Eur. J. Biochem. 96, 373-378 (1979)

Identification of mRNA in the Slime Mold Physarum polycephalum Peter W. MELERA, Joseph P. DAVIDE, and Catherine HESSION Laboratory of R N A Synthesis and Regulation, Sloan-Kettering Institute for Cancer Research, Walker Laboratory, Rye, New York (Received October 24, 1978/February 13, 1979)

Using a differential extraction procedure which had previously been shown to yield one nucleic acid fraction enriched in cytoplasmic RNA and another enriched in nuclear RNA, we have been able to isolate two polyadenylated RNA populations from microplasmodia of Physarum polycephalum. The poly(A)-containing RNA from the cytoplasmic-enriched fraction accounts for approximately 1.2 of the cytoplasmic nucleic acid, has a number-average nucleotide size of 1339 39 nucleotides, and has been shown, in a protein-synthesizing system in vitro, to be capable of directing the synthesis of peptides which have also been shown to be synthesized in vivo by microplasmodia. The poly(A)containing RNA from the nuclear-enriched fraction has a number-average nucleotide size of 1533 f 104 nucleotides and represents a mixture of cytoplasmic and nuclear adenylated RNA molecules. Based upon these observations, we have identified the polyadenylated RNA isolated from the fraction enriched in cytoplasmic nucleic acid as Physarum poly(A)-containing messenger RNA. The acellular slime mold Physarum polycephalum represents an organism whose simple biology, natural mitotic synchrony, and ease of cultivation in the laboratory make it highly desirable for studies of mitotic cycle regulation and the control of differentiation at the molecular level. Although a considerable amount of information is available concerning the identification and characterization of rRNA and tRNA in this organism [l - 61, very little information has been presented concerning its mRNA and nuclear RNA populations, since techniques have not yet been published which have adequately demonstrated the isolation of polysomes and nuclei from which intact RNA has been prepared. This demonstration, although desirable, is not an easy task in this system since substantial nuclease levels and a highly viscous cellular milieu make both the isolation of polysomes and the preparation of nuclei containing intact RNA difficult. In the absence of such techniques, therefore, we have pursued an observation made several years ago [ l ] which indicated that, under the proper conditions of temperature, pH and salt concentration, whole microplasmodia or stationary cultures of Physarum could be differentially extracted to yield fractions enriched in cytoplasmic RNA species and nuclear Ahhreritrtiot7s.

IinRNA, heterogeneous nuclear R N A ; s R N A ,

soluble R N A . D e / i / / i / i o / fAzhl) . u n i t , thc q u a n t i t y of ma[erial contained in 1 ml o f a solution which has an absorbance of 1 at 260 nm, when mea-

sured in a I-cm pathlength cell.

RNA species. This fractionation was shown to be reproducibly obtained and to allow the isolation of undegraded intact RNA, as judged by the presence of high-molecular-weight rRNA precursors in the nuclear-enriched fraction. Using these same basic techniques, we now report the successful isolation of Physarum mRNA from microplasmodial cultures.

MATERIALS AND METHODS Muintenunce of' Cultures and Radioactive Labeling P. polycephalum subline M3CVIIG3 (obtained by sporulation of subline M3CVIIG2) were grown as previously described [I]. Microplasmodia were labeled by addition of radioactive precursors to growing cultures as indicated in the figure legends. Microplasmodia were harvested from mid-log phase cultures by centrifugation, washed once with growth medium, and either used immediately for preparation of cytosol protein (see below) or frozen in liquid nitrogen for extraction of nucleic acids. E.xtraction

of' Nuckic Acid and Isolation

of Poly(A)-Containing R N A

A11 buffers and glassware, etc. were autoclaved before use. Total nucleic acid was extracted from lyophilized microplasmodia using the three-step phenol/

374

dodecylsulfate/bentonite extraction procedure described previously [ ll. Modifications included the elimination of the high salt precipitation step and the inclusion of an additional room temperature extraction of each of the aqueous supernatants with a 50150 (v/v) mixture of phenol reagent [ l ] and chloroform. Nucleic acid from the 4 "C, 25 "C, and 45 "C extracts were collected by precipitation from 73 % EtOH after 18 h at -30°C. Pellets were washed with several volumes of 73 % EtOH containing 0.01 M NaCl, dried and dissolved in an appropriate volume of oligo(dT)cellulose binding buffer consisting of either 0.01 M Tris-HC1, pH 7.5, 0.4 M NaCl and 0.2% sodium dodecylsulfate, or 0.01 M Tris-HC1, pH 7.5, 0.5 M KCl. Samples were then applied to columns of oligo(dT)-cellulose (P. L. Biochemicals, type 7) which had been equilibrated in binding buffer. Equilibration of the columns and chromatography were carried out at 30'C. Nucleic acid solutions were cycled over the column at a flow rate of 0.25 ml/min and after loading, were washed with sufficient binding buffer to ensure that nonbinding RNA had been eliminated. Elution of the bound poly(A)-containing RNA was accomplished by washing with 0.01 M Tris-HC1, pH 7.5, 0.2y4 sodium dodecylsulfate or with the 0.01 M Tris alone. In either case, the eluted RNA was adjusted to 0.4 M NaCl or 0.5 M KCl and recycled over a second oligo(dT) column. This recycling was repeated twice, and the final poly(A)-containing RNA eluate precipitated at - 30°C after addition of 0.1 vol. 20% potassium acetate, pH 5.0, and 2.5 vol. cold 95% EtOH. The poly(A)-containing RNA was then collected by centrifugation in the cold at 16300 x g for 20 min, dissolved in sterile distilled water and its ultraviolet spectrum and specific radioactivity determined. The RNA was then reprecipitated as above and stored either as an ethanol precipitate at - 30 "C or, after recentrifugation, in sterile distilled water at a final concentration of 500 pg/ml at - 70 "C. Preparation of rRNA marker was achieved by high salt precipitation [ I ] of the nucleic acid isolated from the 4 :C extraction. The salt-soluble RNA was collected by centrifugation, dissolved in total nucleic acid buffer (0.01 M Tris-HC1, pH 7.5, 0.05 M NaCl, 0.001 M Na2EDTA and 0.01 M MgCl) and precipitated from EtOH twice before being stored as an ethanol precipitate at - 30 "C.

Forniumide-Sucrose Density Gradient CentriJingation The technique used was that described by Hames and Perry [7] except that we found it necessary to adjust the final concentration of salt in the gradients and sample to 0.05 M in order to obtain adequate resolution of our rRNA markers. Details of our findings concerning the requirement for cation in these

Physarum mRNA

gradients will be presented elsewhere. Linear sucrose density gradients (5 - 20 %, w/v) were prepared in 70 deionized formamide [S] containing 0.05 M NaCI, 0.3 mM Na2EDTA. The final pH of the gradients was the same as the pH of the deionized formamide which was consistently pH 6.0. RNA samples were dried well and dissolved in a final volume of 0.4 ml sample buffer (75 % deionized formamide, 0.05 M NaCl, 0.3 mM Na2EDTA), heated to 70 "C for 2 min, cooled to room temperature and layered onto the gradients which had been formed in polyallomer tubes (Beckman Inst.). Centrifugation was for 20 h at 22°C in the SW40 rotor at 39000 rev./min, after which gradients were fractionated directly into scintillation vials and counted in 10 ml of Biofluor (New England Nuclear) in a refrigerated Packard model 3255, calibrated for dual label (14Ci3H) counting.

Cell-Free Protein Synthesis The micrococcal-nuclease-treated rabbit reticulocyte lysate system described by Pelham and Jackson [9] was used for the protein-synthesis assays in vitro. Nuclease treatment was essentially as reported [9], except that the incubation period was carried out for 20 min at 25 "C before addition of the 'master mix' [9], which was altered so that the final concentration of each of the 19 unlabeled amino acids in the reaction mixture would be 50 pM. In addition, our reaction mixtures contained 5 mM dithiothreitol, 1 mM NazEDTA and 0.2 mM spermidine (final concentrations) [lo]. The radioactive amino acid used in these experiments was either [3H]leucine (New England Nuclear, 115 Ci/mmol) at a concentration of 21 pmol/50-p1 reaction mixture or [35S]methionine (New England Nucelar, 643.4 Ci/mmol) at a concentration of 38 pmol/ 50-p1 reaction mixture. Polyadenylated RNA prepared as described above and stored in sterile distilled water was used at concentrations varying from 0.5 to 1.5 pg/ 50-pl reaction mixture. Assays were allowed to proceed for up to 90 min at 30°C and were terminated and analyzed for amino acid incorporation by procedures described by others [9].

Preparation of Cytosol Proteins, Acrylurnide Gel Electrophoresis and Fluorogruphy [35S]Methionine-labeled cytosol protein was prepared by homogenizing microplasmodial pellets at 4 ° C in 2 ml of a buffer containing 0.01 M potassium phosphate (pH 7.5), 0.005 M KCl, 0.001 M MgCI, 0.002 M dithiothreitol, 15% glycerol and 1 mg/ml methionine. The homogenate was centrifuged at 40500 rev./min in a Beckman rotor type 65 for 2 h at 4 "C and the supernatant (i.e. cytosol) removed and stored at - 30 "C.

P. W. Melera, J. P. Davide, and C. Hession

The dodecylsulfate/polyacrylamide gel electrophoresis techniques used here have been described by Laemmli [ l l ] and the fluorographic methods have been detailed by Bonner and Laskey [12]. Samples of cytosol protein or aliquots of the translation assays in vitro were adjusted to the prescribed concentration of dodecylsulfate, 2-mercaptoethanol and glycerol [ l l ] , boiled for 2-3 min, cooled and layered into the wells of a 0.75-mm slab gel. Electrophoresis was carried out for 5 h at a constant current setting of 20 mA/gel after an initial stacking time of 1 h at 10 mA/gel [13]. After electrophoresis, gels were fixed in 10% (v/v) acetic acid, methanol, or stained with Coomassie blue before fluorography [12].

315 Table 1. Distribution of'poly(A)-containing R N A The table presents representative values obtained from the extraction of one mid-log growth phase shake flask of microplasmodia. Total nucleic acid was extracted with the three-step phenol/dodecylsulfate/ bentonite procedure and poly(A)-containing RNA prepared from each fraction as described in Materials and Methods. The conversion factor used to calculate the weight of RNA from the absorbance at 260 nm was: 1 A260 unit = 40 pg RNA Temp of extraction

"C

4 25 4s

Nucleic acid released

%total 77.5 16.5 6.0

Total amount of nucleic acid

Weight of total nucleic acid

weight

cf total

A260units 100 21 8

mg 4.00 0.84 0.32

Pg 48.2 36.2 9.4

1.2 4.3 3.0

Poly(A)containing RNA -

- -

-

-~

%

RESULTS The data presented in Table 1 are those typically obtained from the extraction of mid-log phase microplasmodia via the techniques described above. The distribution of nucleic acid is very similar to that reported earlier [l], as are the yields. Using each of the nucleic acid extracts obtained at the three temperatures as starting material, poly(A)-containing RNA [experimentally defined for these studies as that alkalinelabile nucleic acid fraction which binds to either oligo(dT)-cellulose or poly(U)-Sepharose under the conditions described] was prepared by three cycles of chromatography over oligo(dT)-cellulose. Although the bulk of the poly(A)-containing RNA was found with the bulk of the nucleic acid in the 4 -C fraction, poly(A)-containing RNA was also found in the 25°C and 45°C fractions and represented in those fractions a greater percentage of the nucleic acid. Since 82 '%;of Physarum nucleic acid is rRNA and 12°,;l is sRNA (4-S + 5-S RNA) [ l ] and since most ( > 95 yo) sRNA is found in the 4"C extracts (Melera, unpublished observations), it can be calculated from the data of Table 1 that the 100 ,4260 units of nucleic acid in the 4 "C extract is composed of approximately 85 A2bo units of rRNA and 15 A260 units of sRNA. If one now makes the reasonable assumption that the large majority of Physarum ribosomes are associated with polyribosomal structures during mid-log phase growth, it can be calculated that the poly(A)-containing RNA found in the 4°C fraction comprises 1.4% of the presumptive polysomal RNA of the 4°C extracts. Although this figure must be considered minimal, it is in agreement with other reports in the literature which have shown that in most cases poly(A)containing RNA, i.e. mRNA, accounts for approximately 1-2% of total polysomal RNA [14-161. Applying similar calculations to the 25 "C and 45 "C extracts and assuming that Physamm DNA, which accounts for 5 of the total nucleic acid [l], is equally distributed between these two fractions, it can be

shown that the 25 "C extract contains 17.75 A260 units rRNA and 3.25 A260 units DNA, whereas the 45°C extract contains 4.75 A260 units rRNA and 3.25 A260 units DNA. Assuming, as above, that the rRNA is polysomal and that 1.4 % of it is poly(A)-containing, the 25 "C extract should have 0.25 A260 unit (10 pg) of poly(A)-containing RNA and the 45 "C extract 0.07 A260 unit (2.8 pg). Since by measurement (Table l), the 25 "C extract contains 36.2 pg poly(A)-containing RNA and the 45°C extract 9.4 pg, then 26.2 pg (36.2- 10 pg) of the poly(A)-containing RNA extracted at 25 "C and 6.6 pg (9.4- 2.8 pg) of the poly(A)containing RNA extracted at 45 "C are most likely not polysomal. Since the 25°C and 45°C extracts are known to be enriched in nuclear RNA, i.e. rRNA precursors, we suggest that the nonpolysomal poly(A)containing RNA of these fractions may represent polyadenylated hnRNA. Since our previous data concerning rRNA and rRNA precursor distribution [l], coupled with the points just presented, indicate that the RNA extracted at 4°C is cytoplasmic, whereas the RNAs extracted at 25 "C and 45 "C contain mixtures of cytoplasmic and nuclear RNA, and since recent data [8] have indicated the presence in 45 "C extracts of poly(A)containing RNA of large molecules not detected in 4 "C extracts, we have chosen to characterize further the adenylated RNA from only the 4 "C and 45 "C nucleic acid extracts. Poly(A)-containing RNA obtained from these extracts was prepared as described in Materials and Methods and subjected to formamide/sucrose density centrifugation. The results obtained from a typical set of gradient fractionations are presented in Fig. 1. The number-average size in nucleotides of the adenylated RNA obtained from three separate experiments was 1339 f 39 for the poly(A)-containing RNA extracted at 4°C and 1533 104 for that extracted at 45 T.

376

Physarum mRNA

3000

-

2600

. c

'E 2203 v)

c

5

-Is

1800

1400

m

1000

600 200

Fig. I . Formamide/sucrose gruriicvir .srtiimenration of poly( A)-containing R N A . Mid-log phase microplasmodia were labeled for 20 min in the presence of 50 pCi/ml [3H]uridine. Polyadenylated R N A from the 4 ° C and 45°C extracts as well as marker R N A was prepared, denatured and sedimented as described in Materials and Methods. Marker R N A was prepared from microplasmodia labeled for 1 h in the presence of 20 pCi/ml [14C]uridine. Final concentration of the adenylated RNAs in sample buffer was less than 10 pg/ml. Size of the poly(A)-containing RNA was estimated from the sedimentation of the included t R N A (4 S) and rRNA (26 S and 19 S) markers and was calculated as the numberaverage size in nucleotides as described by Vournakis et al. [I91 and Timberlake et al. [20].).-( I4C marker; (*---.) poly(A)containing [3H]RNA. (A) 4 ° C extract; (B) 45°C extract

These numbers represent the means and standard deviation obtained from at least five separate determinations and suggest that the 4 "C and 45 "C extracts contain different populations of adenylated RNA molecules. They also show that the size of the adenylated RNA in the 4°C extract is consistent with a variety of published values for the size of eukaryotic mRNA [14,16- 181. We next measured the abilities of the adenylated RNAs extracted at 4 "C and 45 "C to direct the synthesis of protein in vitvo.The results (not shown) indicated that under our conditions of nuclease treatment (see Materials and Methods) the reticulocyte lysate [9] backgrounds were reduced to less than 0.5% of the untreated endogenous levels and that at input RNA concentrations of 20 pg/ml poly(A)-containing RNA extracted at 4 "C stimulated the amino acid incorporation of the system to 30% of its endogenous level in 60 min. During a similar time period at the same input concentrations, 45 "C extract poly(A)-containing RNA stimulated amino acid incorporation to 12'x of the endogenous level; that is, 45 "C extract adenylated RNA stimulated amino acid incorporation to only 40";, of the level achieved by 4 "C extract adenylated RNA. Several reasons for the differential stimulation are possible and include the presence of inhibitors such as rRNA in the 45 ' C extract adenylated RNA, specific degradation of mRNA in the 45°C extracts, and the presence in the 45°C extracts of untranslatable poly(A)-containing RNA. As noted earlier, our previous data concerning the purification of large rRNA precursors from the 45 "C extracts tend to rule out degradation. Also, since 75% of the cellular rRNA

is extracted at 4°C (Table l), the potential for rRNA contamination is far greater in the adenylated RNA of the 4°C extract than of the 45 "C extract. Hence, we suggest that the enhanced stimulation of the reticulocyte lysate system by the 4 "C extract adenylated RNA is due to a greater proportion of translatable RNA in the 4°C extracts as compared to the 45 "C extracts. This interpretation is also consistent with the results of Fig.1 and our recently reported electrophoretic data [8] which show that the 4 "C and 45 "C extract adenylated RNAs represent different, but not necessarily entirely different, nucleic acid populations. To demonstrate that the poly(A)-containing RNA in the 4°C extract was indeed representative of PIzysarum mRNA and to determine if delete the 45 C extract contained some of these molecules, we compared by fluorography the peptides synthesized by microplasmodia in vivo with those synthesized in vitr-o by direction of these adenylated RNAs. The results are presented in Fig. 2 and clearly demonstrate several points. (a) The 4 ' C extract poly(A)-containing RNA is capable of directing the synthesis of specific peptides in vitvo; (b) these peptides are similar in size to those synthesized by the organism in vivo; (c) the 45'C extract poly(A)-containing RNA directs the synthesis of peptides virtually identical to those synthesized by mRNA, demonstrating that this adenylated RNA contains translatable mRNA sequences. Taken together with the other data presented in this report, the results presented in Fig.2 allow us to identify the poly(A)-containing RNA in the 4 ' C extract as Phj.sarum mRNA. They also suggest that the ability of the

311

P. W. Melera, J. P. Davide, and C. Hession

poly(A)-containing RNA in the 45 "C extract to direct protein synthesis in vitro is due to the presence of mRNA in these extracts. As pointed out in the calculations pertaining to Table 1, we can estimate from the relative yields of poly(A)-containing RNA that 2.8 pg of the adenylated RNA in the 45°C extract can be attributed to the presence of mRNA. That 2.8 pg represents 30% of the 9.4 pg of adenylated RNA in these extracts (i.e. this RNA is contaminated to 30% by mRNA), is in good agreement with the estimated 40 % contamination of this poly(A)-containing RNA by mRNA as determined by translational assay.

DISCUSSION

Fig. 2. Comparison of peptides synrliesized in vivo and in vitro by Physarum growth-phase m R N A . Mid-log phase microplasmodia were labeled for 24 h in the presence of 50 pCi/ml [35S]methionine and cytosol protein was prepared as described in Materials and Methods. The cytosol protein preparation (100000 counts/min) was then electrophoresed on a dodecylsulfate/acrylamide slab gel I l l ] , in the other wells of which were similar radioactive amounts taken from the translation mixtures in vitro. The molecular weight markers used in these gels, but not shown, were: ribonuclease A ( 1 3 700), chymotrypsinogen A (25000), ovalbumin (45000) and phosphorylase h (90500). The identity of the 47000-M, peptide, although not yet confirmed, is most likely to be Physarutn actin (Melera, unpublished observations, and Le Stourgeon, personal communication). Lane 1, peptides synthesized in vivo by direction of Physarum mRNA. i.e. cytosol proteins; lane 2, peptides synthesized in v i m by direction of Ph~.sarumpoly(A)-containing R N A extracted at 4 "C; lane 3. peptides synthesized in vitro by direction of Physarum poly(A)-containing R N A extracted at 45'C. Although lanes 2 and 3 display some quantitatively and qualitatively different peptide bands than lane I , it cannot be concluded that different mRNAs are translated in v i m than in vivo. The peptides in lane 1 were labeled in vivo for 24 h and therefore represent only those species with long half lives. Many peptides synthesized by the organism and which accumulate slowly or not all would not be detected under the labeling conditions used

We have presented evidence in this report for the presence of two polyadenylated RNA populations in microplasmodia of Physarum polycephalum. Coupled with other observations [1,8], it appears that the threestep phenol/dodecylsulfate/bentonite extraction procedure is capable of the selective extraction of these two RNA populations from whole microplasmodia. Although the basis of the selection is not entirely clear, it is reproducible and allows one to obtain routinely a cytoplasmic RNA fraction and a nuclear RNA-enriched fraction from which the two adenylated RNA populations can be prepared. Based upon its cytoplasmic location, yield, numberaverage size, and ability to direct the efficient synthesis in vitro of peptides shown to be synthesized by the organism in vivo, the poly(A)-containing RNA extracted at 4 "C has been identified as Pliysarum mRNA. Although we cannot specifically discriminate between cytoplasmic and polysomal adenylated RNA populations, it is apparent by translational assay (Fig.2) that the major mRNAs translated in vivo by growthphase microplasmodia are present in our mRNA preparations. We suggest, therefore, that these preparations are representative of the polysomal poly(A)containing RNA of Physarum microplasmodia. Whereas we have been able to identify the 4°C extract poly(A)-containing RNA as Physarum mRNA, we cannot, in the absence of an assay or a bona fide preparation of nuclei, rigorously identify the 45 ' C extract poly(A)-containing RNA as hnRNA. Although it is clear that this RNA is contaminated with mRNA, it nevertheless shares some of the properties of adenylated hnRNA: (a) it is found in a nuclear RNAenriched fraction; (b) it is adenylated; (c) it displays an average molecular size somewhat larger than mRNA; (d) it can be shown by electrophoresis after denaturation to contain a population of large molecules not found in mRNA [8]. It was mentioned earlier that the size of the poly(A)-containing RNA in the 45 "C extract, 1533 104 nucleotides, should be con-

378

sidered as a minimum value. Taking into consideration the combined data of Table 1 and Fig.2, it is reasonable to suggest that this adenylated RNA is contaminated to approximately 40% by mRNA with an average nucleotide size of 1339 -f 39 (Fig. 1). It can then be calculated that removal of this amount of mRNA from the poly(A)-containing RNA population would leave an adenylated RNA population with the characteristics mentioned here and an average nucleotide size of 1724 nucleotides. Interestingly, this figure is 22% larger than the value for Physarum mRNA and that degree of difference is very similar to the value reported for the difference in size between Dictyostelium hnRNA and its mRNA, i.e. 20% [17]. Clarification of these possibilities awaits the preparation of undegraded hnRNA from Physarum nuclei. This work was supported by grants G M 21863 and CA 08748 from the National Institutes of Health, IN-114 from the American Cancer Society, and by the Annelise Ghika Fund for Cancer Research.

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P. W. Melera, J. P. Davide, and C. Hession, Laboratory of RNA Synthesis and Regulation, Sloan-Kettering Institute for Cancer Research, Donald S. Walker Laboratory, 145 Boston Post Road, Rye, New York, U.S.A. 10580