Isolation and Characterization of Poly (Adenylic Acid) —containing Ribonucleic Acid From Dry Spores of Anemia phyllitidis L. Sw.

Isolation and Characterization of Poly (Adenylic Acid) containing Ribonucleic Acid From Dry Spores of Anemia phyllitidis L. Sw. A. FECHNER and H. SCHRAUDOLF Abt. Allgemeine Botanik, Biologie II, Universitat Ulm, D-7900 Ulm, F.R.G. Received August 10, 1982· Accepted October 14, 1982

Summary About 1 % polyadenylated RNA was isolated from a total RNA preparation of dry spores of Anemia phyllitidis by affinity chromatography on oligo(dT)-cellulose. This fraction contained ribonuclease-resistant poly(A) tracts of 35-220 nucleotides in length. Analysis of base composition from poly(A)-RNA showed a high GMP-content. The poly(A)-containing RNA was proved to be polydisperse and included molecules with mobilities from 5 S to 25 S. An in vitro translation system derived from rabbit reticulocytes was stimulated by adding Anemia poly(A)RNA. It therefore has m-RNA character. Experiments with 7-methylguanosine-5'-monophosphate gave evidence for this m-RNA containing «caps».

Key words: Anemia phyllitidis, Fern, Poly(A).RNA, Spore, Translation.

Introduction Since the discovery of the mRNA character of most poly(A)-RNAs, and since the observation that the poly(A)-sequences stabilize the mRNA molecule in Xenopus oocytes (Huez et aI., 1974; Marbaix et aI., 1975; Nudel et aI., 1976) the existence and function of this type of RNA as preformed information in resting organs of plants as seeds (reviewed by Delseney et aI., 1980/1981), pollen (Frankis and Mascarenhas, 1980) and spores (Van Etten and Freer, 1978) have also been postulated. Although it has been repeatedly discussed that this poly(A)-RNA which is transcribed during late developmental stages of resting organs may be necessary for protein translation during the early germination steps (Spiegel and Marcus, 1975; Delseneyet aI., 1977; Hecker et aI., 1977; Brooker et aI., 1978; Freer and Van Etten, 1978) it is still under discussion whether such proteins indeed are uniquely involved in the germination process (Roberts and Lord, 1979; earlier et aI., 1980; Weir et aI., 1980). There is increasing evidence that at least in seeds the long-lived poly(A)-RNAs might be information carried over from processes of seed ripening (Mori et aI., 1978; Peumans et aI., 1980).

Abbreviation list: GMP, guanosine monophosphate; HPLC, high performance liquid chromatography; m7 G S ,p, 7-methylguanosine-5'-monophosphate; m-RNA, messenger ribonucleic acid; oligo(dT), oligothymidylic acid; poly(A), polyadenylic acid; poly(U), polyuridylic acid.

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The analysis of both synthesis and function of nucleic acids and proteins during early events of seed germination is doubtlessly complicated by the inhomogeneity of the cell population of this organ. Fern spores, even if not homologous in regard to the induction of germination by red irradiation and gibberellin, show physiological regulation processes comparable with those of seed germination. The investigation of the existence and possible function of stored poly(A)-RNA in a unicellular fern spore may therefore provide a model for the more complicated process of seed germination. Although Raghavan (1977) has already proposed that gibberellin activates stable mRNA present in the dry spore of Anemia phyllitidis and therefore provides templates for proteins necessary for early events in germination, the existence of poly(A)-RNA in fern spores has not yet been proven. We report here on the existence and the characteristics of poly(A)-RNA in dry spores of Anemia phyllitidis L.Sw.

Material and Methods Spores of Anemia phyllitidis L. Sw. grown in the greenhouses of the University of Ulm were There was no measurable reduction in harvested in spring 1980 and stored in the dark at 4 the rate of germination during 2 years of storage.

0c.

RNA extraction In a preliminary comparison of the yields of total RNA after extraction with phenol (Kirby, 1964) and diethylpyrocarbonate respectively (Solomosy et aI., 1968), the latter gave higher RNA values. In spite of a better gain of total RNA, relatively higher amounts of poly(A)-RNA were found using phenol for deproteinization in a variation of Kirby's method as proposed by Wollgiehn (1968).

Isolation ofpoly(AJ-RNA For the isolation of the poly(A)-RNA, total RNA (40-60 A260 unitsl ml) dissolved in 10 mM Tris-HCI buffer (pH 7.5) containing O.SM NaCI was submitted to affinity chromatography on oligo(dT)-cellulose as described by Aviv and Leder (1972). Poly(A)-RNA was eluted with 10 mM Tris-HCI (PH 7.5) and precipitated by addition of two volumes of ethanol at -20°C. After washing with 70 % ethanol the precipitate was dried under a stream of nitrogen, dissolved in sterile distilled water and stored at -20 0C. The A2601 A280 and A2601Am ratios of all poly(A)-RNA preparations were in the range of 2.

Electrophoresis Poly(A)-RNA samples were analyzed on cylindrical gels of polyacrylamide/agarose according to the method of Sina and Chin (1979). Pattern identification was done by UV-scanning of the gels.

Size determination For preparation of poly(A)-segments and in vitro methylation with CH)-dimethylsulfate (Amersham-Buchler, specific activity 77.7 GBq· mmol- l ) we followed the method of Arendes et al. (1980). Synthetic poly-AMP preparations of defined length (Miles, average chain lengths 184,146,95,74) were treated in the same manner and used as standards.

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Gels stained with methylene blue were cut into 2 mm slices, incubated for 16 h in 0.5 ml perhydrole and the radioactivity was counted in a liquid scintillation counter after addition of 10 ml of dioxane based scintillation fluid.

Base composition Poly(A)-RNA samples were hydrolyzed in 0.3 M KOH at 37° for 18 h (Smillie and Krotkov, 1960). After neutralization with perchloric acid the hydrolysate was subjected to HPLC on a poBondapak-NH2 column (4 mm . 30 em). Solvent system: A. 0.005 M NHiH2P04 (pH 3.3); B. 0.1 M NHiH 2P04 (PH 4.5). Nucleotides were eluted with a convex gradient (No.7 Waters 660 solvent programmer) from 0 % solvent B to 30 % solvent B. The flow rate was constantly 3.5ml·min- 1• All solvents were degassed by sonication for a minimum of 3 min immediately before use.

In vitro translation The template activity of the poly(A)-containing RNA fraction was assayed in a commercial messenger RNA-dependent translation system prepared from rabbit reticulocytes by Amersham-Buchler. Standard incubation mixtures contain per sample: 8 ILl lysate, 2 ILl 5S)methionine (ca. 185 KBq) and 1 ILl poly(A)-RNA solution. After incubation at 30°C for 60 min a 1 ILl sample was withdrawn and the amount of radioactivity incorporated into proteins was determined as described by Amersham-Buchler.

C

Results

Isolation Total nucleic acid isolated from dry spores of Anemia phyllitidis gives a typical electrophoretic profile as demonstrated in Fig. 1. The total content ranges from 27-300D per g dry weight. The composition of nucleic acids was in conformity with preparations isolated from young prothallia and is shown in Tab. 1. The RNA includes a fraction of poly(A)-RNA which was bound to oligo(dT)-cellulose and eluted with a low salt buffer. The amount of RNA bound to the column on the

255

185

E c

o

55 45

~
~ ~

H

u

1+1

electrophoretiC mobility

Fig. 1: Total RNA was fractionated by electrophoresis on a polyacrylamide/agarose gel and detected by UV-scanning.

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Table 1: Fractionation of nucleic acids by methylated albumin-coated kieselguhr column chromatography. % of total nucleic acid

t-RNA

DNA

r-RNAI r-RNA II

19.5 17.9 15.9 46.1

experimental conditions used varied somewhat for the different spore populations and amounted to about 1 % of the RNA applied.

Size distribution ofpoly(A)-containing RNA The poly(A)-RNA as isolated by affinity chromatography was exposed to electrophoretic separation on agarose polyacrylamide gels. E. coli ribosomal RNA (23 S, 16 S, 5 S) and t-RNA were used as standards. The distribution of poly(A)-RNA on gels was broad (Fig. 2) and extended from 5 S to 25 S. Since the pherograms represent total cell extracts, nuclear RNA may be included, but doubtlessly the portion of this type of heterogeneous RNA should be extremely low in the nuclei of dry and resting organs.

23s

5s

16s

E c

~

C\I

«

L'_-'_____________---'-'+'--l' electrophoretic mobIlity

Fig. 2: Electrophoretic separation of poly(A)-RNA on a gel of polyacrylamide/agarose, detected by UVscanning.

Size determination and base composition ofpoly(A)-RNA In vivo labelling of ripening spores in fern sporophylls is extremely difficult, since the developing spore wall inhibits uptake of labelled precursors at least during the late phase of spore differentiation. Dipping of sporophyll-spines into labelled solutions as well as injection of tracers of high specific activities into the vascular area of sporophylls led to low specific acitivities in spore RNA. For the determination of the size of poly{A) the in vitro labelling method of Z. Pjlanzenphysiol. Bd. 108. S. 419-428. 1982.

Fig. 3: Polyacrylamide gel electrophoretic distribution of polyadenylate segments of poly(A)RNA. Arrows denote the migration of poly(A) segments of known average length (A 184 , A 146 , A9S, A74). Arendes et al. (1980) has therefore been used. CH)-methylated poly(A)-segments of defined size served as standards. Fig.3 demonstrates the distribution of CH} methylated poly(A) segments cut from poly(A}RNA of Anemia phyllitidis spores. Their lengths extended between 35 and 220 nucleotides with preference for higher association rates. These results agree with those obtained by CH)poly(U) hybridization using the method of Bantle et al. (1976) (data not shown). Table 2: Nucleotides of poly(A)-RNA separated by HPLC. mol % AMP

GMP

eMP

UMP

25.7

32.2

21.5

20.5

In spite of the high number of adenylate residues of poly(A}RNAs of the dormant spore the base composition of the respective poly(A)-RNAs is characterized by higher GMP content (Tab. 2). These results differ from findings for the poly(A)RNA isolated from vegetative cells of the gametophyte of Anemia phyllitidis by IQBAL (1977), who found an exceptional 93 % of AMP in hydrolyzates of this nucleic acid fraction. A dramatic change in base composition of the poly(A)-RNA

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could be expected to occur during germination and morphogenesis of prothallia if these data could be verified.

In vitro translation ofpoly(A)-RNA The template activity of the RNA absorbed by oligo(dT)-cellulose was tested in a cell-free system derived from rabbit reticulocytes. At least a part of this fraction has mRNA character. The amount of 5 S) methionine incorporated into trichloroacetic acid-precipitable products was a function of the amount of RNA added to the incubation mixture. Saturation was reached above 0.5 /-!g of poly(A)-RNA. Protein synthesis was stimulated from 10 to IS-fold above background values without added RNA (Fig.4-S). Separation of the translation products on SDS slab gels (Laemmli, 1970) gave more than 50 protein bands (Fig. 6) indicating a complex information content of the poly(A)-RNA of dry spores of Anemia. Using a total RNA preparation gained from this source only very low translation rates are obtained.

e

______ D.

0.125

0.25

0.5

RNA [1J9]

Fig. 4

~-_-_--<>

15

control ___ ---<>-------o--------o

3D

45

Time (min)

60.

75

Fig. 5

Fig. 4 and 5: Translation kinetics of poly(A)-RNA. 4: Activity as a function of RNA concentration. - 5: Time course of protein synthesis. The amount of RNA added was about O.06/lg. Values represent CSS)-methionine incorporated in 1 /ll aliquots in the absence ( 0) or presence ( • ) of RNA.

The capped nature of these templates is supported by the results of incorporation experiments carried out in the presence of 7-methylguanosine-S'-monophosphate 7 (m G 5 I p). This nucleotide is known to inhibit competitively the translation of «capZ. Pjlanzenphysiol. Bd. 108. S. 419-428. 1982.

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220,0 61 ,0 60,0

Fig. 6: Fluorogram of in vitro sy nthesized proteins fractionated by electrophoresis on a SDS-polyacrylamide gel (12.5 %). The contents of the samples are: 1, endogenous activity of the rabbit reticulocyte lysate tanslation system; 2-4, in vitro translation products of poly{A)-RNA from dry spores. The numbers on the left are molecular weights of marker proteins.

36,0

-

18,5 -

2 3 4

ped» RNAs in cell-free systems (Rondinelli et al., 1980). The results in Tab. 3 show the effect of m7 G S ,p, in different concentrations, on the incorporation of eSS)methionine under the conditions described. Discussion The data reported here demonstrate that dry and dormant spores of Anemia phyllitidis contain preformed poly(A)-RNA. Since this fraction is at least partly translated in a cell-free system, the results presented here confirm previous experimental indications of the existence of stored mRNA in fern spores (Schraudolf, 1967; Raghavan, Table 3: Inhibition of incorporation of CSS)-methionine by m7G S, p. incorporation of eSS)-methionine %

o 0.3 0.6 1.25 2.5 5 7

100 83

74

60

52 44 42

1977). Since it is well documented for seeds that protein-synthesizing systems are activated in very early phases of imbibition and that the stored poly(A)-RNA present in dry seeds takes part in this early translation process (Gordon and Payne, 1976; Chandra and Ashul Bak, 1977; Cuming and Lane, 1978; Cears et al., 1979), a broad conformity in regard to early germination processes in seeds and fern spores exists. The same may be considered for pollen (Frankis and Mascarenhas, 1980). However, as long as the nature of the proteins encoded in this stable mRNA is not elucidated,

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the extent to which protein-synthesizing processes necessary and typical for spore ripening are still present in this fraction (Peumans et al., 1980) remains undecided. The properties of the poly(A)-RNA of Anemia spores show broad conformity with the corresponding fraction from seeds. For the latter, poly(A)-RNA rates of 0.3 %-2 % of the total RNA have been reported (Hecker, 1976; Frankis, 1980; Matilla, 1980). Also in comparison with poly(A)-RNA isolated from Anemia, all preparations from seeds (Carlier et al., 1980; Weir et al., 1980; Martin, 1981; Lane, 1981) and other spores (Van Etten and Freer, 1978; Van Etten and Rawn, 1979) show a broad polydispersity of poly(A)-RNA. Size determination of the Poly(A) tracts when determined by the method of Arendes (1980) produced chain lengths for spores of Anemia between 35 and 220 nucleotides. Parallel determination by eH)poly(U) hybridization methods (Bantle, 1975) resulted in a 6 % portion of poly(A) sequences which means an average poly(A) chain length of 90-120 nucleotides. For cotton seeds poly(A) lengths of 75-100 (Hammet, 1975) have been reported while for fungi spores lower values (average for Botryodiplodia theobromae 26 (Van Etten and Rawn, 1979) for Rhizopus stolonifer 31-36 (Van Etten and Freer, 1978)) have been obtained. The chain lengths of poly(A) tracts of Anemia spores and plant seeds seem to show closer conformity. In spite of high poly(A) values of Anemia poly(A)-RNA there still is no evidence for the existence of free poly(A) tracts in spores. Regarding the average length of poly(A) segments which in other objects gave rise to high AMP portions in hydrolysates of the respective RNA (Sieliwanowicz, 1977; Lane, 1981) the high GMP level of poly(A)-RNA of Anemia spores is above all surprising. Hammet (1975) reported comparably high GMP values from poly(A)RNA preparations of cotton seeds which also contain long poly(A)-tracts: our findings thus have parallels in data received from dry seeds. The caps of the mRNA cannot be the explanation of the high level of GMP. In comparison to the number of AMP-nucleotides in poly(A) segments the quantity of GMP-nucleotides present in caps is very low. Nevertheless, this astonishing shift in base composition in favour of GMP will be the subject of special investigations. Acknowledgements The authors wish to thank Mrs. C. Meyer and Mr. K. Russ for excellent technical assistance, as well as Mrs. S. Krug for checking the English version of the manuscript. The experiments have been supported by the Deutsche Forschungsgemeinschaft.

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