./. Nol.

Rid.

(1979) 128. 37 l-395

Fractionation and Functional Analysis of Newly Synthesized Decaying Messenger RNAs from Vegetative Cells of

and

Die tyos telium discoideum CARL MATHEW

PALATNIK~.

ROBERT

V. STORT?

AND ALLAS

.IACOBSOS'

l &pm-tment of Microbioloyy CJtGversity of Massachusetts Medical School Worcester, Mass. 016&j, U.S.A.

2 f~epurtment of Biological C?wn istr!y University of Illinois at the Medical Center Chicago, Ill. 60612, U.X.A. (Received 10 Ma~q IY78, and in revised form

29 September 1978)

\Ve 11i~ve used thermal elntion from poly(U)-Sepharose to separate RN9 from l)ictyostetiwna discoideum into several fractions which differ in their respectivtx poly(A) sizes. We have shown that most newly synthesized poly(b)-containing RNA from vegetative cells of t.his organism contains long poly(A) tracts which sllorten with age, and that these new transcripts can be purified at least tenfold, and perhaps as much as 60-fold, from other cellular messenger RNAs by t.his tt&rlique. We have isolated newly synthesized mRNA and mRN12s of differerlt @y(A) contents and analyzed their translntiorl ttct,ivitics und translation product.s using mKNA-dependent wIleat germ atld rcticulocyt,e lysates and twodimensional gel c,lootropl~orc:sis. Our results demonstrate that translatable RNAs HI’~ not distributed equally amotlpst RNAs of diff+sretlt poly(A) contents; some’ appetzr t,o he relatixrely more abundant in newly synthesized mKKA than in RNAs containing shorter poly(r2) tracts, While others appear to be less abundant,. A comparison of the translation products of newly syntllesized poly(A)-cont,aining RN-A with those of ot.her RNA frttctiorls has led us to suggest that mRNA synthesis is pre-emillent in establishing the frequency distribution of mRNAs in vegettttivr cells of this organism, and that additional minor adjustments are made by differential stabilities. We also have shown that poly(A)-minus RNA cells of this organism codes for only a small number of majot from vegeta.tive proteins. Since sllortening of poly(A) with age: is u common occurrence in cells of higher organisms, t.hermal elution from polylU)-Sepl-Iarose could be a general13 applicable t~echniquc to IKP for eltriching for rnRNBs irtdrtcctl by alterations in tlovf,l[)I)rrlcntal or metitholir states its \vcll as for stlltlyirrg ellkaryotic mRN.4 ux~tabolisln.

1. Introduction -4lthough it. has been several years since the discovery of poly(A) sequences in eukaryotic messenger RNA, the functional significance of these sequences remains to be determined. In spite of this, the presence of poly(A) tract,s on mRNA has greatly facilitated the isolation of eukaryotic mRXA and the study of mRNA metabolism in eukaryotic cells (for reviews see Greenberg, 1975: Molloy $ Puckett, 1976).

372

C. M. PALATNIK,

R. V.

STORTI

AND

A. JACOBSON

Over the past several years a great deal of information has accumulated on mRNA metabolism in Dictyostelium discoideum (for a review see Firtel & Jacobson, 1977). TWO classes of poly(A) sequences are found on Dictyosteliuvn mRNA: a short, transcribed oligo(A),, sequence, and a large, post-transcriptionally added, poly(A) tract. Most mRNAs contain one mole of each class of poly(A). A major fraction of nuclear poly(A)-containing RNA is transported to the cytoplasm and over 90% of t,his material becomes associated with polysomes. The size of the post-transcriptionally added poly(A) sequence in cytoplasmic RNA is initially indistinguishable from that of nuclear poly(A). Wit’hin a short period of time, however, the length of the poly(A) tract begins to shorten and, as we shall later demonstrate, it reaches a steady-state level of about 60 to 65 nucleotides by 6.5 hours. This phenomenon has been well documented in mammalian systems (Sheiness & Darnell, 1973) and may be a general feature of eukaryotic mRNA metabolism. The stability of vegetative mRNA has been measured by a variety of methods and shows first-order decay kinetics with a half-life of 3.5 to 4.0 hours. Most of these conclusions have been drawn from studies involving relatively unfractionated preparations of RNA. While these studies have been valuable, it remains to be determined whether all mRNAs have identical half-lives and whether all mRNAs are polyadenylated and processed in the same way. It is also of paramount importance to study the transcription and metabolism of individual mRNAs, in particular those which are transcribed at specific stages of development in this organism. In this paper, we describe a method for fractionating mRNA on the basis of differences in poly(A) content. We have been using this method to enrich for, and to study, newly synthesized mRNAs and mRNAs of different poly(A) contents, init was originally cluding those devoid of poly(A) (poly(A)- minus RNA). Although believed that all mRNAs, except histone mRNAs (Adesnik et al., 1972; Greenberg & Perry, 1972), were synthesized with long, post-transcriptionally added poly(A) tracts, this belief was questioned by Milcarek et el. (1974) and Nemer et al. (1974), who suggested that a substantial and specific fraction of mRNA in mammalian cells and sea urchin embryos lacked poly(A) tracts. As pointed out by Wilt (1977), however, for the sea urchin studies, no complexity mea,surements of the putative poly(A)-minus mRNAs were made, making it impossible to estimate the number of different gene products specifically lacking poly(A). R ecently, Sonenshein et al. (1976) showed that a poly(A)-minus fraction from mammalian cells was enriched for the translation activity of a protein which had the same molecular weight as actin. Similar results were reported by Hunter & Garrels (1977) and by Kaufmann et al. (1977), and both groups confirmed that the protein was indeed actin. Kaufmann et al. (1977), however. showed that their poly(A)-minus fraction was not as unique as they had originally suspected (Milcarek et al., 1974). Most of the proteins synthesized by this fraction fraction. In Dictyosteliun~, ea’rly could also be detected in their poly(A)- containing experiments suggested that poly(A)-minus RNA coded for the same proteins as these studies all used poly(A)-containing RNA (Lodish et al., 1974). However, oligo(dT)-cellulose to fractionate RNA., s a procedure which could lead to subst,antia,l cross-contamination of poly(A)- minus RNA with mRNA that contains poly(A). For example, Hunter & Garrels (1977) showed that a large fraction of their B-actin translation activity which did not bind to oligo(dT)-cellulose, did bind to poly(U)Sepharose. They were, therefore, unable to state definitively whether the /3-actin or whether the mRNA was completely mRNA was merely deficient in poly(A)

NEWLY

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mItNA

373

As we shall show, the results obtained with Dictyostelium poly(A)poly(A)-minus. minus RNA are very different when analyzed by poly(lJ)-Sepharose, as opposed to oligo(dT)-cellulose chromatography. In addition, we also show that, thermal elution from poly(U)-Sepharose fractionates translation activities for individual mRNA species.

2. Materials and Methods (a) Genera2

methods

D. discoideum st,raiu 9x-3 was used throughorrt, these studies. Cells were gro~vn ill MES-HL-5 medium containing (per 1): 5 g yeast, ext,ract (Difco). 10 g proteose peptom. (Difco), 10 g glucose and 1.3 g MES (2-(N-morpholino) ethane sulfonic acid, monohydrat~e: (~albiochem). Culture conditions and methods of preparation of subcellular fractions ~vrrf’ as previously described by Jacobson (1976). (b) Labeling

of RXA

Some of the experiments reported in this paper required labaling of RNA in ~ivo. RNA was labeled with 32P0, (New England Nuclear) as previously described (Jacobson, 1976). The use of 32P0, was dictated by the kinetics of incorporation of nucleic acid precursors. Alt,hougll it has been shown that there is linear incorporation of 32P04 into poly(A)c,ontaining RNA within 2 min after addition of isotopic label (Firtel et al., 1976; Palatnik & due to slow equilibration with cellular nucleotide pools. (c) Isolation

of nuclei

Nuclei were isolated by differential centrifugation of detergent-lysed cells using H modification of the procedure of Cocucci & Sussman (1970). Cells were harvest,ed b? centrifugation at 500 g for 5 min and then washed twice with 0.1 lb (w/v) NaCI. Cell 0.05 M-HEPES p~~llet,s were resuspended in 10 to 20 vol. of ice-cold lysis buffer containing loo/;, (a/v) sucrose and 2% (v/v) Cemulsol NPTI2 (pH 7.5). 5 mM-magnesium acetate, (Melle-Bezons), and vortexed at 4°C for 45 to 60 s. Debris and unbroken cells \v(‘r(b removed by centrifugation at 400 g for 5 min, and the supernatant was then centrifuged at 2000 g for 5 min. The resulting nuclear pellet was resuspended in lysis buffer, vortexrd and centrifuged at 2000 g. The purity of nuclear preparations and the efficiency of lysis were monitored by phase-contrast, microscopy; contamination with unbroken cells was Iisually less than 0.01 “/b. (d) Isolation

ofpolysomes

Crlls were harvested and washed as in the prot,ocol for nuclear isolation. (Ml pellets w(‘re again resuspended in 10 to 20 vol. of ice-cold lysis buffer and vortexed at 4°C for 45 tcj 60 s. Debris, unbroken cells, mitochondria, nuclei and other vesicular structures were removed by centrifugat,ion at 20,000 g for 15 min. The resulting supernatant was layered over a 1594 to 5076 sucrose gradient in 0.01 M-HEPES (pH 7.5). 0.01 M-MpCI, and 0.01 nr-KCl. Centrifugation in the Beckman SW27 rotor, at 4”C, was for 4 to 5 11 at 27.000 revs/min. Fractions containing polysomes were pooled and used for isolatiorl 01 polysomal RNA. (e) Isolation

of hWA

RN;1 was isolated from cells or subcellular fractions bv extraction with a mixture of phenol, chloroform and isoamyl alcohol as previously described (Jacobson, 1976). Washed cell pellets were resuspended in ice-cold 0.05 M-TriS’HCl (pH 7.5), at a final concn of about 5 x 107 to lO* cells/ml. Cells were lysed by adding sodium dodecyl sulfate to a final concn of 0.5?i,, followed by vortexing. To further inhibit nucleases, dietllylpyrocarbonl~t,f, (East,man Organic Chemicals) was added to a final concn of 1% while mixing was eontinuc>d. One and one-half volumes of a cold mixture of phenol/chloroform/isoamyl alcollc~l

374

C. M. PALATNIK,

R.

V. STORTI

AND

A. JACOBSON

(66 : 33 : 1, by vol.) were subsequently added and vigorous’shaking was continued for 1 to 5 min. Aqueous and organic phases were separated by centrifugation ar 12,000 g for 10 min. The aqueous phase was re-extracted with the phenol/chloroform/iaoamyl alcohol mix at least 3 times, or until there was no longer any detectable material at the interphase found after centrifugation. The RNA was isolated from nuclei by resuspending them in reduced volumes of 0.05 M-Tris.HCl (pH 7.5) and proceeding as described above. The RNA was isolated from polysomes and mitochondria by adding sodium dodecyl sulfate and diethylpyrocarbonate to the sucrose solution and extracting with phenol, chloroform, and isoamyl alcohol as above. After extraction wit11 phenol, RNA was precipitated by the addition of O-2 vol. of 2 Msodium acetate and 2.5 vol. of chilled 95% ethanol and stored at -20°C overnight,. The resulting precipitate was centrifuged at 9000 g for 1 h, dried, resuspended in water and stored at - 80%. The quality of individual RNA preparations was monitored by (1) determining the translation activity (cts/min [35S]methionine incorporation/pg RNA) in mRNA-dependent wheat germ extracts, (2) examining the size distribution of polypeptides synthesized in vitro, by densitometry of l-dimensional polyacrylamide gels, and (3) analyzing the RNA on polyacrylamide gels containing 99% formamide (Jacobson, 1976) by staining with ethidium bromide or by autoradiography. (f) Fractionation

of RNA by thermal elution from poly(U)-Sepharose

Poly(U)-Sepharose (Pharmacia) was swollen in, and washed extensively with, a buffer containing 1 M-NaCl, 5 rnM-Tris*HCI (pH 7.5). The resulting slurry was poured into a water-jacketed column and washed at 25°C with EB buffer containing 90% (v/v) deionized formamide (Matheson, Coleman and Bell), 50 mM-HEPES, 10 mM-EDTA (pH 7.0) and 0.2% sodium dodecyl sulfate. After the EB buffer wash, t,he colmnn was equilibrated with CSB buffer containing 25% formamide, 0.7 M-NaCl, 50 mM-Tris.HCl (pH 7.5) and 10 mMEDTA. Equilibration was monitored by reading the absorbance at 254 nm and by measuring the conductivity of the eluting buffer. The sample was prepared in a buffer containing 1% sodium dodecyl sulfate and 30 mM-EDTA (pH 8.0) and heated to 60°C for 3 min before quick-cooling in a solid COz/ethanol bath. The sample was rapidly brought back to 25”C, diluted &fold with CSB buffer and loaded onto the column. At no time were amounts loaded wllich required more than half the capacity of the column (10 to 15 pg poly(A)-containing RNA/ml of resin). Tile colurnl~ was washed with CSB buffer until the non-binding fraction had eluted (monitored at Azs4 or, with samples labeled with 32P04, by Cerenkov radiation). At this time, CSB buffer was replaced by LS 0.1 M-NaCl, 50 maI-Tris.HCl (pH 7.5) and 10 mMbuffer containing 25% formamide, EDTA. After collecting the material which eluted with LS buffer at 25°C (25°C eluate), the temperature of the column was raised in 10 deg. C increments to 55°C. With each temperature increment a reproducible fraction of the bound RNA eluted from the resin (35 to 55°C eluates). In a final wash at 55”C, the remaining bound RNA was eluted with EB buffer. Individual fractions were made 0.2 M with sodium acetate and precipitated with 2.5 vol. of 95% ethanol. The EB eluate was diluted 4-fold with 0.2 &I-sodium acetate before precipitation with ethanol. Recovered material was always greater than 90% of the input sample. The percentage of material in the \Tarious eluates was det.ermined either by direct counting in a liquid scintillation counter or by determining areas under peaks with a Wang Laboratories digitizer. Quantities determined by these methods were in agreement with recoveries from ethanol-precipitated material. For analytical experiments, a 5-ml column was used and for preparatix-e experiments a 25-ml column. The flow rate was 1 ml/min. We and others (Jacobson, 1976; Wilt, 1977) have found poly(U)-Sepharose to be far superior to oligo(dT)-cellulose for mRNA isolation. The poly(A)-containing RNA which binds to oligo(dT)-cellulose is greatly contaminated with ribosomal RNA (and possibly poly(A)-minus mRNAs as well), which is difficult to remove even with several passages through the column. In addition, with each succeeding passage some degradation OCCURS. Moreover, inefficient binding of poly(A)-containing RNA to oligo(dT)-cellulose complicates the analysis of poly(A)-minus mRNA and can lead to overestimates of the complexity of

SE\VI;LY

SYS’l’HERTZEl)

37.-i

mRru’.-\

this fractioll. 111 contrast to oligo(dT)-cellulose, we 11a\.~ fo~~r~d that polS(A)-cont,airliIlg RNA binds efficiently and with higher specificity t.o poly(U)-Sepharose. Commercial preparations of poly(U)-Sepharose (Pharmacia) retail1 their binding efficiencies for at least 2 years and do not leach significant amounts of bound poly(U) (Jacobson. unpublished experiments). (g) Isolation

and analysis

qf poly(A)

Poly(A) residues were separated from the bulk of the RNA by virtue of their resist~ancc~ to t,lrr combined action of ribonucleases A and T, in 2 x SSC (SSC is 0.15 M-NaCI, 0.015 %Isodium cibrate). An ethanol-precipitated RNA sample was resuspended in 0.1 to 0.3 ml of 2 < SSC containing 10 units RNase T,/ml and 5 pg RNase A/ml. Samples were iitenbated at 37°C for, 30 min. Nuclease digestion was terminated by tile addition of O.!j”,, sodium dodecyl sulfate and 1 mg of proteinase K. A subsequent incubation for 30 min at 37°C was followed by extraction with phenol, and binding to, and elution from, poly(‘c’)Sepharose. The final poly(A) fragments were then precipitatrd from ctllanol in t,hcl prrsrrrc*cs of .50 pg of Escherichia coli transfer RNA. gels prepared in H Poly(A) sequences were analyzed on 10 y0 (w/17) polyacrylamide buffer containing 0.04 M-Tris.HCl (pH 7.3), 0.02 M-sodium acetate. 1 mM-Na-EDTA (pH 7.0) and 0.2% sodium dodecyl sulfate. Electrophoresis was carried out in tile salncs t)uffer at 5 to 10 mA/gel and was terminated when the brompllenol bhlc dye marker \z’ax upprox. 4 rm from t,he bottom of the gel. (I I) Translation

%n. messenger

RNA

-dependent

wheut

germ

lynates

Sa~nplc,s were analyzed iti wheat germ lysates prepared ant1 assayed by a modificat,i~,t~ of thca met~llod of Alton & Lodish (1977). Reactions were incubated at 22 t,o 23°C for 3 II. Reaction mixes contained the following additives : 670 r~~-ATP. 270 @I-GTE’. 16 I~.\Icreatine phosphate, 2.1 pg creatine phosphokinase, 20 m>f-potassium acetate, 800 phi270 ~11 each of 18 amino spermidinc.HCl, I6 mM-HEPES (pH 7.3), 2.7 mM.dithiothrf~itol, acids (minus methionine and cysteine), 160 FM-cysteine, 8 ~1 wheat germ extract atld IO pc(i [.35S]mt?thionine in a final vol. of 25 ~1. While there are some differences bet,ween 0111 assay conditions and those of Alton & Lodish (1977), the major difference is that. O,II' lysattss are made mRNA-dependent by treatment with micrococcal nuclease it1 a fashiori analogous to procedures developed for reticulocyte lysates by Pnlham & *Jackson (I!)ici). .-\fter thawing, CaCl, and micrococcal nuclease (Worthington; NFCP) are added t,o cxxtracts t,o gi1.e final concentrations of 1 mM and 10 rg/ml, respectively. The extracts ar(b tllen incubated at 22 to 23°C for 15 min after which time ethyleneglycol-bis(/%amino. c%hyl ether) N,X’-tetra acetic acid (EGTA) is added to a final concn of 2 mM. Portions arta immediat,ely added t,o individual reaction mixes. Analysis by densitometry of poly . acrylamide gels of translation products using extracts prepared in this manner showed H large number of peaks (bands) in the high molecular weight, range, arid the translatiorl products werr not, skewed toward the low molecular weight range as Ilad been t,ll( RNA (Lodish et al.. 1974). The, siztx of tllra case iti early translations of Dictyostelium translation products was partially dependent on t,he m&hod of RNA preparation, th(t t.mnslation conditions, t,lle preincubation step and thcx micrococcal nllclease t,reatment .\ltllougll the caxtracts treated with micrococcal nuclease arc almost totally mRN.4. tlrpcxrldrnt. a small amollnt of endogenous wctivit,y remains, which ~a11 bc dc~t,t~ctc~d it1 IOII~ ctxposurrs of gels corltaining translation mixes to wlricll Rio Dictyosteli~rm RNA Jla({ l)c%en added. While this appears t,o represent a neglipiblr amount, of hackgrolllld cilld(). g:rtrcoc~s activity, a few low molecular weight bands and OLI(’ higIl molrcular wrigllt harr(l (SP~LFig. 8) still persist irl translat,ion mixes, and art‘ rvidr,nt in translat,ion produ& of RNAs wit,h low translation activities. TVhether ally or all of tllrsc, rc~presc~nt honn ,jit[p I)rotPin syrrt,hesis is not known. (i) Tran,slation

in messenger

RNA-dependent

retic/rlocyte

Iysates

Reticulocyte lysatcs were prepared from anemic rabbits bp the> rnettlod described by \‘illa-Komaroff et al. (1974). New Zealand white rabbits weighing 4 to 6 lb were lnadct :inCrnie hy sllbcntanrotls injection of 1.20/” acet,ylpllerl~lh?rdrazirlcl arcording to t/l{3

376

C. M. PALATNIK,

R. V. STORTI

AND

A. JACOBSOS

following schedule: 2 ml on day 1, 1.6 ml on day 2, 1.2 ml on day 3, 1.6 ml on day 4, and 2 ml on day 5. On days 7, 8 and 9 the rabbits were bled: one ear was swabbed with cotton saturated with xylene, and a single incision using a new razor blade was made in the posterior ear vein about midway along the length of the ear. Each rabbit yielded 50 to 60 ml of blood collected into chilled saline containing 0.001% heparin. The blood was filtered through cheesecloth, then centrifuged at 3500 revs/min for 5 min. The cells were washed by centrifugation 3 times, with the last centrifugation at 7000 revs/min. Packed cells were lysed at 0°C with an equal volume of cold water. After 1 min, the lysate was centrifuged at 15,000 revs/min for 20 min. Portions (0.5 ml) of the supernatant were frozen at - 8O”C, at which temperature activity was stable for several months. The lysates were rendered mRNA-dependent by digestion with the calcium-dependent micrococcal nuclease. Thereafter the nuclease was inactivated by sequestering the calcium with EGTA (Pelham & Jackson, 1976). Protein synthesis assays contained, in a final vol. of 25 ~1, 10 ~1 of nuclease-digested reticulocyte lysate, 80 mM-potassimn acetate, 1.5 mMmagnesium acetate, 500 PM-spermidine (free base), 8 mM-creatine phosphate, 8 mg creatine kinase/ml (Sigma; 155 units,‘mg), 20 mnr-HEPES (pH 7.(i), 2 mM-ditbiothreit,ol and 20 to 40 &i [35S]methionine. Reactions were incubated at 37°C for 40 min. (j) Sodium

dodecyl sulfate/polyacrylamaide

gradient

gel electrophoresis

Portions of reaction mixes were displayed on 6% to 15% linear gradient polyacrylamide gels containing sodium dodecyl sulfate using the procedures of Laemmli (1970) and Studier (1973) and assayed by quantitative fluorography according to t#he procedure of Laskey & Mills (1975). (k) Quantitation Estimates of the percentage Sepharose eluates were made Wang computer.

of actin

of actin in translation products by digitizing densitometer traces

(1) Two-dimensional

polyacrylarnicle

from different of fluorograms

poly(U)using a

gel electrophoresis

Two-dimensional polyacrylamide gel electrophoresis was carried out by a modification of the procedure of O’Farrell (1975) as described by Storti et al. (1978). Samples of extract (3 to 15 ~1) were electrophoresed in 2 mm x 130 mm glass tubes on isoelectric focusing gels containing a pH 5 to 7 gradient. Samples were electrophoresed at 500 V for 16 to 20 h. After electrophoresis, the isoelectric focusing gels were equilibrated for 30 min in 10% (w/v) glycerol, 0.1 M-dithiothreitol, 0.0625 M-Tris.HCl (pH 6.8), and either frozen or electrophoresed directly in the second in solid COz/ethanol and stored at -80°C dimension. Second-dimension electrophoresis was in sodium dodecyl sulfate/l2% polyacrylamide slab gels according to Laemmli (1970), except that the stacking gel contained 2.5 M-urea. The addition of urea facilitated overlaying the stacking gel with 0.1% sodium dodecyl sulfate. The isoelectric: focusing gel was layered on top of the stacking gel and sealed with Gels were analyzed by quantitative fluorograph>i 1% agarose in equilibration buffer. (Laskey & Mills, 1975).

3. Results (a)

Fractionation

of messenger

RNA

by thermal

e&ion

from

poEy(U)-Xepharose

In order to fractionate mRNAs according to poly(A) content, we used the following at, method. Using water-jacketed columns, we bound RNA to poly(U)-Sepharose 25°C in a buffer containing O-7 ivf-NaCl and 25% formamide. To elute the bound RNA, the column was washed at 25”C, 35”C, 45°C and 55°C with a buffer containing O-1 MNaCl and 25% formamide. In a final wash, at 55”C, we eluted the remaining bound RNA with a buffer containing 90% formamide (EB buffer). Figure 1 shows an elution profile from a poly(U)-Sepharose column to which vegetative slime mold cytoplasmic

NEWLY

SYNTHESIZED

377

mH.Sh

0

EB c

9 y

0

?

x lo-’

F

x 10-a

i I .!

I

A

‘\ \

10

20

30

40

50

h 60

70

Fmction

00

90

I/

h-i!L IIO 120

130

number

FIG. 1. Poly(U)-Sepharose thermal elution profile of vegetat,ive rytoplasmic RNA labeled for 1 h with “‘PO,. X total of 50 mCi 3”P0, (carrier-free) w*s added to a 50.ml culture of exponentially growing cells of D. t/iscoirlr;um Ax-R (5.7 x 10s cells/ml). After 1 h, the cells were pelleted, washed with 0.20;, NaCl and lysed with 5 ml of lysis buffer (50 mix-HEPES, pH ‘7.5. 5 mM-magnesium acetate. IO”,, NPTlS). Nuclei and cell debris were pelleted by 2 centrifugat,ionr at 2000 g sucP”sc, 2’:,; Cemulsol for 5 min. Mitochondria were removed by an additional centrifugetion st 15,000 g for 16 min. Thea supernatant was extracted with sodium dodecyl sulfate/diethylpyrocarbonet/phenol/chlorof(,rrn. w(*rc precipitated with et,hanol and chromatographed on poly(U)-Sepharose. Then Z-ml fractions collertetl and 0.5 ml portions were assayed for Cerenkov radiation.

RNA, which had been labeled for one hour with 32P0, (1 mCi/ml). had been bound. As shown. a fract)ion of the bound RNA elutes with each alteration in conditions. As \vill be shown, the profile obtained varied with the cell fraction being analyzed or the labeling conditions utilized, with different. characteristic percentages of bound material eluting in the various fractions. The absolute amount of RNA which binds is also variable and is dependent upon the labeling conditions and the cell fraction being analyzed. RNA from all of these fractions has been analyzed on polyacrylamidc gels containing 99% formamide (Jacobson, 1976) and does contain some ribosomal RNA contamination. Percentage-binding measurements from experiments in which bound RNA wax isolated and rebound to the column, as well as analysis of translation activities in bound and unbound fractions (see Pig. 7), suggest that the enrichment for mRNA is at least 25fold after a single column passage. More accurate estimates of rRNA contamination are presently being made using a recombinant plasmid containing ribosomal DNA sequences.

378

C. M. PALATNIK,

(b) Size of poly(A)

R. V.

STORTI

ANI)

tracts in poly(U)-Sepharose

A. JACOBSON

therm,al eluutes

The size of the poly(A) tracts in the various t’hermal eluat’es was det,ermined in two ways. In the first method, vegetative cells were labeled with 32P04 for one hour, whole cell RNA was extracted, and the RNA was chromatographed as described above. The RNA from each of the eluates was then digested with RNases A and T,, rebound to poly(U)-Sepharose. and subjected to elec6rophoresis on 10% polyacrylamide gels. In the second method, unlabeled whole cell RNA was fractionated by thermal elut’ion from poly(U)-Sepharose, the eluates were digested with RNases A and ?‘r and the digestion products were directly subjected to clect~rophoresis. Gel fract,ions were then hybridized with 3H-labeled poly(U) according to the method of Bishop et al. (1974). Figure 2 is a graph showing the modal poly(A) content of the different eluates. As shown, bot’h methods gave similar results, with poly(A) content increasing with temperature of elution. No detectable poly(A) tracts are present in the non-binding fraction of RNA labeled in vivo, indicative of the high binding efficiency of poly(U)Sepharose. In experiments in which poly(A) tracts were Ctrated with 3H-labeled poly(U), however, poly(A) sequences representing less than lyi of the total poly(A)cont,aining RNA and showing a distribution similar to whole cell RNA were detectable,

Elutlon cmdliions FIG. 2. Poly(A) content of poly(U)-Sepharose thermal eluates. -4 lBO-ml culture of exponentially growing cells (S x 10s cells/ml) was labeled for 1 h with 80 mCi 32P04. Whole cell RNA was extract,ed and chromatographed on poly(U)-Sepharose. Individual fractions were precipitated with ethanol and digested with a combination of RNases A and T,. The poly(A) fragments were precipitated with ethanol and analyzed on 10% polyacrylamide gels. The size of the poly(A) fragments was determined by the method of Jacobson et al. (1974). Since all bound fragments were found to contain ohgo(A tracts, the sum of the oligo(A) and modal poly(A) tracts is plotted as a function of the elution conditionsAlternatively, unlabeled whole cell RNA was chromatographed on poly(U)-Sepharose and individual eluates were digested with RNases A and T, and electrophoresed directly on 10% gels, without rebinding to poly(U)-Sepharose to purify the poly(A). The poly(A) content of the eluates was determined by hybridization of odd-numbered fractions with 3H-labeled poly(U) (Miles) according to the procedure of Bishop et al. (1974). e2P0,-labeled in eiwo poly(A) (0) ; 3H-labeled poly(U) hybridized poly(A) (0). FT, non-binding fraction.

XE:\VLY

suggesting

SYN’J’HESlZEl)

mR?itI

that’ a small percentage (less than lo/,) of the p&y(A)-containing

37!?

KSA ditl

not bind. As predicted from previous experiments (*Jacobson et al., 1974), all of the t’hermal eluates contained oligo(A),, tracts. We have therefore plotted the sum of the modal pal?;(A) sizt, and the oligo(A),, sequence as a function of the various ehnion eonditiotls. Typical sizing experiments which were used to establish this relationship are illustrat,ed in Figure 3. Figure 3 compares the poly(A) content of the EB eluate, charwtwizecl b?- t,he tirst m&hod, with the poly(A) content of whole cell RX,\. characterized by the second method. The results of both se& of experiments arcs plott,ecL ntr bhct same Figure for purposes of comparison. As shown. there is a largta tlifferenct~ in the modal polv(A) content, of these t\vo fract,ions. ‘l’h~ large poly(X) tract of the EB fract,ion has a size of 110 to 115 nucleot,idrs: whereas it,s complrm~t~t ill “st,cLady-statcl” RKA is 60 t ,o C5 I nucleotides in length. The hybridization of 3Hlabeled poly(U) to fractions 48 to 56 does not represent hvbridization to J~ol,v(~\) t,racts and is an artifact caused by layering t*he digested samples directly onto thr pal rather than by re-bintling t,hem to poty(U)-Sepharose. as in the labeling experimerits i,, VI~CO.It is difficult to estimate the size of these fragments: they are probably all less than ten nucleot’ides and most of them are probably less than tivo nuclwtides, Additional portions of some of these fractions were hybridizcad under mow stringwt rondit ions and failed to show detectable hpbridizet,ion. SO t tie?- probahl?; (to not rclprestnt authentic poly(A). .Is a control for possible degradat,ion of poly(A) t,racts during our cllromatographic, procedure. MT have compared the poly(A) tract’s of RNA samples before and aft,(\r poly(r’)-Srpharosf chromatography. For example, as noted above, the modal sizch of tlrrx large poly(A) t,ra&s in nIlfractionated whole cell RNA is 60 to A5 nuclrotidcs. .\s

IO

20

30

40

Slice

number

50

60

FIG. 3. P’oly(A) content. of whole cell RNA and poly(U).Scphe~~se EB eluata. The 10% polyacrylamide gels of the poly(.4) fragments from tho EH elueto (&-a--C ) ~-) from wlls labeled for 1 h with 32P0,, characterized by the method of Jacobson et ~2. (1974) ; and from wholo cell RNA (--e--a---) characterized by the method of Bishop et nl. (1974). The numbers on the Figure refer to lengths of poly(A) tracts determined &q previourly de varh fraction ((area undrr act,i n peak/total area) Y IOO]. F’l’, Iwn-hinrling fmctiou.

&ctrophoresis (Storti et ul.. 1978). A pH 5 to 7 range was ctlosen so as t,o rt:solw potential multiple forms of actin. Figure 11(a) and (b) :I\ mws a comparison betnwn the t,ranslation products produced by both translational systems. For purposes of comparison. some of the major polypeptides produced by bot)h sets of extracts arc\ indicated on the Figure (circled spots). The two spot#s marked with triangles rcst~mblc the actin purified from adult chick skeletal muscle in both molecular weight (42.000, M,.) and isoelectric points. Because of this and t’hc analogy nit’h actin synthesis in vert,ebrat,e cells, we consider it likely that one or bot’h of these proteins is actin. In addiCon. Alton & Lodish (1977) have fingerprinted the only major 42,000 molecular weight, spot which appears on pH 3.5 to 10 two-dimensional gels and found it t,o actin. wrrespond solelp t’o IktyosteZiurn, As can be seen. t’he translation products produced by both set’s of lysates arc remarkably similar. While some extra spots are seen in each set of translations thca major differences are quantitative. For example, the ratio of the two putative actin spots is different in the t,wo sets of translations. We have analyzed the in vitro product,s from both translation systems on two-dimensional gels and have found the same relative distribution of translation activities. Therefore, while each set of translatiorls may not, accurately measure the absolute amount of t,ranslation act,ivity for a given polypeptide, they probably reflect relative differences in RNA concentrations between the different fractions. Since the results and conclusions are comparable. we will onl? shon, the translat,ion product ‘R from the rc&ulocyte lysa&s. Figure 11(c) shows csndogeneous incorporation of [35S]methionine in this syst~em into material which is separated by electrophoresis in the pH 5 t’o 7 range. As shown: t,here is onl!- on(’ broad mdogeneous spot, on these gels. (i) 550~diw~ensional

analysis

of pol?y(A)-rriirms

trnt~slatior~

products

Figure 1 l(d) shows those translation products of polx(A)-minus RNA which aw resolvable in t)he pH 5 t’o 7 range. Since our earlier analysis on one-clirnensional gels suggested that most of the incorporation programmed by this fraction was in hist,oncs, only a small number of major spots is detectable. These spots include t,he two putative a&n spots in thth same rat,io as in the unfract)ionated sarr~pl” shown in Figure 1 l(a).

PH

FIG. 11. Two-dimensional analysis of whoat germ RNA isolated from exponentially growing cells was lysates and the translation product,s wore displayed t,richloroacctio acirl-p~t?ripitahl(, rts/min W&H appliotl

ant1 rot,iculocyte translation products. translated in whoat germ and reticulocyto art 2.tlinwnsionsl gels. A total of 170,000 to gds (a) at111 (h), l!%,OOO to gel (c) (an

I,ntirn retiodocyte reaction mix lacking exogeneous RNA) and 25,000 to gel (d). For puqwsw 111 wrnparison, some spot,s in gels (a) and (b) are marked with aides. The 2 putat,ivc act& spots WV~ madwtl wit)h triangles. (a) Heticulocyte; (b) wheat germ; (c) Ic~t,iclllo~yt,o-en~l~Jg(~t~o~ls; ((I) wt~irtllc~ translation products. c~~t~r--1~~)1~(.2)-nlitl~is

PIG. 12. Two-dimensional analysis of polypeptides programmed by poly(U)-Sepharose fractions. Whole cell RNA from exponentially growing cells was fractionated by thermal elution from poly(U)-Sepharose. The individual fractions were used to program reticulocyte lysates and the translat,ion products were displayed on $-dimensional gels. A total of 165,000 trichloroacetic acid-

I)wcipitablr rtsimin waq applied to each gel. Trannlat,ion aotixrlt it ‘i most ahunrlent~ it) I hfd 55 (’ Iracti~w ( : ) ; activit iw most abundant in the 45°C fraction ( 0) ; ad ivitirs rr1o.d abundant in t h,. ‘15 C” fraction ( 0); actjivitit:s showing the same tlistribution RC;actin ( ‘,,). (a) 25°C’ duat,r~: (tl) 35 ( cdlratc: (c) 45 C! chute; (cl) 55°C’ elnatr.

392

C. M. PALATNIK,

R. V. STORTI

ANI)

A. JACOBSON

A specific form of actin does not appear to be enriched in the translation products of poly(A)-minus RNA from Dictyostelium as has been reported for vertebrate actins (Hunter & Garrels, 1977). None of the major spots appears to reflect a translation activity which is unique to tlte poly(A)-minus fraction. In addition, since most of the major spots appear on the acidic side of the gel, and since these show the same distribution in the binding fractions (see Fig. 12) some of these may be related to actin (e.g. premature termination products or degradation product’s). (j) Two-dimensional

analysis of translation binding fractions

products programmed

by

Figure 12 compares the translation products of the poly(U)-Sepharose binding fractions when analyzed on two-dimensional gels. As shown, the results obtained are entirely consistent with those described earlier (Fig. 8) ; some translation activities are most abundant in the 55°C fraction (marked with open squares), others appear to be at the highest relative concentration in the 45°C fraction (marked with open circles), and still others appear to be most abundant in the 25°C fraction (marked with open diamonds). In addition, a series of spots shows the same behavior as actin (Fig. 10) and, as discussed earlier, may be related to that protein. These spobs are marked with open triangles on the Figure. The major difference between these results and those shown earlier (Fig. 8) is that there appear to be more differences than similarities between the various fractions. All of the spots which could be analyzed appear to be unequally distributed in the different, fractions.

4. Discussion (a) Isolation

of newl?y synthesized

m,esserLger KNA

We have described a procedure for isolating newly synthesized poly(A)-containing RNA from vegetative amoebae of D. discoideum. This met)hod takes advantage of the fact that most newly synthesized poly(A)-containing RNA from this organism contains long poly(A) tracts which shorten with age. Other methods for fractionating mRNAs according to poly(A) content have been published (e.g. see Ihle et al., 1974; Firtel et al., 1976). This is the first study, however, in which fractionation based on poly(A) content has been utilized for the isolation of newly synthesized poly(A)containing RNA. The method is extremely efficient in terms of time, yield and mRNA integrity. In addition, it avoids possible artifacts which might be caused by the use of other theoretically possible methods. For example, nucleotides labeled with mercury (Dale & Ward, 1975) h ave been used to isolate RNAs synthesized in vitro. However, it has recently been shown that this approach can lead to artifacts (Schafer, 1977). In addition, Grainger & Wilt (1976) have used nucleosides labeled with heavy isotopes to characterize newly synthesized RNAs labeled in vivo. However, their methods are difficult to scale up for preparative work and no assays of the functional integrities of their RNAs have been made. Based on the experiments illustrated in Figure 5, the 55°C and EB eluates represent approximately a tenfold enrichment for poly( A)-containing RNA labeled for one hour with 32P04. Additional experiments have shown that the specific activity of the EB eluate remains relatively constant after 15 minutes of labeling, and possibly sooner, while the 45°C eluate shows an increasing specific activity during this same time. By extrapolating back to shorter labeling times the enrichment increases. Figure 5(c) and (d) shows that 1.6 to 1.7% of the steady-state poly(A)-containing RNA elutes

NEWLY

SYNTHESIZED

mRN.1

393

in the EB fract,ion. If this fraction were all the newly synthesized mRNA. then the tlnrichment could be as much as 60-fold. Since the shortening of poly(A) with age is a common occurrence in cells of higher organisms (Sheiness & Darnell: 1973). the met,hods we describe should be directly applicable to other eukaryotic systems with the possible exception of systems in which there is extensive turnover of pol,v(A) in the cytoplasm (Dolecki et al., 1977; Wilt, 1977). (b) Relative contributions of messenger RNA stability and messenger RNA synthesis in determining messenger RNA abundance classes Our resulbs show that most newly synthesized poly(A)-containing Rn’A contains poly(A) tracts of 110 to 115 nucleotides which are subsequent’ly metabolized to a steady-stat,e level of 60 to 65 nucleotides. Since thermal elution from poly(U)Sepharose fractionates RNA on the basis of its poly(A) content. it also appears to fractionate most poly(A)-containing RNA on t’he basis of it,s age. This enables us to compare RNAs of different ages and make predictions regarding the relative cont’ributions of mRNA stability and mRNA synthesis in determining the frequency tiistribut’ion of mRNAs in vegetative cells of this organism. For example. if some mRNAs are more stable than others, we would expect them to represent a larger fraction of “aged” poly(A)-containing RNA, while if some were less stable, we would expect, them to represent a larger fraction of newly synthesized poly(A)-containing RNA. Comparison of the translation products of the eluates suggestIs that, mRh’rZ synthesis is pre-eminent in establishing mRNA abundancies. since the protein synt,hetic pattern produced by the newly synthesized fraction is very similar to that of the ot)her fractions. Most poly(A)-containing RNAs appear to be prese& in similar concent,rations in each of the eluates. Firt,el and co-workers (reviewed by k’irt,el &I, .Jacobson, 1977) have hybridized poly(A)-containing RKA from regrt,ative nuclear and cyt’oplasmic fractions, in vast excess, to complementary DNA made against vegetative, poly(A)-containing cytoplasmic RNA and found very similar abundancies and complexities in the two fractions. Their data t,herefore are consistent with mRNA synt’hesis being pre-eminent in establishing mRNA abundancies in vegetative cells of t#his organism. In addition, analysis of pulse-labeled and steady-stat,e mRNA on polyacrylamide gels (Ward, Lane, Palatnik & Jacobson. manuscript in preparat*ion) also suggests t,hat) t’here are no radical changes in the distribution of mRNA during aging. (c) Multiple

classes of messenger RNA

The relative translation activity for some polypeptides is greatly diminished in aging poly(A)-containing RNA while for others it increases. It is possible that there is some relationship between these results and different mRNA stability classcxs analyzed by labeling experiments (Singer $ Penman, 1973; Puckett et al, 1975). Our labeling experiments, however, reflected the behavior of several thousand different RNA species, present in different relative concentrations, while the translation c,xperiments represented functional assays of a very small fraction of the total RNA population. The unequal distribution of some translatable RNAs in the various fractions could, therefore, also be explained by some mechanism which recognizes specific mRNAs and maintains their poly(A) contents within finite ranges, or by some mechanism which synthesizes specific mRNAs with distinct, poly(A) lengths. What,(tvpr the explanation for the differences in distribution bet,wt:en t,ranslatable RNAs in 15

494

C. M. PALATNIK,

R. V. STORTI

ANI)

A. JACOBSON

the various fractions, we are forced to conclude that there are multiple classes of mRNA in Dictyostelium: (1) those which preferentially have long poly(A) tracts: (2) those which are more abundant in steady-state RNA than in newly synthesized RNA ; (3) poly(A)-minus mRNAs ; (4) mRNAs which predominantly have oligo(A) tracts. Some members of this last class may be mitochondrial in origin. Our experiments have demonstrated that mitochondrial poly(A)-containing RNA in slime molds, like that of other eukaryotes (Avadhani et al., 1973; Hirsch et al., 1974), contains short poly(A) tracts. We have not been able to show, however, that mitochondrial mRNAs are translated by our cell-free systems.

(d) Poly(A)-minus

messenger RNA

The translational complexity of the fraction which does not bind to poly(U)Sepharose appears to be extremely low. The major translation products are actin and, probably, histones. This result is very different from early results in which RNA was fractionated by oligo(dT)-cellulose chromatography (Lodish et al., 1974) and probably reflects the greater binding efficiency of poly(U)-Sepharose for poly(A)containing RNA. Because of the presence of transcribed oligo(A),, sequences on most Dictyostelium mRNAs, we do not know, however, if similar results would be obtained with poly(A)-minus RNAs from other systems. The question of a specific class of poly(A)- minus actin mRNA has been recently raised by studies in mammalian systems (Sonenshein et al., 1976; Hunter & Garrels, 1977; Kaufmann et al., 1977). As shown in Figure 10, actin represents about 4% of the [35S]methionine incorporation programmed by poly(A)-minus RNA from vegetative cells of Dictyostelium., with most of the remaining incorporation in the putative histone bands. The methionine content of actin in other eukaryotic systems (Carst’en & Katz, 1964), however, is about 3.5 to 4*0-fold higher than that of histones (Busch, 1965). Therefore, if the various mRNAs in the poly(A)-minus fraction are translated RNA of with equal efficiencies, actin mRNA represents about 1 y0 of the translatable that fraction. We have shown that a small percentage of the poly(A)-containing RNA (less than 1%) can be found in the poly(A)-minus fraction. Since actin mRNA is the most abundant mRNA in Dictyostelium, (Jacobson et al., 1975: Alton & Lodish, 1977), this contamination could account for a large fraction of the observed actin synthesis in the non-binding fraction. Other poly(A)-containing RNAs may also be in the non-binding fraction, but at concentrations too low to allow translation by our in vitro system. Finally, we could never rule out the possibility of a slight loss of poly(A) during our extraction procedure. We therefore cannot state with certainty that there is a definitive class of poly(A)-minus actin mRNA in vegetative cells of Dictyostelium. We also cannot rule it out, especially since most or all of the spots apparent in Figure 11(d) show the same distribution as the actin spots in the bound fractions (see Figs 10 and 12). We thank Cheryl T. Mabie, Carol Wilkins and Anne Capone for excellent technical assistance and Marion Dorscheimer for patience and expertise in the preparation of this manuscript. We are particularly grateful to Ray White and Michael Rrenner for their advice and criticism. This work was supported by grants (to A. a.) from the National Science Foundation and the American Cancer Society. T-70 authors (C. M. P. and. E V. S.) are postdoctoral fellows of the National Institutes of Health. The other author (A. J.) is a recipient of a Faculty Research Award from the American Cancer Society.

NEWLY

SYNTHESIZE11

mRS.1

3!Ki

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