Plant Cell Reports

Plant Cell Reports (1982) 1 : 165-167

© Springer-Verlag 1982

Stability of Poly(A)+RNA in Nucleate and Anucleate Cells of Acetabularia Klaus Kloppstech and Hans-Georg Schweiger Max-Planck-Institut ffir Zellbiologie, 6802 Ladenburg bei Heidelberg Received January 29, 1982; March 3, 1982

Abstract

Nethods

The s t a b i l i t y o f poly(A)+RNA was compared in n u c l e a t e and a n u c l e a t e cells of Acetabularia. While i n the absence o f t h e n u c l e u s p o l y ( A ) + R N A e x h i b i t s a pronounced s t a b i l i t y , it is s i g n i f i c a n t l y l e s s s t a b l e in the presence of tile nucleus.

Cells of Acetabularia (A.) mediterranea were grown under c o n d i ~ s ~ w h i c h have--been d e s c r i b e d p r e v i o u s l y ( S c h w e i g e r 1969; B e r g e r and S c h w e i g e r 1980). C e l l s o f 25 to 30 mm l e n g t h , p r i o r to cap f o r m a t i o n , were used f o r the e x p e r i m e n t s . A t o t a l o f 4000 c e l l s were i n c u b a L e d ii~ 9roups o f 200 c e l l s per 10 ml o f E r d - S c h r e i b e r - m e d i u m (ESM) in the p r e s e n c e o f 3.7 MBq ( 3 H ) - u r i d i n e (spec. a c t . 1.1 TBq/mmol ) and 20 pg/ml r i f a m p i c i n f o r 24 h and t h e r e a f t e r t r a n s f e r r e d to f r e s h n o n - r a d i o a c t i v e medium. S i x days a f t e r i n c u b a t i o n , h a l f o f the c e l l s were l i g a t e d in groups o f 5 c e l l s by a t h i n t h r e a d 5 mm above the r h i z o i d and t h e r h i z o i d s were removed by a m p u t a t i o n . D u p l i c a t e samples o f 100 n u c l e a t e and a n u c l e a t e c e l l s r e s p e c t i v e l y were t a k e n a t the i n d i c a t e d t i m e s and f r o z e n as w e l l as s t o r e d in l i q u i d n i t r o g e n .

Introduction A n u c l e a t e c e l l s o f A c e t a b u l a r i a are c a p a b l e of performing morphogenesis over a period o f more than f o u r weeks a f t e r removal o f the n u c l e u s (H~mmerling 1 9 3 2 ) . At the m o l e cular level, i t has been s h o w n t h a t a t l e a s t some p r o t e i n s which are d e f i n i t e l y coded f o r by the n u c l e a r genome, i . e . , the i s o enzymes o f m a l a t e d e h y d r o g e n a s e (MDH) ( S c h w e i g e r e t a l . 1 9 6 7 ) , are s y n t h e s i z e d in the absence o f t h e n u c l e u s f o r s e v e r a l weeks. T h e r e f o r e , i t has been c o n c l u d e d t h a t mRNA i s e x t r e m e l y s t a b l e i n a n u c l e a t e A c e t a bularia cells. After implantation of a heterologous nucleus i n t o an a n u c l e a t e c e l l f r a g m e n t , two s t r i k ing changes can be o b s e r v e d : f i r s t , isozymes which are c h a r a c t e r i s t i c f o r the n u c l e u s donor s p e c i e s a p p e a r and second, the isozymes which are s p e c i f i c f o r the cytoplasm acceptor species disappear ( S c h w e i g e r 1 9 6 9 ) . This a p p a r e n t p a r a d o x m i g h t be e x p l a i n e d by d i f f e r e n t stabilit i e s o f mRNA in n u c l e a t e and a n u c l e a t e cells. The v e r i f i c a t i o n o f an i n f l u e n c e o f the n u c l e u s on the s t a b i l i t y o f RNA would be o f g r e a t i n t e r e s t w i t h r e s p e c t to t h e i n s i g h t i n t o t h e r e g u l a t i o n o f gene e x p r e s s i o n in the e u k a r y o t i c c e l l . For t h i s r e a s o n , we e s t i m a t e d the s t a b i l i t y of poly(A)+RNA in n u c l e a t e and e x p e r i m e n t a l l y enucleated cells of Acetabularia.

A l l samples were p r o c e s s e d a t the same t i m e . For t h i s p u r p o s e , 0.5 ml o f b u f f e r (0.1 M NaCl; 0.05 M T r i s / H C l , pH 7 . 4 ; 0.05 M EDTA; 2% SDS) and I mg o f E. c o l i r i b o s o m e s were f r o z e n and ground i n l i q u i d n i t r o g e n . T h e r e after, 100 c e l l s were added, ground to powder and t r a n s f e r r e d to 10 ml o f the same b u f f e r c o n t a i n i n g 200 ~g/ml p r o t e i n a s e K. The s u s p e n s i o n was r a p i d l y b r o u g h t to room t e m p e r a t u r e by s h a k i n g i n a w a t e r bath a t 60 ° C. The phenol e x t r a c t i o n was p e r f o r m e d as d e s c r i b e d ( K l o p p s t e c h and S c h w e i g e r 1975). The s u p e r n a t a n t o f the f i n a l c h l o r o f o r m e x traction step was s u p p l e m e n t e d w i t h 1/10 volume o f 4 M NaCl and poly(A)+RNA was bound to 0.2 g o l i g o ~ d T ) c e l l u l o s e Purification o f the poly(A)TRNA f o l l o w e d t h e p r o c e d u r e d e s c r i b e d ( K l o p p s t e c h and S c h w e i g e r 1975) e x c e p t t h a t NaCl was s u b s t i t u t e d f o r KCI and 0.2% SDS was used t h r o u g h the e n t i r e p r o c e d u r e . The poly(A)+RNA was f i n a l l y eluted with 6 x I ml o f d e s t i l l e d w a t e r . One hundred pg tRNA was added to d u p l i c a t e 2 ml a l i q u o t s o f the combined f r a c t i o n s and then 2 ml o f 100%

0 7 2 1 - 7 7 1 4 / 8 2 / 0 0 0 1 / 0 1 6 5 / $ 01.00

166 TCA was added. The p r e c i p i t a t e was c o l l e c t e d on c e l l u l o s e nitrate filters, w h i c h were then d r i e d and c o u n t e d . The amount o f p o l y ( A ) + per c e l l was e s t i m a t e d by h y b r i d i z a t i o n of the phenol e x t r a c t e d RNA w i t h t r i t i a t e d polyuridylic a c i d a c c o r d i n g to the method o f S u l l i v a n and Roberts 1973. P o l y a d e n y l i c a c i d was used to c a l i b r a t e the determinations. Results In o r d e r to d e t e r m i n e the s t a b i l i t y o f nuc l e a r RNA in a n u c l e a t e c e l l s , i t was n e c e s s a r y to make sure t h a t the t r a n s p o r t a t i o n of the n e w l y s y n t h e s i z e d , l a b e l e d RNA from t h e n u c l e u s to the c y t o p l a s m was e s s e n t i a l l y comp l e t e d b e f o r e the assay was p e r f o r m e d . For t h i s r e a s o n , the r h i z o i d s c o n t a i n i n g the n u c l e i were removed n o t e a r l i e r than six days a f t e r t h e l a b e l i n g ( K l o p p s t e c h and Schweiger 1975). S u r p r i s i n g l y , despite this rather long period of chase, in all experiments w i t h n u c l e a t e c e l l s a s l i g h t ~ncrease i n the r a d i o a c t i v i t y o f the p o l y ( A ) - R N A fraction was o b s e r v e d d u r i n g the two weeks a f t e r t h e removal o f the n u c l e u s ( F i g . I ) . In the p o l y ( A ) - R N A f r a c t i o n no such i n c r e a s e was f o u n d . T h i s r e s u l t m i g h t i n d i c a t e t h a t p a r t o f t h e RNA o f the n u c l e u s becomes p o l y adenylated only very slowly.

&O

"~"

3.0

E

60

x

8

~.o

2.o

In n u c l e a t e c e l l s , the p o l y ( A ) + R N A w h i c h had been p u l s e - l a b e l e d d u r i n g the 25-30 mm stage remained s t a b l e o v e r a p e r i o d o f a t l e a s t t h r e e weeks, b e f o r e i t s t a r t e d to d e c l i n e with a half-life o f a b o u t ten days. The decay o f t h i s l a b e l e d f r a c t i o n of poly(A) + RNA c o i n c i d e d w i t h cap f o r m a t i o n In cont r a s t to the n u c l e a t e c e l l s , pol;(A)+RNA in enucleated cells exhibited only a slight d e c l i n e d u r i n g a seven weeks p e r i o d o f obs e r v a t i o n ( F i g . I ) . W h i l e in a n u c l e a t e c e l l s p o l y ( A ) RNA was r a t h e r s t a b l e , the r a d i o activity o f the p o l y ( A ) - R N A f r a c t i o n dec l i n e d to a b o u t one t h i r d o f the o r i g i n a l v a l u e ( d a t a n o t shown) w i t h i n seven weeks. A similar loss of radioactivity was observed in n u c l e a t e c e l l s . S i n c e the p o l y ( A ) RNA i n c l u d e d the t o t a I e x t r a c t a b l e cellular RNA minus the p o l y ( A ) ' R N A we c a n n o t e x c l u d e the p o s s i b i l i t y that different populations o f t h i s RNA e x h i b i t d i f f e r e n t stabilities. For i n s t a n c e , i t m i g h t be t h a t the mRNA i n the p o l y ( A ) - RNA f r a c t i o n a l s o remains s t a b l e in anucleate cells. However, one c o u l d e x p e c t t h a t t h i s poly(A)-mRNA does n o t a c c o u n t f o r more t h a n a b o u t I% o f the ~ o t a l c e l l u l a r RNA as i s the case f o r poly(A)TmRNA and t h e r e f o r e escaped d e t e c t i o n . A c c o r d i n g to our observations, the amount o f p o l y ( A ) i n the h i g h m o l e c u l a r w e i g h t RNA f r a c t i o n increased up to the p e r i o d o f cap f o r m a t i o n ( F i g . 2 ) , w h i c h c g r r e s p o n d e d to an i n c r e a s e i n the poly(A)'RNA fraction s i n c e the average l e n g t h o f the p o l y ( A ) c h a i n i n d i f f e r e n t developmental s t a g e s was between 50 and 100 n u c l e o t i d e s ( K l o p p s t e c h and S c h w e i g e r 1 9 7 5 ) . T h i s s t a t e m e n t i s s u p p o r t e d by the f a c t t h a t the UV a b s o r p t i o n p r o f i l e s o f the o l i g o ( d T ) cellulose eluate fraction increased with d e v e l o p m e n t on a per c e l l b a s i s . S h o r t l y a f t e r the h i g h e s t l e v e l o f p g l y ( A ) had been reached ( F i g . 2) the p o l y ( A ) ' R N A s y n t h e s i z e d a t an e a r l i e r stage of development was r a p i d l y degraded i n the n u c l e a t e c e l l s .

z,,.O

~

11)

3.0

2.0 =~ 1'0

2'0 time

3'0

z.'o

SO

(days)

Fig. 1 Stability of poly(A)+RNA in nucleate and a n u c l e a t e c e l l s o f A c e t a b u l a r i a m e d i terranea After labeling with (3H)-uridine f o r 24 h on day O, the c e l l s were kept i n n o n - r a d i o a c t i v e medium. H a l f o f the c e l l s were e n u c l e ated on day 9. Samples o f 2 x 100 c e l l s were frozen in liquid n i t r o g e n at d i f f e r e n t time~ and l e n g t h and r a d i o a c t i v i t y o f the p o l y ( A ) T RNA f r a c t i o n was e s t i m a t e d . The a r r o w i n d i c a t e s where cap f o r m a t i o n s t a r t s . •

•

O

O

•

•

[]

[]

poly(A)+RNA in nucleate cells poly(A)+RNA in anucleate cells length of nucleate cells length of anucleate cells

/

~.0

E ~

.--

31)

cJ, x

z1)

./ 10

2'0 3'0 (mm )

4'0

length

Fig. 2 Poly(A) content of cells of different stages of development For each d e t e r m i n a t i o n the RNA was e x t r a c t e d from 100 c e l l s by phenol c h l o r o f o r m ( K l o p p s t e c h and S c h w e i g e r 1975) and h y b r i d i z e d to (3H)-polyuridylic a c i d . The p o l y ( A ) c o n t e n t was e s t i m a t e d as r a d i o a c t i v i t y resistant to t r e a t m e n t w i t h RNAase ( S u l l i v a n and R o b e r t s 1973).

167 Discussion I t has been shown t h a t p o l y ( A ) + R N A i s more s t a b l e in a n u c l e a t e than i t i s i n n u c l e a t e c e l ~ s . In the p r e s e n c e o f the n u c l e u s , p o l y (A) RNA w h i c h had been s y n t h e s i z e d d u r i n g the 25 mm s t a g e d e c l i n e s q u i t e r a p i d l y d u r i n g cap f o r m a t i o n . I t r e m a i n s an open' question whether this labilization of poly (A)-RNA can be a t t r i b u t e d to a d i r e c t or to an i n d i r e c t i n f l u e n c e o f the n u c l e u s . One p o s s i b l e e x p l a n a t i o n c o u l d be tha t the i n c r e m e n t i n the s t a b i l i t y of poly(A)'RNA i s the r e s u l t o f a l o w e r r a t e o f r e i n i t i a t i o n o f mRNA d u r i n g t r a n s l a t i o n in anucleate c e l l s . This interpretation seems to be unlikely f o r the f o l l o w i n g reason: It is known from s t u d i e s on r e t i c u l o c y t e s that the i n i t i a t i o n but not the elongation, i s the r a t e - l i m i t i n g s t e p i n p r o t e i n synt h e s i s ( L o d i s h and Desalu 1 9 7 3 ) . N u c l e a t e as w e l l as a n u c l e a t e c e l l s e x h i b i t a simil a r r a t e o f g r o w t h ( F i g . I ) and a s i m i l a r i n c r e a s e i n t o t a l p r o t e i n ( C l a u s s and Werz 1961) or i n p a r t i c u l a r p r o t e i n s l i k e MDH (Schweiger et a l . 1967). T h e r e f o r e , r e i n i tiation s h o u l d n o t be s i g n i f i c a n t l y lower in anucleate than in nucleate cells, especially i f one c o n s i d e r s t h a t i n c o n t r a s t to n u c l e a t e c e l l s the amount o f 8OS r i b o s o m e s d e c l i n e s s l o w l y a f t e r removal o f the nucleus. A second e x p l a n a t i o n f o r the d e c r e a s e o f stability o f p o l y ( A ) + R N A i n the p r e s e n c e o f the n u c l e u s m i g h t be t h a t the p o l y ( A ) c h a i n becomes s h o r t e n e d f a s t e r i n n u c l e a t e t h a n i n a n u c l e a t e c e l l s and as a consequence the p o l y ( A ) ~ R N A w o u l d no l o n g e r be de-tected by the chosen method o f i s o l a t i o n with oligo(dT) cellulose. This interpretation i s a l s o improbable, since although in nucleate cells of Acetabularia the r a t e o f s y n t h e s i s i s slightly reduced a t the end o f the v e g e t a t i v e phase ( K l o p p s t e c h and Sc h w e i g e r 1977) the amount o f p o l y ( A ) + R N A w h i c h can be i s o l a t e d by the above m e n t i o n e d method, s t i l l i n c r e a s e s up to a t l e a s t the s t a g e o f cap formation (Fig. 2). A t h i r d e x p l a n a t i o n i s based on the f a c t t h a t the t o t a l amount o f p o l y ( A ) ~ R N A i n nucleate cells is not substantially differe n t from t h a t i n a n u c l e a t e c e l l s . This means t h a t c o r r e s p o n d i n g to the r a t e o f synthesis of poly(A)RNA in nucleate cell~ a s i m i l a r r a t e o f d e g r a d a t i o n o f p o l y ( A ) RNA must o c c u r . P o s s i b l y , t h i s i n c r e a s e d r a t e o f degradation reflects a higher percentage of mRNA w h i c h i s n o t bound to r i b o s o m e s and t h e r e b y n o t p r o t e c t e d from d e g r a d a t i o n ,

w h i l e in a n u c l e a t e c e l l s the mRNA becomes reinitiated a t a maximal r a t e . An i n t e r e s t i n g q u e s t i o n w h i c h can be r a i s e d i n t h i s c o n n e c t i o n i s w h e t h e r i n ~ u c l e a t e c e l l s the n e w l y s y n t h e s i z e d p ~ l y ( A ) RNA or the a l r e a d y preexistent poly(A)-RNA is preferentially d e g r a d e d . Even i f t h i s d e g r a d a t i o n happens in a non-preferential way, i . e. the chances f o r d e g r a d a t i o n o f both t y p e s o f p o l y ( A ) are s i m i l a r , a f t e r a c e r t a i n p e r i o d o f time the p r e e x i s t e n t p o l y ( A ) + R N A would be r e p l a c e d by n e w l y s y n t h e s i z e d p o l y ( A ) + R N A . The adv a n t a g e o f such a w o r k i n g h y p o t h e s i s would be t h a t i t c o u l d a l s o e x p l a i n the abovem e n t i o n e d r e s u l t s c o n c e r n i n g the d i s a p p e a r ance o f an o l d isozyme p a t t e r n i n h e t e r o logous nucleo-cytoplasmic hybrids (Schweiger et a l . 1 9 6 7 ) . H y b r i d i z a t i o n experiments with specific cDNAs m i g h t h e l p to check t h i s working hypothesis.

Acknowledgements: We are g r a t e f u l to Mrs. Mo S c h i m b o r s k i f o r e x p e r t t e c h n i c a l a s s i s t a n c e and to Dr. R. Shoeman f o r c r i t i c a l suggestions during p r e p a r a t i o n o f the m a n u s c r i p t .

References B e r g e r S, S c h w e i g e r HG (1980) I n : G a n t t E (ed) Handbook o f P h y c o l o g i c a l Methods Cambridge U n i v e r s i t y P r e s s , pp 47-57 Clauss H, Werz G (1961) 162-165 H~mmeriing 0 (1932) 42-61

Z

Bioi

Naturforsch Zentraibl

K l o p p s t e c h K, S c h w e i g e r HG (1975) tiation 4: 115-123

16 b: 52:

Differen-

K l o p p s t e c h K, S c h w e i g e r HG (1977) I n : Woodcock CL:: (ed) P r o g r e s s i n A c e t a b u l a r i a R e s e a r c h , Academic P r e s s , New ¥ ~ San F r a n c i s c o , London, pp 19-32 L o d i s h HF, Desalu 0 (1973) 248:3520-3527 S c h w e i g e r HG (1969) Immunol. 50: 1-36 S c h w e i g e r PIG, M a s t e r N a t u r e 216: 554-557 S u l l i v a n N, R o b e r t s 12: 2395-2403

Curr

J Top

Biol

Chem

[licrobiol

RWP, Werz G (1967) WK (1973)

Biochemistry