22 A3

PER 97

Non-cell-type

D15

EB4 * *

GM55b 9 * •

Prestalk GM27 •

D14

D11

1A

3B

PER79 • • • «

5D •

5G#

2D

f H •Fig. 1. The decay of specific mRNAs following disaggregation. Cells were labelled between 13h and 17 h of development,then disaggregated and shaken fast in suspension culture to maintain them as single cells. Poly (A)+ RNA was isolated atvarious times and then hybridized to dot blots of DNA (2/ig per dot) from constitutive, non-cell-type-specific, prestalk andprespore clones as indicated. The dots for each clone, left to right, represent hybridization to the poly(A)+ RNA isolated at0, 30, 60, 90 and 120 min of incubation in shaking suspension culture.


personal communication); GM55b, GM27 (Mangiarottietal. 1980); A3 (Mangiarotti et al. 1982, 1983); 1A, 3B,2C, 2D, 4D, 2H (Corney et al. 1989)] are degraded upondisaggregation. The rate of degradation is specific foreach prespore mRNA, some mRNAs decaying with ahalf-life of 30min or less. In addition to the clonesindicated in Fig. 1, constitutive mRNAs CZ5 and CZ12(Mangiarotti et al. 1982, 1983) and prestalk mRNAs Bl(Mangiarotti etal. 1982) and Per74 (R. Giorda; unpub-lished observations) were also examined and found tobe stable to disaggregation whilst prespore mRNAsEB5 (E. Barklis, personal communication) 6C, IF, 3F,7F, 1H and II (Corney et al. 1989) were all unstable(data not shown). Thus, of the mRNAs we haveexamined, the only class that are unstable upon disag-gregation are those which accumulate preferentially inprespore cells during development.

Prespore destabilization is reversed by reaggregationIf cells dispersed from tight aggregates and kept insuspension for 30 min (a time period sufficient to halvethe amount of prespore mRNAs) are replated on filtersat a concentration of 107 cells cm"2, they reaggregate toform visible mounds in less than 15 min. In principle itshould be possible to monitor the effects of reaggrega-tion upon mRNA stability by the 32P labelling protocolused in Fig. 1. However, in practice, we were unable toobtain sufficient incorporation of 32P into poly(A)+

RNA to allow this experimental approach to succeed.We therefore chose the alternative approach of follow-ing the decay of unlabelled mRNAs by Northern blotanalysis. For this protocol, it is necessary to inhibitfurther mRNA synthesis. The antibiotic nogalamycin isideal for this purpose; at 200figmV1 nogalamycin,RNA synthesis both by cells developing normally on

30Time (min)

60

Fig. 2. Inhibition of RNA synthesis by nogalamycin. Cellsat 15 h of development were either allowed to continuedevelopment unperturbed (•) or in the presence of200^gmP1 nogalamycin (O). Other cells weredisaggregated at 15 h and placed in suspension culture. Onehalf of the suspension was shaken in PDF (A) and theother in PDF containing 200/igml"1 nogalamycin (A).[3H]uracil was added to each cell fraction and samplestaken at the time points shown. The data represent [3H]radioactivity in TCA-insoluble material.

filters and by disaggregated cells incubated in shakingsuspension is inhibited greater than 98% (Fig. 2).Furthermore, nogalamycin does not alter the rate ofdecay of mRNA in disaggregated cells (see Fig. 3below) and so has no effect on the intrinsic stability ofthese mRNAs.

To examine the effects of reaggregation on mRNAstability, cells allowed to develop for 17 h on filters weredissociated and maintained in suspension for 30min.The suspension was then divided into three fractions.

B C

0 1 2 3 4 1 2 3 4 1 2 3 4

SC79

D19

A3

GM55b

Fig. 3. mRNA stabilization on reaggTegation. Cells at 17 h of development were disaggregated, kept in suspension for30 min, and then divided into three fractions. Fraction A was kept shaking without further addition and fraction B wasshaken after the addition of nogalamycin to 200 figmP1. Cells from fraction C were collected by centrifugation and replatedon filters in the presence of 200 jigmF1 nogalamycin. Sample 0 was taken at the time of cell disaggregation. For each offractions A, B and C, samples were also taken at the time of replating (sample 1), and then at 30 min (sample 2), 60 min(sample 3) and 90 min (sample 4) later. Northern blots of RNA isolated from each sample and probed with SC79, D19, A3and GM55b [32P] DNA are shown.


One fraction (Fig. 3A) was kept shaking withoutfurther manipulations. To a second fraction (Fig. 3B)was added 200/igmJ~1 nogalamycin and shaking wasthen continued. Finally, cells from the third fraction(Fig. 3C) were harvested by centrifugation and replatedon niters at 107 cells cm"2 in the presence of 200 pig ml"1

nogalamycin. Samples were taken at intervals and poly(A)+ RNA extracted from each was examined byNorthern blot analysis using a labelled DNA probefrom SC79 (a constitutive mRNA), and prespore D19,A3 and GM55b probes. The constitutive mRNA, SC79,was stable under the three conditions tested and there-fore not affected by either disaggTegation or reaggre-gation of the cells. In contrast, the three presporemRNAs decayed rapidly upon disaggregation, a resultin keeping with the data of Fig. 1. The presence ofnogalamycin did not affect this rapid decay (comparefractions A and B in Fig. 3). However, upon reaggrega-tion, the prespore mRNAs D19, A3 and GM55b whichhad not yet decayed remained undegraded over thenext 90 min. Reaggregation therefore stabilized the

residual prespore mRNA against degradation, that is,the disaggregation-induced decay of prespore mRNA isreversible.

Destabilized prespore mRNAs remain inpolyribosomesThe data reported in Fig. 3 suggest that although theaverage half-life of a given species of mRNA is greatlyreduced in disaggregated cells, each mRNA moleculeremains unaltered until a sudden event leads to its rapiddegradation. In agreement with this interpretation, wefind that prespore mRNAs known to be sensitive to celldisaggregation remain in polyribosomes during decay indissociated cells. The evidence for this is shown inFig. 4. Lysates of cells allowed to develop normally onfilters for 17 h or disaggregated at this time and shakenin suspension for 30 min were centrifuged on sucrosedensity gradients (Fig. 4A). Fractions corresponding tolarge polyribosomes (L; over 6-7 ribosomes permRNA), small polyribosomes (S; 6-7 ribosomes per

M

EcoCM

Development

Disaggregatedcells

Bottom Top

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U)

0)Q

T IS L S M

SC79 D19

(A

O

/>

L S M L S M1 rL S M"L S M

I. I; ' j'

EB4 PER97

BFig. 4. Sensitive mRNA remains in polyribosomes in disaggregated cells. (A) Polysome profiles from cells at the 15 h stageof development or disaggregated at this stage and maintained in shaking suspension for 30 min are shown. (B) RNAextracted from the three regions of the gradients indicated in A were analysed by Northern hybridization with DNA fromthe indicated clones. In the case of Northerns to be hybridized with D19, EB4 and Per97 probes, the loading for the(Disagg) samples was approx. fourfold greater than for the (Dev.) samples to compensate for mRNA decay. Both in normaldeveloping cells (Dev.) and disaggregated (Disagg.) cells the RNAs are found associated with large (L; greater than 6-7ribosomes per mRNA) and small (S; up to 6-7 ribosomes per mRNA) polysomes. A negligible amount is present in 80Smonosomes (M) and lighter material.


Development

BDisaggregated

cells

SC79

D19

EB4

PER97

0 2 4 6 8

Hours30 60 90 120

Minutes

Fig. 5. Comparison of mRNA decay in developingand disaggregated cells. (A) Nogalamycin (finalconcentration 200 ng ml ) was added to cells onniters at the 15 h stage of development. RNA fromcells harvested at the indicated times was thenanalysed by Northern blot hybridization to DNAfrom clone SC79 (constitutive) and clones D19, EB4and Per97 (prespore-enriched). The slight increasein EB4 mRNA at 2h may reflect incompleteinhibition of transcription of this particular mRNAin this experiment. (B) Cells at 15 h of developmentwere disaggregated and kept in shaking suspensionin the presence of 200/igmP1 nogalamycin. RNAfrom cells harvested at the indicated times wasanalysed by Northern blot hybridization as describedin A.

mRNA), single 80S monosomes and lighter material(M) were pooled for RNA extraction. The RNAs wereanalysed by Northern blot hybridization with cloneSC79 (constitutive) and EB4, D19 and Per97 (presporeenriched). Both in normal developing cells and in cellsdisaggregated and maintained in suspension for 30min,all four species of mRNA are found entirely in thepolyribosome fraction (Fig. 4B). This argues that for agiven population of mRNAs destabilized by disaggre-gation, individual mRNAs remain functional in proteinsynthesis until they are suddenly degraded.

The use of nogalamycin verifies that disaggregationdramatically reduces prespore mRNA half-livesPrevious studies have used different experimental pro-tocols to measure mRNA half lives in normal develop-ment and after disaggregation. The conclusion thatdisaggregation leads to mRNA destabilization hasnever been verified using the same measurement pro-cedure on both sets of cells. Since nogalamycin stronglyinhibits RNA synthesis in both developing and disag-gregated cells (Fig. 2) without inhibiting mRNA decay(Fig. 3), we have been able to use this drug to comparemRNA half-lives directly in each experimental situ-ation. Fig. 5 follows the decay of several mRNAs byNorthern blot analysis after the addition of nogalamy-cin to cells allowed to develop normally on filters for13 h (Fig. 5A) and to cells disaggregated at this timeand maintained in shaking suspension (Fig. 5B). Thehalf-lives of the mRNAs examined in normal develop-ing cells range from 4-6 h, while in disaggregated cellsthey fall to about 30 min for the prespore enriched D19,EB4 and Per97 mRNAs. The constitutive SC79 mRNA(Fig. 5B) and prestalk-enriched 5G mRNA (data notshown) remain relatively unaffected by the disaggre-gation.

RNA synthesis is probably not required for mRNAdestabilizationNogalamycin does not affect the decay of prespore A3,D19, EB4, GM55b or Per97 mRNAs upon disaggre-gation (Figs 3 and 5) despite the fact that it inhibitsRNA synthesis by disaggregated cells essentially com-pletely (Fig. 2), strongly suggesting the RNA synthesisis not required for mRNA destabilization.

This result is in direct contrast to the conclusions ofAmara & Lodish (1987) that blocking RNA synthesisdoes block the destabilization of prespore mRNAs.These workers used actinomycin D and daunomycin toinhibit RNA synthesis rather than nogalamycin. Sincethe phenomenon may be prespore mRNA-specific, it isimportant to examine the decay response of the sameprespore mRNAs to each of these drug regimes. Fig. 6follows the effect of actinomycin D and daunomycin onthe disaggregation-induced decay of four presporemRNAs (D19, EB4, Per97 and A3) which we havealready examined using nogalamycin (Figs 3 and 5).Cells were allowed to develop on filters for 15 h, thendissociated and maintained in suspension in the absence(Fig. 6A) or presence (Fig. 6B) of 125/xgml~1 actino-mycin D and 250/zgmP daunomycin, the concen-trations previously employed by Amara & Lodish(1987). Total RNA extracted from samples taken atintervals during the incubation were analysed by North-ern blot hybridization. Whereas the prespore-enrichedD19, EB4, Per97 and A3 mRNAs decayed upon incu-bation of disaggregated cells in buffer alone asexpected, none of these mRNAs decayed in the pres-ence of the antibiotic mixture. Since nogalamycinappears to inhibit RNA synthesis completely yet notaffect the decay of the D19, EB4 and Per97 mRNAs(Fig. 5), it seems probable that RNA synthesis is notrequired for mRNA destabilization and that the blockin decay caused by actinomycin D and daunomycin is a

to

3

I

C C

II'Z 3O CO<0 ^

D19

0 30 60 0 30 60

MinutesFig. 6. Effect of actinomycin and daunomycin on mRNAdecay in disaggregated cells. Cells at 15 h of developmentwere disaggregated and maintained in shaking suspension inPDF without drugs or in PDF-EDTA containing125 n% ml"1 actinomycin D and 250/igml"1 daunomycin.Total RNA from samples taken at the indicated times wasanalysed by Northern blot hybridization to DNA fromclones D19, EB4, Per97 and A3.

secondary effect. Alternatively, however, it is conceiv-able that a small subclass of RNAs exist whose synthesisis required for destabilization and that only actinomycinD and daunomycin inhibit their synthesis.

Discussion

The first indication that dissociation of Dictyosteliumcell aggregates leads to destabilization of a class ofmRNAs was obtained by Chung et al. (1981). Theycompared the average rate of decay of total mRNA innormal developing cells, treated with actinomycin Dand daunomycin to block transcription, to the decay ofboth total mRNA and a single species of mRNA indisaggregated cells in the absence of drugs. Since, as weshow here and has been reported elsewhere (Amara &Lodish, 1987), actinomycin and daunomycin stabilizemRNA in disaggregated cells, one cannot exclude thepossibility that these drugs have a similar effect onnormal developing cells. As an alternative approach,we studied the incorporation of 32P into specificmRNAs during development and the decay of the samelabelled mRNAs in disaggregated cells (Mangiarotti etal. 1982). Although it was not possible to obtain anaccurate measure of mRNA half-life in the developingcells, from our data it was apparent that cell dissociation


destabilized a class of mRNAs by at least a factor of 5.This conclusion was questioned by Casey et al. (1983).From a similar analysis on total polyadenylated mRNA,their study found that most mRNA is relatively stableand a minor fraction is unstable both in aggregated anddisaggregated cells. They suggested that the latterfraction might include the specific mRNAs studied by usand that we may have overestimated their stability innormal developing cell aggregates.

In a more recent study, Manrow & Jacobson (1988)used a 32P pulse-chase protocol to analyse the rate ofdecay of individual mRNA species both in aggregatedcells and in cells disaggregated in the presence andabsence of cAMP. During the chase in aggregated cells,the amount of 32P label decreased slowly in some of themRNA studied while it remained constant or evenincreased in other mRNAs. In cells disaggregatedwithout cAMP, the amount of 32P label decreased veryslowly in constitutive and non-cell-type-specificmRNAs but decayed with half-lives of 20 to 30 min incell-type-specific mRNAs, at least during the first1-1-5 h after disaggregation. These results are quanti-tatively similar to those previously reported by us(Mangiarotti et al. 1982). However, since Manrow &Jacobson (1988) accept that their 32P chase was notcompletely effective in aggregated cells, it is difficult toquantify the effect of cell disaggregation on mRNAstability on the basis of their data. Nevertheless, Man-row & Jacobson (1988) conclude that cell-type-specificmRNAs do indeed undergo 'a short-term labilization'upon disaggregation in the absence of cAMP, in agree-ment with the data presented by us here and elsewhere(Mangiarotti et al. 1982, 1983, 1985). It is thereforecurious and apparently contradictory that these authorsalso state that the decrease in mRNA abundance upondisaggregation reflects their 'inherently fast normalrates of decay'.

Manrow & Jacobson (1988) also question our earlierfinding that cAMP stabilizes cell-type-specific mRNAsin disaggregated cells, a conclusion we based not onlyon pulse-chase data but also on studies using approachto steady-state labelling (Mangiarotti et al. 1983, 1985).In fact, the quantitative effect of cAMP on mRNAstability is difficult to evaluate from their data since theeffectiveness of the chase varied between aggregatedand disaggregated cells and some prespore mRNAsexhibited first order decay in the disaggregated cells inthe presence of cAMP but biphasic decay in the absenceof cAMP. Nevertheless, all the prespore mRNAs testeddecayed at a substantially lower rate in the 2 hourperiod following disaggregation when incubated in thepresence of cAMP than in its absence. The heterogen-eity of the kinetics of mRNA decay observed byManrow & Jacobson (1988) may indicate that the effectof cAMP on mRNA stability varies with differentmRNAs.

As we show in this paper, the uncertainties inherentin previous radiolabelling studies of mRNA decay canbe eliminated by the use of nogalamycin, which has noeffect upon the half-lives of mRNA in disaggregatedcells. Since the mRNA species that we have studied are


in the same physical state, undergoing translation inpolysomes, both in aggregated and disaggregated cells,it is very unlikely that mRNA stability is altered by thesame drug in aggregated cells. Nogalamycin can there-fore be used to measure the effect of cell disaggregationon mRNA half-life directly. From the data reportedhere, it appears that our previous estimates (as well asthose of Chung et al. 1981) were correct; the half-life ofthe prespore mRNAs examined decreases from 4-6 h innormal developing cells to 20-30 min upon disaggre-gation. It is interesting that if disaggregated cells areallowed to reaggregate after the rapid decay of presporemRNAs has already begun, the decay is promptlyhalted. This observation strengthens the relationshipbetween the multicellular state and mRNA stability.These data are also in line with our previous report thatsome mRNAs are transcribed from the beginning ofdevelopment but do not accumulate in the cell becausethey are unstable. Their stability increases and theybegin to accumulate at the aggregation stage (Mangiar-otti etal. 1985).

The fact that reaggregation promptly halts anyfurther rapid decay of sensitive prespore mRNAs pro-vides some insight into the mechanisms involved. Thedata rule out the possibility that upon disaggregation allmolecules of a sensitive mRNA species are modifiedirreversibly, for example by shortening of the 3' poly(A) tail or removal of 5' terminal sequences, and thatthis then eventually leads to degradation of all of themRNA molecules at a common rapid rate. Rather, itseems likely that whatever factors cause the preferentialdegradation of this class of mRNA, they act on themRNA molecules at random, quickly causing thedegradation of each molecule soon after it is selected.By this model and taking into account molecules not yetselected, the degradation event itself must be extremelyrapid, yielding an mRNA half-life substantially lessthan the 20-30 min measured for that mRNA popu-lation as a whole. This model of disaggregation-induceddecay is in line with our other finding that all moleculesof sensitive mRNA left at a given time in disaggregatedcells are associated with polyribosomes, presumablystill functioning in protein synthesis.

The nature of the event that selects individualmRNAs for rapid degradation is still unknown. Ournogalamycin data indicate that RNA synthesis is notrequired to trigger this event. If this is correct, theblocking action of actinomycin D and daunomycin mustbe a secondary effect due to the action of these drugs onaspects of cell metabolism other than transcription. Wecannot exclude the remote possibility that nogalamycinmay fail to inhibit the synthesis of a minor species ofRNA, essential for induction of mRNA decay, which isinhibited by actinomycin D and daunomycin. However,it is difficult to envisage how RNA synthesis can besufficiently rapid to play a major role in inducing thedestabilization of sensitive mRNAs, which begins only10 min after disaggregation (Amara & Lodish, 1987).

One way in which destabilization of sensitive mRNAsmight occur is via their exclusion from polyribosomes.In this case, the primary control would be at the level of

mRNA translation rather than mRNA stability per se.This possibility is not excluded by our data which showonly that, if such an event occurs, it does not involve allmolecules of sensitive mRNA at the time of celldispersion, but must occur progressively. Cyclohexi-mide reduces the rate of mRNA decay upon celldisaggregation (Amara & Lodish, 1987; P. Morandini,G. Mangiarotti and A. Ceccarelli, manuscript in prep-aration) suggesting either that the translation of par-ticular pre-existing mRNA is necessary to induce thedecay of sensitive prespore mRNAs or that the move-ment of ribosomes along the sensitive mRNAs them-selves is required for destabilization. Interestingly, thedegradation of histone mRNA is also prevented byinhibiting protein synthesis (Sive et al. 1984; Stimac etal. 1984) and has been shown to depend on thetranslatability of the histone mRNA itself (Sive et al.1984). Similarly, destabilization of tubulin mRNAdepends upon recognition of the first four amino acidsof cv-tubulin as they emerge from the ribosome (Yen etal. 1988). However, the role of translation in Dictyo-stelium mRNA decay is stiil unclear. Thus puromycin,which inhibits protein synthesis in Dictyostelium cellsalmost as well as cycloheximide (Amara & Lodish,1987), and canavanine, [an arginine analogue whichleads to the synthesis of faulty proteins (P. Morandini,G. Mangiarotti and A. Ceccarelli, manuscript in prep-aration] fail to prevent the decay of sensitive Dictyo-stelium mRNAs upon disaggregation in contrast to theeffect of cycloheximide. The demonstration that duringdisaggregation the residual mRNA is still associatedwith polysomes is in contrast to the data of Steel &Jacobson (1987) on ribosomal protein mRNA early indevelopment and so probably represents a differentmechanism of post-transcriptional control.

Mehdy et al. (1983) have asserted that disaggregationleads to rapid loss of both prespore- and prestalk-enriched mRNAs although one of the two prestalkmRNAs examined, 16-G1 (pst-cath), seemed fairlystable. Manrow & Jacobson (1988) also found that threeprestalk mRNAs were destabilized upon disaggre-gation. Barklis & Lodish (1983) and Chishoim et al.(1984) noted that prestalk-enriched mRNAs fell intotwo distinct classes. Prestalk I mRNAs (e.g. Cl, Dlland D14) are already expressed in preaggregation cellsand are fairly stable upon cell disaggregation whereasprestalk II mRNAs (e.g. Al and PL1) are expressedonly later in development and become unstable upondisaggregation. Of the clones examined in the presentstudy, the major characteristic that appears to correlatewith mRNA sensitivity to disaggregation is cell-speci-ficity, not the time of expression. Thus some of theprestalk-enriched clones (Per74, Per79, 5D) are lategenes (Giorda, R. & Mangiarotti, G., unpublishedobservations; Corney et al. 1989) but their stability islargely unaffected by disaggregation. In contrast, all theprespore-enriched mRNAs examined were sensitive tocell dissociation. How general the correlation betweencell specificity and sensitivity to disaggregation is wecannot say. However, we would suggest that constitut-ive and many prestalk-enriched genes may be con-


trolled only at the level of transcription whereas pre-spore cells appear to have developed a second majortype of control at the post-transcriptional level for allgenes expressed preferentially in this cell type. At-tempts to analyse this latter regulation using in vitrosystems are now in progress.

This work was supported by funds from Italian CNR(Progetto finalizzato di Ingneria Genetica e Gruppo Nazio-nale di Biologia Molecolare, Cellulare e Evolutiva) andM.P.I. We thank E. Barklis for the generous gift of clonesEB4, Per79 and Per97.

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(Accepted 13 April 1989)

analysis of specific mrna destabilization during dictyostelium development · mrna destabilization...

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