the cap and poly(a) tail function

8/2/2019 The Cap and Poly(a) Tail Function

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10.1101/gad.5.11.2108Access the most recent version at doi:1991 5: 2108-2116Genes Dev.

D R Gallietranslational efficiency.The cap and poly(A) tail function synergistically to regulate mRNA

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T h e c ap a n d p o l y ( A ) t a il f u n c t i o ns y n e r g i s t i c a l l y t o r e g u l a t e m R N At r a n s l a t i o n a l e f f i c i e n c yD a n i e l R . G a l l i eDep artm ent of Biochemistry, Un iversity of California, Riverside, California 92521 USA

T h e c a p s t r u c tu r e a n d t h e p o l y ( A ) t a i l ar e i m p o r t a n t r e g u l a to r y d e t e r m i n a n t s i n e s t a b l i s h i n g t h e t r a n s l a t i o n a le f fi c ie n c y o f a m e s s e n g e r R N A . A l t h o u g h t h e m e c h a n i s m b y w h i c h e i t h e r d et e r m i n a n t f u n c ti o n s r e m a i n sp o o r l y c h a r a c te r i z e d , t h e i n t e r a c t i o n b e t w e e n t h e p o l y( A ) t a i l- p o l y ( A ) - b i n d i n g p r o t e in c o m p l e x a n d e v e n t so c c u r r i n g a t t h e 5 ' t e r mi n u s d u r i n g t r a n s l a t i o n i n i t i a t i o n h a s b e e n a n i n t r i g u i n g p o s s i b i l i t y . I n t h i s r e p o r t , t h em u t u a l d e p e n d e n c e o f t h e c a p a n d t h e p o l y (A) t a i l wa s s t u d i e d . P o l y ( A) + a n d p o l y ( A ) - l u c i f e r a s e (Luc) m R N A sg e n e r a te d i n v i t r o c o n t a i n i n g o r l a c k i n g a c a p w e r e t r a n s la t e d i n v iv o i n t o b a c c o p r o t o p l a s ts , C h i n e s e h a m s t e ro v a r y c e l l s , a n d y e a s t f o l l o w i n g d e l i v e r y b y e l e c t r o p o r a ti o n . T h e p o l y( A ) t a i l -m e d i a t e d r e g u l a t i o n o ft r a n s l a t i o n a l e f f i c i e n c y w a s w h o l l y d e p e n d e n t o n t h e c a p f o r f u n c t i o n . M o r e o v e r , c a p f u n c t i o n w a s e n h a n c e do v e r a n o r d e r o f ma g n i t u d e b y t h e p r e s e n c e o f a p o l y ( A) t a i l . T h e r e l a t i v e d i f f e r e n c e s i n s t a b i l i t y b e t we e n t h em R N A s c o u l d n o t a c c o u n t f o r t h e s y n e r g i s m . T h e s y n e r g i s m b e t w e e n t h e c a p a n d p o l y ( A ) t a i l w a s n o to b s e r v e d i n y e a s t c e l l s i n w h i c h a c t i v e t r a n s l a t i o n h a d b e e n d i s r u p t e d . I n a d d i t i o n , t h e s y n e r g i s m w a s n o to b s e r v e d i n i n v i t r o t r a n s l a t i o n l y s a t e s . T h e s e d a t a d e mo n s t r a t e t h a t t h e c a p a n d t h e p o l y ( A) t a i l a r ei n t e r d e p e n d e n t f o r o p t i m a l f u n c t i o n i n v i v o a n d s u g g e s t t h a t c o m m u n i c a t i o n b e t w e e n t h e t w o r e g u l a t o r yd e t e r m i n a n t s m a y b e i m p o r t a n t i n e s t a b l i s h i n g e f f i c i e n t t r a n s l a t i o n .[Key Words: T r a n s l a t i o n a l c o n t r o l ; m R N A ; p o ly ( A ) t a il ; c a p ; g en e e x p r e s s io n ]Received July 22, 1991; revised version accepted August 11, 1991.

S i n c e t h e i r d i s c o v e r y , b o t h t h e c a p a n d t h e p o l y ( A ) t a i lh a v e b e e n i m p l i c a t e d a s b e i n g i n v o l v e d i n th e r e g u l a t i o no f t r a n s l a t i o n a l e f f i c i e n c y a n d m e s s a g e s t a b i l i t y ( F u r u i -ch i e t a l . 1977 ; F i l i powi cz 1978 ; B aner j ee 1980 ; B r awer -m an n 1981 ; Gr e en e t a l . 1983 ; R ho ads 1988 ; S onenber g1 9 8 8 ; B e m s t e i n a n d R o s s 1 9 8 9 ; J a c k s o n a n d S t a n d a r t1 9 9 0; M u n r o e a n d J a c o b s o n 1 9 9 0 a, b ). R e c e n t l y , w e d e m -o n s t r a t e d t h a t a l t h o u g h t h e a d d i t i o n o f a p o l y ( A ) t a i l t op o l y ( A ) - m R N A i n c r e a s e d b o t h i t s t r a n s l a t i o n a l e f f i -c i e n c y a n d s t a b i l i t y i n p l a n t a n d a n i m a l c e l l s, t h e p o l y (A )t a i l w a s p r e d o m i n a n t l y a r e g u l a t o r o f t r a n s l a t i o n a l e f f i -c i enc y ( Ga l li e e t a l . 1989, 1991). C on s i de r ab l e ev i de nceh a s a c c u m u l a t e d r e c e n t l y t o s u p p o r t t h i s v i e w . T h ep o l y ( A ) - b in d i n g ( P A B ) p r o t e i n , a h i g h l y c o n s e r v e d p r o -t e i n i n euka r yo t es ( Gr ange e t a l . 1987) , med i a t e s t he r eg-u l a t i o n a s s o c i a t e d w i t h t h e p o l y ( A ) t a i l a n d i s e s s e n t i a lf o r v i ab i l i t y i n yeas t ( S achs e t a l . 1987) . S uppr es so r mu -t a n t s w h i c h o v e r c o m e t h e l o s s o f P A B p r o t e i n a f f e c t 60 Sr i b o s o m a l s u b u n i t b i o g e n e s i s o r f u n c t i o n ( S a c h s a n dDavi s 1989) . I n an i n v i t r o s t udy , exogenous o l i go( A) r e -d u c e d t h e t r a n s l a t i o n o f c a p p e d, p o l y (A ) + m R N A i n r ab -b i t r e t i c u l o c y t e l y s a t e , p o s s i b l y b y c o m p e t i n g f o r P A Bp r o t e i n . M o r e o v e r , a t c e r t a i n c o n c e n t r a t i o n s , e x o g e n o u so l ig o ( A I c o u l d s t i m u l a t e t h e t r a n s l a t i o n o f c a p p e d ,p o l y ( A ) - m R N A i n t rans ( M unr oe and Jacobson 1990a) .I t h a s b e e n p o s t u l a t e d , t h e r e f o r e , t h a t i n t e r a c t i o n .be-

t w e e n t h e p o l y ( A ) t a i l (a s m e d i a t e d b y P A B p r o t e in ) a n dt h e 6 0 S s u b u n i t i s i m p o r t a n t i n c o n t r o l l i n g 8 0 S i n i t i a -t i o n c o m p l e x f o r m a t i o n ( S a c h s a n d D a v i s 1 9 8 9 ) . W e h y -p o t h e s i z e d t h a t t h e p o ly ( A ) t a i l - P A B p r o t e i n c o m p l e xm a y n o t b e l i m i t e d t o i n te r a c t i o n w i t h t h e 6 0 S s u b u n i t,b u t m a y b e i n v o l v e d i n e a r l i e r e v e n t s i n t r a n s l a t i o n i n i -t i a t io n , s u c h a s c a p r e c o g n i t i o n . T o t e s t t h i s p o s s i b i l i t y ,w e s y n t h e s i z e d m R N A s i n v i t r o a s p o l y (A ) + o r p o l y ( A ) -t h a t w e r e e i t h e r c a p p ed o r u n c a p p e d a n d d e t e r m i n e dt h e i r t r a n s l a t i o n a l c o m p e t e n c e i n v i v o i n t o b a c c o p r o t o -p l a s ts , C h i n e s e h a m s t e r o v a r y ( C H O ) c e ll s , a n d y e a s t f ol -l o w i n g d e l i v e r y u s in g e l e c t r o p o r a t i o n . U s i n g t h e f i r e f l yl uc i f e r a se (Luc) r e p o r t e r g e n e, t h e k i n e t i c s o f t r a n s l a t i o nc a n b e f o l l o w e d , a n d f o r t h e f i r s t t i m e , t h e s y n e r g i s mb e t w e e n t h e c a p a n d t h e p o l y ( A ) t a i l i n t h e i r r o l e a s re g -u l a t o r s o f t r a n s l a t i o n a l e f f i c i e n c y c a n b e d e m o n s t r a t e d i nv i vo .

R e s u l t sT h e f u n c t i o n o f t h e ca p a n d p o l y ( A ) t a i l a re m u t u a l l yd e p e n d e n t a s re g u l a to r s o f t r a n s l a t i o n a l e f f i c i e n c yi n t o b a c c oT h e d e b a t e c o n c e r n i n g t h e f u n c t i o n a l r o l e o f t h e p o l y ( A )t a i l in i n c r e a s i n g e x p r e s s i o n h a s n o t y e t b e e n r e s o l v e d . In

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Cap and poly(A) tail function synergistically

plants, however, the poly(A) tail has a dual cytoplasmicregulatory role: mRNA s contain ing a poly(A)s o tail aretwo- to threefold more stable than their poly(A)- coun-terparts and translational efficiency is markedly stimu-lated (Gallie et al. 1989). Because of this demons tratedinvolvement in regulating translation, we wished to ex-amine whether there might be a functional interactionbetween the cap at the 5' terminus of an mRNA and thepoly(A) tail. To directly assess the effect of the cap onpoly(A) tail function, Luc mRNA constructs were syn-thesized in vitro as four different species: uncappedpoly{A)-, uncapped poly(A) +, capped poly(A)- , andcapped poly(A) + mRN A. The poly(A) + Luc mRNAswere synthesized from a T7-Luc vector containing apoly(A)s o sequence, there by ensuring product ion of LucmRNA with a uniform poly(A) tail length. Moreover, theconditions used for the in vitro synthesis of capped RNAresult in mRNA that is uniformly capped (Yisraeli andMelton 1989).

To test the in vitro synthesized mRNAs in vivo, 1 ~geach of the four Luc mRNAs was electroporated intotobacco protoplasts and the cells incubated overnight,sufficient time to ensure complete degradation of theLuc mRNAs. In tobacco protoplasts, luciferase expres-sion was 1.5-fold greater from uncapped, poly(A) + LucmRNA t han from uncapped, poly(A)- Luc mRNA (Table1). An excellent correlation can be made between theimpact of the poly(A) tail on LUC expression and itseffect on the physical half-life of the Luc transcript. Thehalf-l ife of poly(A) + Lu c mRNA was 1.4-fold longer thanthat for the corresponding po ly(A)- mRN A (Fig. 1), ingood agreement with earlier measurement s (Gallie et al.1989, 1991). In contrast to the small impact of thepoly(A) tail on uncapped Luc mRNA, the addition of apoly{A) tail increased expression dra matically whe nadded to the capped form of the mRNA. Expression fromthe poly(A) + mRNA was 21-fold greater than thepoly(A)- mRNA (Table 1). The poly(A) tail, however,increased capped Luc mRNA half-life only 1.9-fold (Fig.1), which is insufficient to account for the effect of thepoly(A) tail on capped Lu c mRNA translation. Althoughthe poly(A) tail does stabilize Luc mRNA whether it iscapped or not, it is only when the mRNA is capped that

Figure 1. In vivo message stabilization conferred by a cap anda poly(A) tail in tobacco. (A) uncapped Luc mRNA; {B) Un-capped Luc-Aso mRNA; (C) cappedLuc mRNA; (D) capped Luc-Aso. Aliquots of protoplasts electroporated with 5 ~g of eachmRNA were removed at the time indicated at the top of eachlane. RNA half-life analysis was carried out as described in Ma-terials and methods.

its second and more important function, that is increas-ing translational efficiency, is apparent.Examination of the data from the perspective of thecap demonstrates the requirement for a poly(A) tail forthe cap to function optimally. Addition of a cap in-creased expression 21-fold for poly(A)- Lu c mRNA butincreased expression 297-fold for poly(A) + Lu c (Table 1).Since the addition of a cap stabilized poly(A)- andpoly(A) + Luc mRNA to approximately the same extent,the effect of the cap on Luc mRNA half-life could notaccount for the difference in cap activity. We conclude,therefore, that although a cap does increase translationalefficiency in the absence of a poly(A) tail, its function isenhanced by an order of magnitude by a poly(A) tail.The monomethylated form of the cap (mTGpppG) wasused throughout these experiments. To examine the roleof the methyl group in the synergistic interaction withthe poly(A) tail, capped (GpppG) Luc mRNAs were testedin tobacco protoplasts. The interdependence betweenthe cap and the poly(A) tail was observed with thesemRNAs, illustrating that, although the methyl group in-creases the synergism, it is not absolutely required (Ta-ble 1). We have repeated these experiments more than 15times to rule out potential variation in protoplast qual-ity. With each repetition, the interdependence was ob-served. We conclude, therefore, that the cap and poly(A)tail interact synergistically: In its capacity as a regulator

Tabl e 1. Synergism of cap and poly(A) during translation in tobacco protoplasts electroporated with in vitro-synthesizedLucmRNAmRNA Luciferase activity(light unit/mg protein) Relative effect ofpoly(A) tail on expression Relativeeffect of cap on expressionUncapped

Luc 2,941Luc-Aso 4,480Capped (GpppG)Luc 35,442Luc-Aso 406,878Capped (mTGpppG)Luc 62,595Luc-Aso 1,331,917

1 11.51 1211.51 2121

91 9

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G a l l i c

o f t r a n s l a t i o n a l e f f i c i e n c y , t h e p o l y ( A ) t a i l i s d e p e n d e n to n t h e c a p f o r f u n c t i o n ; t h e a c t i v i t y o f t h e c a p i s n o te n t i r e l y d e p e n d e n t o n , b u t i s e n h a n c e d b y , a p o l y ( A ) t a i l .

I f r e g u l a t i o n o f t r a n s l a t i o n a l e f f i c i e n c y b y t h e p o l y ( A )t a i l i s s t i m u l a t e d b y a c a p , a n i n c r e a s e i n t h e r a t e o fa p p e a r a n c e o f l u c i f e r a s e s h o u l d b e o b s e r v e d . A s i m i l a ri n c r e a s e i n t h e t r a n s l a t i o n a l e f f i c i e n c y s h o u l d b e d e -t e c t e d f o r t h e p o l y ( A ) s t i m u l a t i o n o f c a p f u n c t i o n . T oa d d r e s s t h i s p o s s i b i l i t y , t h e s a m e f o u r Lu c m R N A c o n -s t r u c t s t e s t e d a b o v e w e r e e l e c t r o p o r a t e d i n t o t o b a c c op r o t o p l a s t s , a l i q u o t s o f c e l l s w e r e t a k e n a t t i m e i n t e r -v a l s , a n d a s s a y e d f o r l u c i f e r a s e a c t i v i t y . L u c i f e r a s e a c t i v -i t y w a s p l o t t e d a s a f u n c t i o n o f t i m e [ F i g . 2 ) a n d t h et r a n s l a t i o n a l e f f i c i e n c y f o r e a c h c o n s t r u c t d e t e r m i n e df r o m t h e f i r s t d e r i v a t i v e o f e a c h c u r v e { F ig . 2 ). L u c i f e r a s ei s a s t a b l e p r o t e i n u n d e r t h e s e c o n d i t i o n s ; t l A = 2 0 h r a t2 4 ~ i n to b a c c o ( D .R . G a l l i e , u n p u b l . ); t h e r e fo r e , it s e n -z y m e a c t i v i t y a c c u r a t e l y r e f l e c t s L U C p r o t e i n p r o d u c -t i o n . T h e t r a n s l a t i o n a l e f f i c i e n c y o f u n c a p p e d , p o l y ( A ) +L u c m R N A w a s e q u i v a l e n t t o t h a t o f t h e u n c a p p e d ,p o l y ( A ) - m R N A . I n c o n t ra s t , t h e a d d i t io n o f a p o l y (A )t a i l t o c a p p e d Lu c m R N A i n c r e a s e d t r a n s l a t i o n a l e f f i -c i e n c y 1 5 - f o l d , d e m o n s t r a t i n g t h a t t h e p o l y ( A ) t a i l n o to n l y r e g u l a t e s t r a n s l a t i o n a l e f f i c i e n c y i n t o b a c c o b u t r e -

q u i r e s a c a p f o r f u n c t i o n . I n t h i s e x p e r i m e n t , a s b e f o r e ,c a p f u n c t i o n w a s s t i m u l a t e d b y a p o l y (A ) t a i l. A l t h o u g hc a p p e d , p o l y ( A ) - Lu c m R N A w a s t r a n s l a t e d 2 3 - f o l dm o r e e f f i c i e n t l y t h a n t h e u n c a p p e d f o r m ; t h e t r a n s l a -t i o n a l e f f i c i e n c y o f t h e p o l y ( A ) + Lu c m R N A w a s 3 6 5-f o l d g r e a t e r f o r t h e c a p p e d v e r s u s t h e u n c a p p e d f o r m ( F ig .2 ) . T h e p h y s i c a l h a l f - l i f e o f a n m R N A m a y n o t b e a n a c -c u r a t e m e a s u r e m e n t o f i t s f u n c t i o n a l h a l f - l i f e . T h e l o s so f a f e w n u c l e o t i d e s a t e i t h e r t e r m i n u s w o u l d g o u n d e -t e c t e d b y N o r t h e m a n a l y s i s. F o r e x a m p l e , a t r a n s cr i p tw h i c h h a d l o s t j u s t t h e c a p t h r o u g h e x o n u c l e a s e a t t a c kc o u l d n o t b e r e s o l v e d f r o m t h e " fu l l - l eng th" t r a n s c r i p tp o p u l a t i o n i n p h y s i c a l h a l f - l i f e d e t e r m i n a t i o n s b u tw o u l d b e s i g n i f i c a n t l y c o m p r o m i s e d i n i t s t r a n s l a t i o n a lc o m p e t e n c y . A s t h e p h y s i c a l h a l f - l i f e f o r u n c a p p e d L ucm R N A w a s 3 1 r a i n , s u c h d e c a p p e d t r a n s c ri p t s a r e n o ti m m e d i a t e l y d e gr a de d . A b e tt e r m e a s u r e o f t h e l o n g e v i t yo f a m e s s a g e i s i t s f u n c t i o n a l h a l f - l i f e , d e f i n e d a s t h et i m e r e q u i r e d , f o l l o w i n g m R N A d e l i v e r y , t o r e a c h 5 0 %o f t h e fi n a l l e v e l o f p r o t e i n p r o d u c ed f r o m a g i v e n m R N Ac o n s t r u c t . F u n c t i o n a l h a l f - l i f e , t h e r e f o r e , m e a s u r e s t h el e n g t h o f t i m e o v e r w h i c h t h e m R N A i s a c t i v e l y e n ga g e di n t r a n s l a t i o n , i n c o n t r a s t t o t h e p h y s i c a l h a l f - l i f e , w h i c h

F i g u r e 2 . T h e e f f e c t o f a c a p a n d p o l y { A ) t a i lo n t h e t r a n s l a t io n e f f i c i e n c y a n d f u n c t i o n a lh a l f - l i f e o f Luc m R N A i n t o b a cc o . A l i q u o t s o fp r o t o p l a s t s e l e c t ro p o r a t e d w i t h u n c a p p e d ( A) o rc a p p e d ( B ) Luc m R N A s w e r e t a k en a t t i m e i n -t e r v a ls a n d a s s a y e d ; d a t a w e r e p l o t t e d a s l u-c i f e r as e s p . a c t . v s . t i m e . T h e t r a n s l a t i o n a l e f f i -c i e n c y f or ea c h m R N A c o n s t r u c t w a s d e te r-m i n e d f r o m m a x i m u m r at e of t h e fi rs td e r i v a t i v e o f e a c h c u r v e . T h e f u n c t i o n a l h a l f -l i f e i s d e f i n e d a s t h e t i m e r e q u i r e d t o r e a c h 5 0 %o f t h e m a x i m u m a c c u m u l a t i o n o f l u c if e r as e a c-t i v i t y .

2 8 - -2 6 - -2 4 - -2 2 - -

r o 20--v- -Xc : 18 --o 1 6 -EL~ m 1 4 - -N 12-_5~ 1 0 - -c-

~ 8--6 -4 -2 - -

m R N AU n c a p p e dL u cL u c - A 5oC a p p e dL u c

L u c - A 5 o

A 2 8 0 0 -2 6 0 0 -24OO -2 2 0 0 - -

~ o 2 0 0 0 - -. ~ 1 8 0 0 - -

1 6 0 0 - -EL1 4 0 0 - -1 2 0 0 - -1 0 0 0 - -

L ~ 8 0 0 --

6 0 0 - -L U C - A 5 0 4 0 0 -

A A A

L U C 2 0 o -I I I I I I

5 0 1 0 0 1 5 0 2 0 0 2 5 0 3 0 0 0

B

= L U C

I I I I 5 0 1 0 0 1 5 0 2 0 0 2 50 3 0 0M i n u t e s M i n u t e s

Translat ionalEf f iciency(Light u n i t s /m g m i n )

9 28 8

2 1 6 03 2 1 0 0

R el a t i ver a t e o ft ranslat ion

10 9 6

11 5

F u n ct i o n a lm R N AHalf-life( m i n )

72 6

3 36 3

R el a t i vem R N AHalf - l i fe

13 7

11 .9

M a x i m u mA ccu m u l a t i o n R e l a t i ve(Light uni ts/ level fmg protein) expression

9 4 0 13 2 0 0 3 4

1 0 0 , 0 0 0 12 , 8 0 0 , 0 0 0 2 8

2 1 1 0 GENES & DEVELOPMENT

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serves only as a measure of an mRNA's physical longev-ity without regard to its translational competence.The functional half-life of the uncapped poly(A) + LucmRNA was 3.7-fold longer than the correspondingpoly(A)- mRN A (Fig. 2). Increased message st abilityshould not affect the rate of translation but will directlyaffect the final level of protein produced. This 3.7-foldincrease in half-life correlated well with the 3.4-fold in-crease in the maximum accumulation of luciferase pro-duced from the uncapped, poly(A) + mRNA when com-pared to its poly(A)- counterpar t (Fig. 2). The transla-tional efficiencies for the uncapped mRNAs, it will berecalled, were equivalent. The functional half-life for thecapped, poly(A) + mRN A was 1.9-fold greater than for thecorresponding poly(A)- mRNA. However, a 28-fold dif-ference in the maximum accumulation of luciferase ac-tiv ity was measured (Fig. 2). The effect that the poly(A)tail has on the maximum accumulation of a protein is afunction of its impact on the translational efficiencymultiplied by its impact on mRNA stability. The differ-ence in the translational efficiencies between thecapped, poly(A) + and poly(A)- mRNAs (14.8-fold)andtheir functional half-life measurements (1.9-fold) ac-counts completely for this 28-fold difference.The functional half-life for three of the Luc mRNAswas just 60% of the physical half-life, while for un-capped, poly(A)- Luc mRNA, it was only 23%, resultsreproducibly obtained in subsequent half-life measure-ment experiments. These data suggest that functionalinactivation of the mRNA was occurring more quicklythan could be detected by the physical half-life measure-ments. The functional half-life of a message, therefore,was a more rigorous assessment of the inherent stabilityof a message t han physical half-life measurements. How-ever, neither the physical nor functional half-life mea-surements could account for the synergism between thecap and poly(A) tail.

Synergism between the cap and poly(A) tail is presentin vivo in animal cells but not in an in vitro lysateTo determine whether the interdependence between thecap and the poly(A) tail was common among higher eu-karyotes, we examined Luc mRNA translation in Chi-nese hamster ovary (CHO) cells. In electroporated CHOcells, addition of a poly(A) tail increased expression 8.5-fold for uncapped Luc mRNA but 156-fold when Luc

Cap and po ly (A) ta i l funct ion synerg i st i ca l ly

mRNA was capped (Table 2), demonstrating that al-though the poly(A) tail is partially functional in the ab-sence of a cap, its function is stimula ted by more than anorder of magnitude when the transcript is capped. As intobacco, the poly(A) tail in CHO cells increases LucmRN A half-life only 1.7-fold (Gallie et al. 1991). The capwas almost entirely dependent on the poly(A) tail forfunction. Addition of a cap increased translation ofpoly(A)- Luc mRNA 2.7-fold but increased by 50-foldthe translation of the poly(A) + counterpart.The translational efficiency and functional half-lifewere also measured for the Luc mRNAs in CHO cells(Fig. 3). As seen in tobacco, the presence of poly(A) taildid little to increase the rate of translation or the func-tional half-life of uncapped Luc mRNA. Only when thetranscript was capped was the poly(A) tail effective inincreasing the translat ional efficiency. Similarly, the caprequired a poly(A) tail to function as a regulator of trans-lational efficiency. The synergism was observed for asecond gene, the uidA gene from Escherichia coli (datanot shown), demonstra ting that the synergism is not spe-cific to eukaryotic genes in general or the luciferase genein particular.An in vitro translation lysate was used to examinewhether the synergism observed in vivo could occur invitro. The same preparations of Luc mRNAs used for thein vivo analysis in CHO cells were translated in reticu-locyte lysate. Cap stimulation of translation was thesame for both poly(A) + and poly(A)- Luc mRNAs andthe poly(A) tail had virt uall y no effect (Fig. 4). Nei the rthe cap nor the poly(A) tail increased message stability invitro, verifying earlier observations for this lysate (Furui-chi et al. 1977). Moreover, no synergism was observedbetween the cap and the poly(A) tail in vitro. The lack ofsynergism may be a consequence of the failure of thelysate to reflect an in vivo environment in a number ofways: Translation was an order of magnitude less cap-dependent in vitro than in vivo; the poly(A) tail failed tofunction in vitro; and the cap and poly(A) tail did notincrease message stability in vitro.

Synergism between the cap and the poly(A) tail/n Saccharomyces cerevisiae is influenced by the stateof translational competencyTo extend our observations to lower eukaryotes, we es-tablished the appropriate conditions for mRNA delivery

T a b l e 2. S y n e r g i s m b e t w e e n c a p a n d p o l y ( A ) t a i l d u r i n g t ra n s l a t i o n i n C H O 0 01 c e l l s e l e c t ro p o r a t e d w i t h Luc m R N A

m R N A L u c i f er a s e a c t i v i t y( l ig h t u n i t / r a g p r o t ei n ) Re la t ive e f fec t o f po ly (A) ta i l on e xpress ion Relative effect of cap on expressionUncappedLuc 160,000 1Luc-Aso 1,360,000 8.5CappedLuc 437,000 1Luc-Aso 68,000,000 156

1

2.7 50

GENES & DEVELOPMENT 2111

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Gall ie

i n t o y e a s t u s i n g e l e c t r o p o r a t i o n ( J .G . E v e r e t t a n d D . R .Ga l l i e , i n p r ep . ) . T he yeas t ce l l s wer e f i r s t sphe r op l as t eda n d a l l o w e d t o r e c o v e r fo r 9 0 m i n b e f o r e e le c t r o p o r a t i o nt o a l l o w a c t i v e t r a n s l a t i o n t o r e s u m e . T h e a d d i t i o n o f ap o ly ( A ) t a il t o u n c a p p e d L u c m R N A r e s u l te d i n a 3 1 -f o ldi n c r e a s e i n L U C e x p r e s s i o n ( T a b l e 3 ). H o w e v e r , e x p r e s -s i o n w a s i n c r e a s e d b y 2 0 1 - f o l d w h e n a p o l y ( A ) t a i l w a sa d d e d t o c a p p e d L u c m R N A . T h e s e d a t a i l l u s t r a t e t h a tt h e p o l y ( A ) t a i l c a n i n c r e a s e e x p r e s s i o n i n t h e a b s e n c e o ft h e c a p b u t i s f u n c t i o n a l l y s t i m u l a t e d b y a c ap . I n y e a s t ,t h e c a p i s f u n c t i o n a l l y a c t i v e i n L u c m R N A l a c k i n g ap o l y( A ) t a il , b u t i s e n h a n c e d w h e n t h e m R N A i s p o l y a d -e n y l a te d . T h e s y n e r g i s m o b s e r v e d i n h i g h e r e u k a r y o t e si s, t h e re f o r e , a l s o p r e s e n t i n a l o w e r e u k a r y o t e s u c h a s S .ce rev i s i ae , b u t i n t e r e s t i n g l y , t o a l o w e r e x t e n t .B e c a u s e s p h e r o p l a s t i n g d i s r u p t s a c t i v e t r a n s l a t i o n i ny e a s t , i t w a s o f i n t e r e s t t o d e t e r m i n e w h e t h e r t h e s y n e r -g i s m b e t w e e n t h e c a p a n d p o l y (A ) t a i l w o u l d b e s i m i l a r l yd i s r u p t e d . L U C e x p r e s s i o n f r o m y e a s t e le c t r o p o r a t e d i m -m e d i a t e l y f o l l o w i n g s p h e r o p l a s t i n g w a s s i g n i f i c a n t l y r e -d u c e d w h e n c o m p a r e d t o t h e s a m e s p h e r o p l a st p r ep a ra -t i o n a l l o w e d a 9 0 m i n r e c o v e r y b e f o re e l e c t r o p o r a t i o n( T a b le 3 ), v e r i f y i n g t h a t a c t i v e t r a n s l a t i o n i s d i s r u p t e d b ys p h e r o p l a s t i n g . T h e f u n c t i o n o f a p o l y ( A ) t a i l w a s n o t

a d v e r s e l y a f f e c t e d w h e n a d d e d t o u n c a p p e d L u c m R N A ,n o r w a s c a p f u n c t i o n a f f e c t e d w h e n a d d e d t o p o l y ( A ) -L u c m R N A . H o w e v e r , th e s y n e r g i s m n o r m a l l y o b s e r v e di n s p h e r o p l a s t s e n g a g e d i n a c t i v e t r a n s l a t i o n w a s n o t d e -t e c t e d i n t h e n e w l y s p h e r o p l a s t e d y e a s t . T h e s e d a t a s u g -g e s t t h a t t h e s y n e r g i s m b e t w e e n t h e c a p a n d t h e p o l y ( A )t a i l i s l o s t w h e n a c t i v e t r a n s l a t i o n i s d i s r u p t e d b ys p h e r o p l a s t i n g a n d i s o n l y r e g a i n e d w h e n a c t i v e t r a n s l a -t i o n r e s u m e s .D i s c u s s i o nS i n c e t h e d i s c o v e r y t h a t R N A s t e r m i n a t e i n a p o l y( A ) t a i l(Darnel l e t a l . 1971; Edmonds et a l . 1971; Lee et a l . 1971)a n d t h e s u b s e q u e n t d i s c o v e r y t h a t t h e 5 ' t e r m i n u s i scapped ( R eddy e t a l. 1974) , t he cen t r a l r o l e t ha t t hes er e g u l a t o r y e l e m e n t s p l a y i n e s t a b l i s h i n g e f f i c ie n t e x p r e s -s i o n h a s b e e n w e l l d o c u m e n t e d ( F i li p o w i c z 1 9 7 8;B r a w e r m a n n 1 9 8 1 ; R h o a d s 1 9 8 8 ; S o n e n b e r g 1 9 8 8 ; B e r n -s t e i n a n d R o s s 1 9 8 9; J a c k s o n a n d S t a n d a r t 1 9 9 0 ; M u n r o eand Jacobson 1990b) . T he r o l e o f t he cap a s t he r ecogn i -t i o n s i t e f o r b i n d i n g t h e e u k a r y o t i c i n i t i a t i o n f a c t o r ( eI F)4 E , a s u b u n i t o f e I F - 4 F , h a s b e e n d e m o n s t r a t e d a s a ne a r l y a n d e s s e n t i a l s t e p i n t r a n s l a t i o n i n i t i a t i o n ( F il ip -

F i g u r e 3 . T h e e f f e c t o f a c a p a n d p o l y ( A ) t a i lo n t h e t r a n s l a t i o n e f f i c i e n c y a n d f u n c t i o n a lh a l f - l i f e o f Luc m R N A i n C H O c e ll s. A l i q u o t so f p r o t o p l a s t s e l e c t r o p o r a t e d w i t h u n c a p p e d ( A)o r c a p p e d { B ) Luc m R N A s w e r e ta k e n a t ti m ei n t e r v a l s , a s s a y e d , a n d t h e v a l u e s d e t e r m i n e da s d e s c r i b e d i n F i g . 2 .

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F i g u r e 4 . A n a l y s i s o f t h e s y n e r g i s m b e t w e e n acap and po ly (A) ta i l in r e t icu locy te ly sa te . Thet rans la t ion o f 200 ng o f each Luc m R N A c o n -s t r u c t w a s f o l l o w e d b y a s s a y i n g a l i q u o t s f o r l u -c i fe ra se ac t iv i ty .

owi cz 1978 ; B aner j ee 1980 ; A l t mann e t a l . 1987 , 1989 ;R h o a d s 1 9 8 8 ; S o n e n b e r g 1 98 8 ). G e n e t i c a n d b i o c h e m i c a le v i d e n c e s u g g e s t s t h a t t h e P A B p r o t e i n , w h i c h b i n d s t ot h e p o l y ( A ) t a i l , m a y i n t e r a c t w i t h t h e 6 0 S r i b o s o m a ls u b u n i t a s a m e a n s b y w h i c h t h e t r a n s l a t i o n a l e ff i ci e n c yo f a m e s s a g e m a y b e r e g u l a t e d ( S a c h s a n d D a v i s 1 9 8 9 ;M u n r o e a n d J a c o b s o n 1 9 9 0 a ) .

O u r i n v i v o s t u d i e s i n b o t h h i g h e r a n d l o w e r e u k a r y -o t e s s u g g e s t t h a t a n i n t e r d e p e n d e n c e e x i s t s b e t w e e n t h ec a p a n d t h e p o l y ( A ) f o r e f fi c i e n t f u n c t i o n a t t h e l e v e l o fr e g u l a t i n g t r a n s l a t i o n a l e f f ic i e n c y . B e c a u se o f t h e i r d u a lr e g u l a t o r y r o l e s , t r a n s l a t i o n a l e f f i c i e n c y a n d m e s s a g es t a b i l i t y w e r e b o t h i n c r e a s e d b y t h e a d d i t i o n o f e i th e r

t h e c a p o r p o ly ( A ) t a il . H o w e v e r , t h e s y n e r g i s m w a s o b -s e r v e d o n l y a s a f u n c t i o n o f t h e t r a n s l a t i o n a l e f f ic i e n c y .T h e s t ab i l i z i ng e f f ec t o f a cap and po l y ( A) t a i l t oge t he r ,

a l t h o u g h n o t s y n e r g i s t i c , w a s a d d i t i v e . T h e c a p i n c r e a s e dm e s s a g e s t a b i l i t y t w o - t o f o u r fo l d , i n g o o d a g r e e m e n tw i t h o t h e r s t u d i e s w h i c h e x a m i n e d t h e s t a b i l iz a t i o n a f-f o r d e d b y a c a p . R e o v i r u s m R N A w a s f o u r f o l d m o r e s t a -b l e w h e n c a p p e d th a n w a s u n c a p p e d m R N A i n m i c r o i n -j e t t e d Xenopus o o c y t e s a n d t h r e e f o l d m o r e s t a b l e i nw h e a t g e r m l y s a t e ( F u r u i c h i e t a l . 1 9 7 7 ) . C h i c k e nl y s o z y m e m R N A w a s s t a b i l iz e d f o u rf o ld b y t h e a d d i t i o no f a m o n o m e t h y l a t e d c a p an d t w o f o l d b y a d i m e t h y l a t e dc a p i n m i c r o i n j e c t e d o o c y t e s ( D r u m m o n d e t a l . 1 9 8 5 ) .

T a b l e 3. Synergism between cap and poly(A) tail during translation in yeast is dependent on translational competency

m R N A

Y e a s t e l e c t r o p o r a te d a f t e r 9 0 m i n o f r e co v e r yf o l l o w i n g s p h e r o p l a s t i n g Y e a s t e l e c t r o p o r a t e d i m m e d i a t e l yf o l l o w i n g s p h e r o p l a s t i n gluc i fe ra se ac t iv i ty re la t ive e f fec t r e la t ive e f fec t o f luc i fe ra se ac t iv i ty re la t ive e f fec t

( l igh t un i t / r a g o f po ly (A) ta i l c ap on ( l igh t un i t / r ag o f po ly (A) ta i lp ro te in ) on exp ress ion exp ress ion p ro te in ) on exp ress ion re la t ive e f fec t o fc a p o n e x p r e s s i o n

U n c a p p e dLucLuc-AsoC a p p e dLucL u c - A s o

26,930 1 1844,410 31100,170 1 3 .720,141,220 201

3,700 11 160, 730 43

41,620 124 1 ,854,320 45

1112

G E N E S & D E V E L O P M E N T 2 1 1 3

http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://www.cshlpress.com/http://www.cshlpress.com/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://www.cshlpress.com/http://genesdev.cshlp.org/


8/10

Ga l l i e

P o l y h e d ro s i s v ir a l m R N A w a s t w o f o l d m o r e s t a b le w h e nc a p pe d t h a n w a s u n c a p p ed m R N A i n w h e a t g e r m l y s a t e( Sh i mo t o h n o e t a l . 1 9 7 7 ) . Th e p o l y ( A) t a i l i n c r e a s e dm R N A s t a b i l i t y a p p r o x i m a t e l y t w o - t o t h re e f o ld , i n go o da g r e e m e n t w i t h o u r p r e v i o u s o b s e r v a t i o n s { G a l l i e e t a l .1 9 89 , 1 9 9 1) . Th e f u n c t i o n a l h a l f - l i fe m e a s u r e m e n t s f o rth e Lu c c o n s t r u c t s w e r e s h o r t e r i n p l a n t c e l l s t h a n i na n i m a l c e l l s, a s w a s r e p o r t e d p r e v i o u s l y f o r t h e p h y s i c a lh a l f - l if e me a s u r e m e n t s ( Ga l l i e e t a l. 1 9 9 1) . I n t h i s r e g a rd ,i t is i n t e r e s t i n g t o n o t e t h a t a d d i t i o n o f a c ap o r a p o l y (A)t a i l h a d a g r e a t e r e f f e c t o n Lu c m R N A s t a b i l i t y i n p l a n tce l l s .

O n e e x p l a n a t i o n f o r th e l o w f u n c t i o n a l a c t i v i t y o f t h ec a p o r p o l y ( A) t a i l wh e n e i t h e r wa s p r e s e n t a l o n e i n ac o n s t r u c t m i g h t h a v e b e e n t h a t t h e Lu c m R N A i s t o or a p i d l y d e g r a d e d t o a l l o w t h e c a p o r p o l y ( A) t a i l a n o p -p o r t u n i t y t o f u n c t i o n . H o w e v e r , t h e f o u r Lu c m R N A sw e r e t r a n s l a t i o n a l l y a c t i v e f o r 3 0 - 2 0 0 m i n i n t o b a c c oa n d 1 5 0 t o ov e r 3 0 0 m i n i n C H O c e ll s , d e p e n d i n g o n t h ec o n s t r u c t . S i n c e l u c i f e r a s e a c t i v i t y c a n b e d e t e c t e dw i t h i n 3 m i n f o l l o w i n g e l e c t r o p o r a t i o n ( D .R . G a l li e , u n -p u b l. ) , o n e c y c l e o f t r a n s l a t i o n i s c o m p l e t e d wi t h i n t h i st i m e . T r a n s l a t i o n o f c a p p e d Luc m R N A w a s m a r k e d l ye n h a n c e d o v e r th a t s e e n fo r u n c a p p e d m R N A , e v e n a tt h e e a r l i e s t t i m e p o i n t , d e m o n s t r a t i n g t h a t t h e c a p i sr e c o g n i z e d a n d f u n c t i o n a l e v e n d u r i n g t h e f i r s t r o u n d o ft r a n s l a t io n . A s t h e Lu c m R N A s a r e s u f f i c i e n t l y s t a b l e t oa l l o w m u l t i p l e r o u n d s o f t r a n s l a t io n , t h e c a p a n d p o ly ( A )t a i l h a v e a m p l e o p p o r t u n i t y t o f u n c t i o n b e f o r e m R N Ad e g r a d a t i o n o c c u r s .

I f c a p f u n c t i o n i s d e p e n d e n t o n a p o l y ( A) t a i l f o r o p t i -m a l f u n c t i o n , h o w d o e s t h e c a p f u n c t i o n i n n a t u r a l l yo c c u r r i n g p o ly { A ) - m R N A s ? I n p l a n t s , t h e o n l y k n o w np o l y ( A ) - m R N A s a r e p l a n t v i ra l m R N A s , f o r ex a m p l e,t o b a c c o m o s a i c v i r u s ( TM V ) . W e h a v e d e m o n s t r a t e d t h a tt h e T M V 3 ' - u n t r a n s l a t e d r e g i o n ( 3 ' U T R ) i s t h e f u n c -t i o n a l e q u i v a l e n t o f a p o l y ( A) t a i l , s e r v i n g t o ma r k e d l yi n c r e a s e t h e t r a n s l a t i o n a l e f f i c ie n c y o f c h i m e r i c m R N Ac o n s t r u c t s ( Ga l l i e a n d W a l b o t 1 9 9 0 ) . W e h a v e o b s e r v e dt h e s a m e i n te r d e p e n d e n c e b e t w e e n t h e T M V 3 ' U T R a n dt h e c a p t h a t w e h a v e s e e n b e t w e e n t h e c a p a n d p o l y ( A )t a i l ( D . R . Ga l l i e , i n p r e p . ) . Th e s e d a t a s u g g e s t t h a t a l -t h o u g h e f f ic i e n t t r a n s l a t i o n o f T M V m R N A i s n o t d e -p e n d e n t o n a p o l y ( A ) t a i l , t h e s y n e r g i s m b e t w e e n t h et e r m i n a l r e g u l a t o r y e l e m e n t s i s n e v e r t h e l e s s i n v o l v e d .

M u c h o f o u r u n d e r s t a n d i n g o f t h e r e g u l a t o r y f u n c t i o no f t h e c a p - - e I F - 4 F a n d p o l y ( A) t a i l - PAB p r o t e i n c o m-p l e x e s h a s b e e n m a d e u s i n g c e l l l y s a t e s . A l t h o u g h a n i nv i t r o a p p r o a c h h a s b e e n u s e f u l i n a n a l y z i n g t h e b i n d i n go f t h e s e r e g u l a t o r y p r o t e i n s w i t h t h e i r t a r g e t s i t e s, i t h a sn o t d e m o n s t r a t e d t h e i n t e r d e p e n d e n c e b e t w e e n t h e c a pa n d t h e p o l y ( A) t a i l . As we i l l u s t r a t e i n t h i s r e p o r t , t h er e a s o n f o r t h i s i s t h a t a n i n v i t r o l y s a t e d o e s n o t e x h i b i tt h e s y n e r g i s m . W h a t m i g h t b e t h e n a t u r e o f t h i s d e fi -c i e n c y ? Th e i mp a c t o f t h e c a p o r t h e p o l y ( A) t a i l o n e x -p r e s s i o n i n v i v o i s n o t r e f l e ct e d i n v i t ro . T r a n s l a t i o n w a sa n o r d e r o f m a g n i t u d e l e s s c a p - d e p e n d e n t i n v i t r o ( re t ic -u l o c y t e l y s a t e ) t h a n i n v i v o ( CHO c e l l s ) . Th e p o l y ( A) t a i lh a d v i r t u a l l y n o i m p a c t o n t r a n s l a t i o n i n v it r o . S t u d ie sd e m o n s t r a t e , h o w e v e r , t h a t e I F -4 F a n d P A B p r o t e i n d o

b i n d t o t h e c a p a n d p o l y ( A) t a i l , r e s p e c t i v e l y , i n v i t r o(Sachs e t a l . 1987; Rh oad s 1988; Son enb erg 1988) . If inf a c t t h e p o l y ( A) t a i l - PAB p r o t e i n a n d c a p - - e I F - 4 F d o i n -t e r a c t s o t h a t t h e y a r e f u n c t i o n a l l y a c t i v a t e d , a f a c t o rm e d i a t i n g t h e i n t e r a c t i o n m a y b e m i s s i n g o r n o n f u n c -t i o n a l i n t h e i n v i t r o s y s t e m s . T h e f a c t o r m i g h t b e as i n g l e p r o t e i n , s u c h a s a n i n i t i a t i o n f a c t o r , o r mi g h t b e af r a m e w o r k , s u c h a s t h e c y t o s k e l e t o n . C y t o s k e l e t o n - a s -s o c i a t e d p o l y s o me s h a v e b e e n o b s e r v e d ( Le n k e t a l . 1 9 7 7 ;Ce r v e r a e t a l . 1 9 8 1 ; Ho we a n d He r s h e y 1 9 8 4 ) a n d i t h a sb e e n s h o w n t h a t e I F -4 E p r e f e r e n t i a l ly a s s o c i a t e s w i t h i n -t e r me d i a t e f i l a me n t s ( Zu mb e e t a l . 1 9 8 2 ) . As s o c i a t i o n o fm R N A w i t h t h e c y t o s k e l e t o n m a y b e a n e c e s s a r y p r e -r e q u i s i t e f o r t h e s y n e r g i s m b e t w e e n t h e t w o t e r m i n a lr e g u l a t o r y d e t e r m i n a n t s t o t a k e p l a c e .T h e s y n e r g i s m o b s e r v e d b e t w e e n t h e c a p a n d p o l y ( A )t a i l s u g g e s t t h a t t h e s e t w o r e g u l a t o r y d e t e r m i n a n t s w o r ki n c o n c e r t t o m e d i a t e t h e r e g u l a t i o n a s s o c i a t e d w i t he a c h . O n e f u n c t i o n o f t h e p o ly { A ) t a i l - P A B p r o t e i n c o m -p l e x m a y b e t o f a c i li t a t e i n i t i a t i o n f a c t o r o r 4 0S s u b u n i ti n t e r a c t i o n w i t h t h e m R N A . T h e p o t e n t i a l f or i n t e r a c -t i o n b e t w e e n P A B p r o t e i n a n d t h e 6 0 S s u b u n i t ( S ac h sa n d Da v i s 1 9 8 9 , 1 9 9 0 ) s u p p o r t s t h e i d e a t h a t t h e p o l y ( A)t a i l - P A B p r o t e i n c o m p l e x m i g h t b e i n v o l v e d i n s e v e r a le a r l y e v e n t s d u r i n g t r a n s l a t i o n i n i t i a t i o n . A l t h o u g h t h em e c h a n i s m r e m a i n s t o be d et e r m i n ed , t h e 3 ' te r m i n u sc e r t a i n l y p l a y s a p i v o t a l r o l e i n t h e r e g u l a t i o n o f e v e n t so c c u r r i n g a t t h e 5 ' t e r m i n u s . C u r r e n t m o d e l s c o n c e r n i n gt r a n s l a t i o n i n i t i a t i o n a r e o f t e n d e c r i b e d a s a s e r i e s o fs t e p s ( Rh o a d s 1 9 8 8 ; So n e n b e r g 1 9 8 8 ) . A mo d e l d e s c r i b -i n g e f fi c i e n t i n i t i a t i o n o f t r a n s la t i o n , h o w e v e r , m a y b eo n e n o t s o m u c h s e q u e n t i a l , a s o n e i n w h i c h t h e p a r t i c -i p a n t s , f o r e x a m p l e , t h e c a p , e I F 's , r i b o s o ma l s u b u n i t s ,a n d t h e P A B p r o t e i n - p o l y ( A ) t a i l, f u n c t i o n i n c o n c e r t .

Ma ter ia ls a n d m eth o d sP l a s m i d c o n s t r u c t s a n d i n v i t r o t r a n s c r i p ti o nT he Luc an d uidA constructs have been described previously(Gallie et al . 1989, 1991). The 3'-untran slated region from theLuc construct has been removed for this s tudy. The T7-basedconstructs were linearized with the appropriate restriction en-zyme and in vi t ro t ranscript ion of the DNA was carr ied out asdescribed (Yisrael i and Me lton 1989). For poly(A) + mR NA s,plasmid l inearizat ion with DraI results in a poly(A)5o tail ter-mina t ing in three uridylate residues. V ir tual ly 100 % of themRNA synthesized as capped mRNA is capped (Yisrael i andMelton 1989). The integ ri ty and quanti tat ion of RNA were de-termined by formaldehyde-agarose gel electrophoresis as de-scribed (Melton et al . 1984). Equivalent am ounts of each m RN Aconstruct w ere used for the in vivo analyses.

I n v i t r o t r a n s l a t i o nTwo hundred nanograms of each Luc mRNA cons t ruc t wastranslated in rabbit ret iculocyte lysate according to the recom-mend ations of the supplier {Boehringer Mannheim ), w ith th eexception th at a complete m ix of nonradiolabeled amino acidswas used. Aliquots were removed at t ime intervals and frozenon dry ice. The extent of t ranslat ion was d etermined by assayingeach aliquot for luciferase activity as described below.

2114 GENES & DEVELOPMENT

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Cap and po ly (A) ta i l funct ion synerg i st i ca l ly

Preparation and electroporation conditionsP r o t o p l a s t m e d i a a n d i s o l a t i o n m e t h o d s f o r t o b ac c o { cv . X a n t h i )were a s desc r ibed (F romm e t a l . 1987 ), excep t tha t 1 .0% C y to -la se (Geneco r ) was u sed in p lace o f Rhozyme . Tobacco p ro to -p la s t s were e lec t ropo ra ted wi th a Gene Zapp e r ( IBI ) by u s ing 300V and 500 txF capac i tance .

C H O c e l l s w e r e c o l l e c t e d f r o m f l a s k s b y b r i e f t r y p s i n i z a t i o n ,wash ed twic e wi th phosph a te -bu f fe red sa l ine (P BS) , and e lec t ro -po ra ted in P BS wi th 400 V and 250 txF capac i tance .

Elec t ropo ra t ion o f yeas t has been desc r ibed in de ta i l e l se -whe re ( J .G . Eve re t t and D.R. Ga l l ic , in p rep .) . S t ra in CRY1 M atacan 1-100 ade 2-1 his 3-11,15 leu 2-3, 112 trp 1-1 ura 3-1, ade r iva t ive o f W303a , was gene r a ted by R. F u l le r and p rov ided byA. S achs . Br ie f ly , mid - log -phase S. cerevisiae, g r o w n i n Y P Dme d ium (1% yeas t ex t rac t , 2% Bac to -pep tone , 2% g lucose ) , wass p h e r o p l a s te d w i t h Z y m o l y a s e - 1 0 0 T (I C N ) i n 5 0 mM T r i s (p H7 .5 ), 1 mN MgC12 , 30 mM d i th io th re i to l , 15 mM ~-m ercap to -e t h a n o l , 1 M s o r b i t o l f or 3 0 r a i n , a n d s u b s e q u e n t l y w a s h e d t w i c ei n t h e s a m e b u f f e r. T h e s p h e r o p l as t s , a l l o w e d t o r e c o v e r i n Y P Dm e d i u m s u p p l e m e n t e d w i t h 1 M s o r b i t o l f o r 9 0 m i n , w e r ew a s h e d t w i c e w i t h 1 M s o r b it o l . O n e - h u n d r e d a n d e i g h t y m i -c r o l i t e r s o f y e a s t w a s m i x e d w i t h t h e t e s t m R N A a n d e l e c t r o -porat ed us ing 80 0 V, 21 IxF capa citan ce, 100011.

F o r t im e - c o u r s e e x p e r i m e n t s , a l i q u o t s o f c el l s w e r e r e m o v e da t t i m e i n t e r v a l s, c o l l e c te d b y c e n t r i f u g a t i o n , a n d f r o z e n u n t i la s sayed . Er ro r in these expe r imen ts i s 15%. Dose - re sponse ana l -y s i s o f RNA e lec t ro po ra t io n i s l inea r th ro ugh 30 ~xg o f inpu tmRNA {Ga l l ie e t a l . 1989 ) .

mRNA stabilityAliquo ts o f tobacco p ro top la s t s e lec t ropo ra ted wi th 5 ~g o f eachm R N A w e r e r e m o v e d a t t i m e i n t e r v a l s , t h e c e l l s c o l l e c t e d b yc e n t r i f u g a t io n , a n d t o t a l R N A p u r i f ie d a s d e s c ri b e d I C h o m c z y n -s k i a n d S a c c h i 1 9 8 7) . T h e R N A w a s d i s p l a y e d o n a f o r m a l d e-h y d e - a g a r o s e g e l , f o l l o w e d b y N o r t h e r n t r a n s f e r , a n d p r o b e dw i t h anti-Luc R N A . T h e r e g i o n o f t h e m e m b r a n e r e p r e s e n t i n gt h e f u l l - l e n g t h f o r m o f Luc m R N A w a s c u t fr o m t h e m e m b r a n e,coun ted , and the log m o f the va lu es p lo t ted a s a func t ion o ft ime . k , the s lope o f the bes t - f i t l ine th rough the da ta po in t s ,w a s u s e d t o c a l c u l a t e t h e h a l f - l i f e a c c o r d i n g t o t h e e q u a t i o ntl/2 = 0 .693 /k .

Analysis of luciferase acti vityF o l l o w i n g i n c u b a t i o n , t h e c e l l s w e r e h a r v e s t e d a n d r e s u s p e n d e din luc i fe ra se a s say bu f fe r [25 mM Tr ic in e (pH 7 .5 ), 15 mM MgCI2 ,7 mM f~ -mercap toe thano l , 2 mM ATP ] , son ica ted fo r 5 sec , andt h e c e l l de b r i s p e ll e t e d . L u c i f e r a se a c t i v i t y w a s m e a s u r e d u s i n g0 .5 mM luc i fe r in in luc i fe ra se a s say bu f fe r and a Mono logh t2 0 1 0 L u m i n o m e t e r ( A n a l y t i c a l L u m i n e s c e n c e L a b o ra t o ry ) . P r o-t e i n c o n c e n t r a t i o n w a s d e t e r m i n e d u s i n g t h e B i o - R a d p r o t e i nassay k i t .

A c k n o w l e d g m e n t sW e t h a n k J . T r a u g h f o r c r i t i c a l r e a d i n g o f t h e m a n u s c r i p t , C .B y u s f o r t h e C H O c e l l l i n e , a n d M . K o b a y a s h i f o r t e c h n i c a la s s i s tance .

T h e p u b l i c a t i o n c o s t s o f t h i s a r t i c l e w e r e d e f r a y e d i n p a rt b yp a y m e n t o f p a ge c h a rg e s . T h i s a r t i c l e m u s t t h e r e f o r e b e h e r e b ym a r k e d " a d v e r t i s e m e n t " i n a c c o r d a n c e w i t h 1 8 U S C s e c t i o n1734 so le ly to ind ica te th i s f ac t .

R e f e r e n c e sA l t m a n n , M . , C . H a n d s c h i n , a n d H . T r a ch s e l . 1 9 87 . m R N A c a p-

b i n d i n g p r o t e i n : C l o n i n g o f t h e g e n e e n c o d i n g p r o t e i n s y n -t h e s i s i n i t i a t i o n f a c t o r e I F - 4 E f r o m S a c c h a r o m y c e s c e r e v i -s iae . Mol. Cell. Biol. 7: 998-1003 .

Al tm ann , M . , N . S onenbe rg , and H. Trachse l . 1989 . Tra ns la t ioni n S a c c h a r o m y c e s c e r e v i s ia e : I n i t i a t i o n f a c t o r 4 E - d e p e n d e n tce l l - f ree sy s tem. Mol. Cell. Biol. 9 : 4467 -4472 .Bane r jee , A .K. 1980 . 5 ' -Te rmina l cap s t ruc tu re in euca ryo t icm e s s e n g e r r i b o n u c l e i c a c id s . Bacteriol. Rev. 4 4 : 1 7 5 - 2 0 5 .

Bernstein , P . and J . Ross. 1989. P o ly (AI, po ly (A) b ind in g p ro te ina n d t h e r e g u l a t i o n o f m R N A s t a b i l i t y . Trends Biochem. Sci.1 4 : 3 7 3 - 3 7 7 .Braw erman n , G . 1981 . The ro le o f the po ly (A) sequence in mam -m a l i a n m e s s e n g e r R N A . CRC Crit. Rev. Biochem. 10: 1-38.

Cerve ra , M. , G . Drey fuss , and S . P en ma n . 1981 . Messenge r RNAi s t r a n s l a t e d w h e n a s s o c i a t e d w i t h t h e c y t o s k e l e t a l f r a m e -w o r k i n n o r m a l a n d V S V - i n f e ct e d H e L a c e l ls . Cell 2 3 : 1 1 3 -120.C h o m c z y n s k i , P . a n d N . S a c c h i . 1987. S i n g l e - s t e p m e t h o d o fR N A i s o l a t i o n b y a c i d g u a n i d i u m t h i o c y a n a t e - p h e n o l - c h l o -ro fo rm ex t rac t ion . Anal Biochem. 162 : 156 -159 .

Darne l l , J .E . , R . Wal l , and R. J . Tu sh in sk i . 1971. A n a d e n y l i ca c i d - ri c h s e q u e n c e i n m e s s e n g e r R N A o f H e L a c e l l s a n d i t sp o s s i b l e r e la t i o n s h i p t o r e i t e r a t e d s i t e s i n D N A . Proc. Natl.Acad. Sci. 68 : 1321 -1325 .

Dru mm ond , D .R. , J . Arm s t rong , and A. Co l man . 1985 . Th e e f-f e c t o f c a p p i n g a n d p o l y a d e n y l a t i o n o n t h e s t a b i l i t y , m o v e -m e n t a n d t r a n s l a t i o n o f s y n t h e t i c m e s s e n g e r R N A s i n X e -nopus oocy te s . Nucleic Acids Res. 13 : 7375 -7394 .

Edmon ds , M. , M.H. Vaugh an J r ., and H. Nakaz a to . 1971 . P o ly -a d e n y l i c a c i d s e q u e n c e s i n t h e h e t e r o g e n e o u s n u c l e a r R N Aa n d r a p i d l y - l a b el e d p o l y r i b o s o m a l R N A o f H e L a c e i l s : P o s -s ib le ev idence fo r a p recu rso r r e la t ions h ip . Proc. Natl. Acad.Sci. 6 8 : 1 3 3 6 - 1 3 4 0F i l ipowicz , W. 1978. F u n c t i o n s o f t h e 5 ' - t e r m i n a l m 7 G c a p i ne u k a r y o t i c m R N A . FEBS Lett. 9 6 : 1 - 1 1 .

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Ga l l ie , D .R. , W .J . Lucas , and V. Walbo t . 1989. V i s u a l i z i n gm R N A e x p r e s s i o n i n p l a n t p r o t o p l a s t s : F a c t o r s i n f l u e n c i n ge f f i c i e n t m R N A u p t a k e a n d t r a n s l a t i o n . Plant Cell 1: 3 0 1 -311.Ga l l ie , D .R. , J .N . F ede r , R .T. S ch imke , and V. Walbo t . 1991.P o s t - t r a n s c r i p t i o n a l r e g u l a t i o n i n h i g h e r e u k a r y o t e s : T h ero le o f the repo r te r gene in con t ro l l ing exp ress ion . Mol. Cert.Genet. 228 : 258 -264 .

Grange , T . , C . Mar t in s de S a , J . Oddos , and R. P ic te t . 1987 .H u m a n m R N A p o l y a d e n y la t e b i n d i n g pr o t ei n : E v o l u t i o n a r yc o n s e r v a t i o n o f a n u c l e i c a c i d b i n d i n g m o t i f . Nucleic AcidsRes. 15 : 4771 -4787 .Green , M.R. , T . Man ia t i s , and D.A. Me l ton . 1983 . Human~ - g l o bi n p r e - m R N A s y n t h e s i z e d in vitro i s a c c u r a t e l ys p l ic e d i n X e n o p u s o o c y t e n u c l e i . Cell 3 2 : 6 8 1 - 6 9 4 .

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the cap and poly(a) tail function

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