Kinetin modifies the secondary structure of poly(A)RNA in cucumber cotyledons

Plant Science, 52 (1987) 67-72 Elsevier Scientific Publishers Ireland Ltd.

67

K I N E T I N M O D I F I E S THE S E C O N D A R Y S T R U C T U R E OF p o l y ( A ) R N A IN C U C U M B E R COTYLEDONS GRZEGORZ JACKOWSKI% ARTUR JARMOLOWSKI b and ALICJA SZWEYKOWSKAa aDepartment of Plant Physiology and bDepartment of Biopolymer Biochemistry, Faculty of Biology, Adam Mickiewicz University, 61-712 Poznafi (Poland)

(Received March 3rd, 1987) (Revision received May 18th, 1987) (Accepted June 4th, 1987) Evidence has been obtained for a presence of double-stranded regions in poly(A)RNA from cucumber cotyledons, as well as for an engagement of the poly(A) segment in the formation of these regions. Kinetin treatment of the cotyledons leads to an increase in degree of the secondary structure of poly(A)RNA, to a slight increase in the length of the poly(A) tail and to a considerable decrease in its involvement in base pairing, thus making the poly(A) segments more 'unmasked'. Key words: kinetin; poly(A)RNA; secondary structure; cotyledons; Cucumis sativus L.

Introduction A m o r e d e t a i l e d i n f o r m a t i o n c o n c e r n i n g the s e c o n d a r y s t r u c t u r e of m R N A is p r a c t i c a l l y limited to d a t a o b t a i n e d for r a b b i t globin m R N A s [1]. In addition, some k n o w l e d g e exist a b o u t g e n e r a l c o n f i g u r a t i o n of m R N A f r o m various, almost exclusively animal sources [2-5]. V e r y little is k n o w n a b o u t the s e c o n d a r y s t r u c t u r e of p l a n t m R N A [6]. T h e r e f o r e , the first a i m of o u r s t u d y w a s to i n v e s t i g a t e t h e degree of the secondary structure of p o l y ( A ) R N A f r o m c u c u m b e r cotyledons. As it is s u g g e s t e d t h a t the s e c o n d a r y struct u r e of m R N A p l a y s a role in the t r a n s l a t i o n efficiency [1] a n d t r a n s l a t i o n a c c u r a c y [7], fact o r s t h a t m o d u l a t e this s t r u c t u r e m a y be inv o l v e d in the m e c h a n i s m s c o n t r o l l i n g p r o t e i n s y n t h e s i s a n d cell d e v e l o p m e n t . It h a s b e e n f o u n d e a r l i e r in o u r l a b o r a t o r y [8-10] t h a t cyt o k i n i n t r e a t m e n t of c u c u m b e r c o t y l e d o n s affects t r a n s l a t i o n a c t i v i t y of p o l y r i b o s o m e s a n d of p o l y ( A ) R N A in vitro. T h e r e f o r e , t h e sec-

Abbreviations: MVC, mixed vanadyl complex; 3H-poly(U), poly(5-3H)uridylic acid; poly(A), polyadenylic acid.

ond aim of the p r e s e n t s t u d y w a s to test the effect of k i n e t i n on the s e c o n d a r y s t r u c t u r e of p o l y ( A ) R N A p o p u l a t i o n u s i n g the s a m e p l a n t material.

Materials and m e t h o d s Materials Seeds of c u c u m b e r (Cucumis sativus L. cv. Monastyrski) were surface-sterilized with 5% c a l c i u m h y p o c h l o r i t e for 5 min, s o w n on w a t e r - s a t u r a t e d tissue p a p e r a n d g r o w n in d a r k n e s s for 6 days. C o t y l e d o n s w e r e excised from e t i o l a t e d seedlings a n d p r e i n c u b a t e d , w i t h the a d a x i a l s u r f a c e down, in P e t r i dishes on w a t e r - s a t u r a t e d filter p a p e r for 2 1 h . One of the two c o t y l e d o n s of e a c h s e e d l i n g w a s n e x t t r a n s f e r r e d and i n c u b a t e d on filter p a p e r s a t u r a t e d w i t h w a t e r ( c o n t r o l ) a n d the o t h e r on filter p a p e r s a t u r a t e d w i t h 0.1 m M k i n e t i n solution. A f t e r 3 h of i n c u b a t i o n the c o t y l e d o n s w e r e b l o t t e d dry a n d frozen in solid CO2. All i n c u b a t i o n s w e r e in d a r k n e s s , a n d man i p u l a t i o n s w i t h c o t y l e d o n s u n d e r dim g r e e n light. T h e t e m p e r a t u r e for g r o w t h a n d incubation w a s 25°C. E x p e r i m e n t s a n d e s t i m a t i o n s were p e r f o r m e d 3-7 times.

0618-9452/87/$03.50 ~ 1987 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

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Isolation of poly(A)RNA Poly(A)RNA was isolated by the modified phenol method followed by oligo(dT)-cellulose affinity "chromatography as described previously [9,11] with the following modifications. The homogenization buffer was supplemented with 10 mM MVC as RNAse inhibitor, prepared according to Berger and Birkenmeier [12] and stored at -18~'C under argon. The excess of MVC was removed during deproteinizations with phenol-chloroform mixture containing 0.1% oxychinoline. MVC nearly doubled the yield of poly(A)RNA when expressed per fresh weight of cotyledons and increased it about 3.5 times when expressed as percent of total RNA. After deproteinization, the final water-phase was heated at 60:'C for 2 m i n and quickly cooled prior to batch-wise binding to oligo(dT)cellulose. In preliminary experiments, the RNA not bound to oligo(dT)-cellulose, subjected to poly(U)-Sepharose chromatography, revealed no traces of polyadenylated RNA. Therefore, it has been assumed that poly(A)RNA population from cucumber cotyledons does not contain significant quantities of RNA with poly(A) segments shorter than 20 nucleotides which are reported [13,14] as binding to oligo(dT)-cellulose with low effectiveness. In the case of our material, the oligo(dT)-cellulose chromatography proved thus to be suitable for isolation of poly(A)RNA without introducing significant changes in the distribution of different lengths of poly(A). A total pool of cotyledonary poly(A)RNA represents a heterogenous population of molecules. Therefore all the data presented in this paper refer to average values for the mRNA population.

Analysis of poly(n)RNA

the

secondary

structure

of

A procedure described by Favre et al. [13] was adopted with few modifications. A sample of 0.0530 A2~0 units of poly(A)RNA in 50 mM cacodylate buffer (pH 7.00) containing 75 ]riM NaC1 in thermostated (25~'C) cuvette of

Hitachi MPF-4 spectrofluorometer, was titrated with 2 pl portions of 200 t~M ethidium bromide. After each addition of ethidium bromide, the solution was stirred with a platinum stirrer, and after l m i n the fluorescent signal was read (exc. 520 nm, era. 608 nm). As a standard, 0.0530 A260 units of yeast tRNA were analysed.

Determination of poly(A) content in poly(A) RNA Two methods were employed to determine the content of poly(A) not engaged in the secondary structure of poly(A)RNA: (a) Hybridization with 3H-poly(U). Poly (A)RNA was hybridized with 3H-poly(U) as described earlier [9], with a modification consisting in increasing the temperature of the assay to 30°C. (b) Fluorimetric titration. A modified procedure of Trapy et al. [6] was employed. A sample of 0.0530 A2~0 units of poly(A)RNA (in the 50mM cacodylate buffer and 75mMNaC1) was titrated with 200gin ethidium bromide until a quasisaturation point of fluorescence was achieved. An overdose of ethidium bromide was then introduced and 3 ttl portions of synthetic poly(U) (1 A26o unit/ml) were added until a plateau of the fluorescence curve has been reached. The content of poly(A) not engaged in the poly(A)RNA secondary structure was calculated from the amount of poly(U) needed to reach the plateau and from the results of a parallel titration of synthetic poly(A) preparation.

Estimation of the engagement of the poly(A) chain in the secondary structure of poly(A) RNA. Hybridizations of the poly(A)RNA with 3Hpoly(U) were compared before and after poly(A)RNA denaturation by heat (90'C, 5 min) or 75% formamide (37°C, 5 rain) treatments. Prior to hybridization, to avoid an inhibitory effect of the denaturant [14], samples containing poly(A)RNA and formamide were diluted so as the final concentration of the formamide amounted to 5%.

69

Reagents Synthetic poly(A), 33 nucleotides standard length, was purchased from Miles Laboratories, poly(U) from Calbiochem (U.S.A.), and 3H-poly(U) from Amersham (England). Results

The extent poly(A)RNA

120

Z L./

of

secondary

structure

of

The extent of secondary structure of poly(A)RNA was probed by fluorimetric titration with ethidium bromide. At low RNA and ethidium bromide concentrations, in a moderate ionic strength solution, ethidium bromide selectively intercalates into helical regions of RNA, while electrostatic binding to RNA is absent or very weak [5,13]. Ethidium bromide intercalated into double-stranded regions fluorizes, upon excitation with green light, several times more intensively than as a free compound or bound to RNA electrostatically. Sequential addition of small portions of ethidium bromide to RNA solution will thus lead to a gradual increase in fluorescence as a result of the dye intercalation into double-stranded regions, until all binding sites are occupied. Beginning with this concentration of ethidium bromide (the quasisaturation point), further addition of the dye causes an increase in fluorescence due exclusively to a fluorescence of ethidium bromide itself, i.e. identical as during addiLidn of ethidium bromide to the RNAfree buffer. Figure 1 shows the results of fluorimetric titration of poly(A)RNA from control and kinetin-treated cotyledons, as well as of a reference preparation - yeast tRNA - the secondary structure of which is well known. The information concerning the extent of secondary structure was taken from comparison of fluorescence intensity of both poly(A)RNAs at quasisaturation point for tRNA (3.2 ItM) or, alternatively, from direct comparison of quasisaturation points of both poly(A)RNAs. Using both criteria it has been shown that poly(A)RNA from kinetin-treated cotyledons has a more complicated secondary structure

K~ , , ~

8O

=~ 60 0 u.

4O

2° ~ E B ,

0.8

, , , , ,

1.6 2.4 3.2 4.0 ETHIDIUM BROMIDE (yM)

Fig. 1. Examination of the secondary structure of poly(A)RNA by fluorimetric titration with ethidium bromide (EB). Poly(A)RNAs were isolated from cotyledons treated with water (control) or 0.1 mM kinetin. Samples of 0.0530 A26o units of poly(A)RNA or yeast tRNA were titrated with 2-#1 portions of 200 p m ethidium bromide, and fluorescences of the analyzed samples and of the dye alone were determined. The fluorescence intensity is presented in relative units taking as 100 the fluorescence of tRNA at 3.2 ttm of EB (the quasisaturation point).

t h a n poly(A)RNA from control cotyledons. As a result of kinetin action, the extent of secondary structure of poly(A)RNA population in the cotyledons became similar to that of tRNA in which about 70% of nucleotides are found in double-stranded regions.

The poly(A) content and its engagement in the secondary structure of poly(A)RNA The addition of synthetic poly(U) to poly(A)RNA, containing ethidium bromide introduced in big excess over quasisaturation point, results in an intercalation of the excessing ethidium bromide into newly formed poly(A)-poly(U) hybrids. Thus introduction of small portions of poly(U) gives rise to a new increase in fluorescence intensity until all intercalation sites in poly(A)-poly(U) are occupied by ethidium bromide. The amount of poly(U) required to reach a plateau of

70

fluorescence depends on the content of poly(A) accessible to poly(U) in poly(A)RNA. This content can be evaluated on the basis of a standard titration of known quantity of synthetic poly(A). As shown in Fig. 2, this amount is higher for poly(A)RNA from kinetin-treated cotyledons than from control cotyledons, thus demonstrating in the kinetin poly(A)RNA a higher content of poly(A) suitable for interaction with synthetic poly(U). Poly(A) content in poly(A)RNA can be al-

180

POLY(A)

t60

140, z

o

u~ 120

U

0100 -i u.

ternatively evaluated by hybridization of poly(A)RNA with 3H-poly(U) and comparing the results with hybridization of 3H-poly(U) with known amounts of standard poly(A) preparation. The evaluations based on fluorimetric and hybridization assays proved to be very similar (Table I). The more efficient interaction of poly(A)RNA from kinetin-treated cotyledons with poly(U) can be due to poly(A) tails longer than in control poly(A)RNA or to a smaller engagement of the poly(A) tails in the secondary structure of poly(A)RNA. To decide between these two possibilities, poly(A)RNAs denatured by heating or formamide treatment were hybridized with 3Hpoly(U). The hybridization results for denatured poly(A)RNAs were compared with those for non-denatured preparations. Heat treatment did not bring about a complete denaturation of poly(A)RNA as judged by slightly lower hybridization values than in the case of formamide-denatured poly(A)RNA (Table II). Data presented in Table II demonstrate that disruption of secondary structure of the control poly(A)RNA led to a high,

80 5 EB 60

20

40

60

80

1OO 1 2 0 1 4 0

1~O

POLY( U )~A260 UNITSxl04/rnl Fig. 2. Determination of the poly(U)-accessible poly(A) content in poly(A)RNA by fluorimetric titration. Poly(A) RNAs were isolated from cotyledons treated with water (control) or 0.1 mM kinetin, Samples of 0.0530 A26o units of poly(A)RNAs and of 0.0050 A2~0 units of synthetic poly(A) were titrated in the presence of 6.4 ~M ethidium bromide (EB) with 2-gl portions of synthetic poly(U) solution (1A2eo unit/ml). The 6.4pM EB alone was titrated for a control. The fluorescence intensity was measured in relative units taking the fluorescence of the control poly(A)RNA sample in the presence of 6.4 gM EB as equal 100. In the standard titration, 0.0098 A26o units of synthetic poly(U) were used to obtain complete hybridization with 0.0050 Ae~o units of synthetic poly(A).

Table I. Effect of kinetin on poly(U)-accessible poly(A) content in poly(A)RNA. Poly(A)RNAs were isolated from cotyledons treated with water (control) or 0.1 mM kinetin. The poly(A) contents were estimated by 3H-poly(U) hybridization or by fluorimetric titration with poly(U) in the presence of ethidium bromide. The contents were calculated from results of standard determinations in which a known amount of synthetic poly(A) was used. Samples of 1 ng of synthetic poly(A) hybridized with 12(~240 cpm of :~H-poly(U), and 0.0050 A2eo units of synthetic poly(A) with 0.0048~.0098 A2eo units of synthetic poly(U). Values represent means of 7 experiments with the use of 3H-poly(U) hybridization (differences significant at 0.025 probability level) and of 3 experiments with the use of fluorimetric titration. Treatment

H20 Kinetin

% poly(A) content ~H-poly(U) hybridization

Fluorimetric titration

6.6 14.0

4.6 11.2

71 T a b l e II. Effect of kinetin on the engagement of the poly(A) segment in the secondary structure of poly(A)RNA. Poly(A)RNAs were isolated from cotyledons treated with water (control) or 0.1 mM kinetin, aH-poly(U) hybridizations were performed with native preparations or with preparations denatured with heat or formamide. Estimations were made in 7 experiments with similar results, values in the table represent results of one representative experiment. Per cent contents of hybridizable poly(A) were calculated from results of standard hybridization with synthetic poly(A); the radioactivity of the hybrid of 1 ng of synthetic poly(A) with aH-poly(U) amounted to 171 cpm. Treatment of poly(A)RNA

Treatment of cotyledons H20

None Heat Formamide

Kinetin

cpm

%poly(A)

cpm

% poly(A)

1160 2910 3520

6.7 17.0 20.5

3000 3980 4040

17.4 23.1 23.4

2.5-3-fold increase in 3H-poly(U) hybridization. In kinetin poly(A)RNA this increase was considerably lower (1.4-fold). One should conclude t h a t in the kinetin-treated cotyledons, as compared with control ones, poly(A) segments were less engaged in base pairing and formation of helical regions. The higher values obtained in poly(A) estimations in hybridization experiments with the use of native, non-denatured kinetin poly(A)RNA, were thus to a considerable extent a result of lower participation of poly(A) segments in the secondary structure of this poly(A)RNA. However, even after denaturation, hybridization of the kinetin poly(A)RNA was somewhat higher which indicated a somewhat higher length of its poly(A) tail t h a n in control poly(A)RNA. The curves of fluorimetric titration of poly(A)RNA with poly(U) have a convex shape (Fig. 2). This also might suggest a considerable engagement of poly(A) segments in secondary structure of poly(A)RNA, which is to a certain degree inhibitory to poly(A)-poly(U) hybridization. Discussion

The majority of existing data pertaining to secondary structure of mRNA come from experiments in which - similarly as in our study phenol was used as a deproteinizing factor

during the isolation of poly(A)RNA. Some authors consider phenol to be an aggressive factor able to accelerate hybridization [18], as well as an aggregation of RNA molecules [19]. Other authors, however, regard phenol as factor merely stabilizing pre-existing duplexes [20], e.g. it is known that phenol stabilizes RNA to thermal denaturation [21]. The question is thus controversial, however, irrespective of possible changes during the isolation procedure, differences in secondary structure of poly(A)RNA preparations from control and from kinetin-treated cotyledons indicate that cytokinins may be regarded as factors affecting structure of the mRNA population. Phenol was also used for RNA isolation in the study of Trapy et al. [6] who reported that 60% of nucleotides in the poly(A)RNA from a plant tissue - root meristem of V i c i a f a b a were in the base-paired regions. As shown in our experiments, double-stranded regions occur also in poly(A)RNA from cucumber cotyledons. Kinetin treatment of the cotyledons leads to a considerable increase in the extent of secondary structure of the mRNA population, so that about 70% of nucleotides in the poly(A)RNA preparation is engaged in basepairing. Kinetin causes also a slight increase in the length of poly(A) tails. This finding is new for cytokinins, but was previously described for

72 g i b b e r e l l i n s [22]. T h e m o s t d r a m a t i c e f f e c t o f kinetin on poly(A) segments in cucumber cotyledons was, however, a change in their involvement in secondary structure of poly(A) RNA. T h e p a r t i c i p a t i o n o f p o l y ( A ) i n t h e seco n d a r y s t r u c t u r e o f a n i m a l R N A h a s b e e n suggested by results of some investigations [2-4,23]. T h i s s e e m s t o b e t r u e a l s o f o r p l a n t mRNA, as shown in our experiments based on denaturation of poly(A)RNA and 3H-poly(U) hybridization. In kinetin-treated cotyledons, the engagement of poly(A) in the base-pairing in the population of poly(A)RNA appeared to be significantly lower than in the control material. Thus kinetin seems to be a factor increasing the content of double-stranded regions in poly(A)RNA population and at the same time d e c r e a s i n g t h e p a r t i c i p a t i o n o f t h e p o l y ( A ) segments in base-pairing.

References 1 J.N. Vournakis and C.P.H. Vary, Dev. Genet., 4 {1984) 313. 2 W.R. Jeffery and G. Brawerman, Biochemistry, 14 (1975) 3445. 3 M.T. Hsu and M. Coca-Prodos, Nature, 280 (1979) 339. 4 A.M. Ladhoff, I. Uerlings and S. Rosenthal, Mol. Biol. Rep., 7 (1981) 101. 5 G. Albrecht, A. KrSwczyfiska and G. Brawerman, J. Mol. Biol., 178 (1984) 881.

6 G. Trapy, A. Favre and R. Esnault, Plant Mol. Biol., 1 (1981) 53. 7 E.G. Shpaer, Nucl. Acid Res., 13 (1985) 275. 8 E.A. GwSid/ and A. Wo~.ny, Physiol. Plant., 59 (1983) 103. 9 G. Jackowski, J. Legocka and A. Szweykowska, Acta Physiol. Plant., 6 (1984) 181. 10 J. Legocka, Biochim. Physiol. Pflanzen, 182 (1987) 299307. 11 J. Legocka, B. Waliszewska-Wojtkowiak, J. Okularczyk, E.A. GwSid/and A. Szweykowska, Acta Physiol. Plant., 6 (1984) 173. 12 S.L. Berger and C.S. Birkenmeier, Biochemistry, 18 (1979) 5149. 13 M.O. Cabada, C. Darnbrough, P.J. Ford and P. Turner, Dev. Biol., 57 (1977) 427. 14 P.A. Gerdes, and G. Kidder, Can. J. Biochem., 57 (1979) 1305. 15 A. Favre, U. Bertazzoni, A.J.M. Berns and H. Bloemendal, Biochem. Biophys. Res. Commun., 56 (1974) 273, 16 I.C. Bathurst and M.G. Smith, Biochim. Biophys. Acta, 669 (1982) 84. 17 C. Urbanke, R. Romer and G. Maass, Eur. J. Biochem., 33 (1973) 511. 18 D.E. Kohne, S.A. Levison and M.J. Byers, Biochemistry, 16 (1977) 5329. 19 M. Nomura and V.A. Erdmann, Nature, 228 (1970) 744. 20 K.S. Miller, V. Zbrzezna and A.O. Pogo, J. Mol. Biol., 177 (1984) 343. 21 A.O. Pogo, in: E. Busch (ed.), The Cell Nucleus 8, Academic Press, New York, 1981, p. 331. 22 L.D. Wasilewska and K. Kleczkowski, Eur. J. Biochem., 66 (1976) 405. 23 J.W. Kulkosky, W.M. Wood and M. Edmonds, Biochemistry, 24 (1985) 3678.