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Counterproductive transcriptional and translational regulation of elongation factor 1-α synthesis during early development in sea urchins
DEVELOPMENTAL BIOLOGY 142, 486-488 (1990)
Counterproductive Transcriptional and Translational Regulation of Elongation Factor 1-c Synthesis during Early Development in Sea Urchins M A R G A R E T T R U S C H E L P E E L E R , 1'2 L E S L I E K E L S O - W I N E M I L L E R , M I N G - F A N W U , J A M E S K . S K I P P E R , AND M A T T H E W M. W I N K L E R
Center for Developmental Biology, Department of Zoology, University of Texas at Austin, Austin, Texas 78712 Accepted August 13, 1990 We have isolated a cDNA clone encoding elongation factor 1-a (EFI-a) and used probes prepared from this cDNA to measure levels of E F I - a transcripts during early development. We also determined the fraction of E F I - a transcripts in polysomes during this time period. Following the blastula stage there is a s h a r p increase in the amount of E F I - a mRNA. This pattern of accumulation is similar to other previously described sea urchin mRNAs. However, while the level of E F I - a mRNA is increasing, the fraction of E F I - a mRNA in polysomes decreases. Thus, there is an apparently counterproductive decrease in efficiency of recruitment into polysomes occurring concurrently with an increase in the overall amount of E F I - a mRNA. © 1990AcademicPress, Inc. INTRODUCTION
The a subunit of eukaryotic elongation factor i (EF1a) functions in the elongation step of protein synthesis, where it mediates the binding of a charged tRNA molecule into the P site of the ribosome. This protein has been cloned from several diverse species and exhibits a high degree of sequence conservation (Hovemann et al., 1988; Nagata et al., 1984; Brands et al., 1986). Recently, it has been shown t h a t in Xenopus, the mRNA encoding E F I - a is a major new transcription product after the midblastula transition, and t h a t the message partitions into polysomes to a high degree, indicating active synthesis of EFI-~ protein (Kreig et al., 1989). We have isolated a cDNA clone corresponding to E F I - a from the sea urchin Strongylocentrotus purpuratus. The mRNA for E F I - a undergoes a 10-fold increase in abundance of the message beginning at the late blastula state. However, this message appears to have an unusual p a t t e r n of translational regulation, such t h a t there is a decrease in the association of the message with polysomes accompanying the increase in message abundance. Thus, there is apparently only an estimated 3-fold increase in the synthesis of E F I - a protein in sea urchins despite a 10fold increase in mRNA abundance during development. MATERIALS AND METHODS
The E F I - a clone was isolated from a blastula stage S. purpuratus (Lambda Zap; Stratagene) cDNA library (a
1 To whom correspondence should be addressed. 2 Current address: D e p a r t m e n t of Biology, Susquehanna University, Selinsgrove, PA 17870. 0012-1606/90 $3.00 Copyright© 1990by AcademicPress, Inc. All rights of reproductionin any form reserved.
gift from Dr. William Lennarz). The library was screened with 32p-labeled Xenopus E F I - a cDNA (a gift from Dr. Paul Krieg). For Northern blots, 5.0 ~g of total RNA, isolated from postmitochondrial supernatants, was separated on agarose gels containing formaldehyde, blotted onto Hybond N nylon membranes, and hybridized as described previously (Kelso-Winemiller and Winkler, submitted for publication). The autoradiograms obtained from the blots were scanned using a densitometer and the area under the peaks was integrated. To correct for possible loading errors, the Northerns were stripped and reprobed with a probe made from a X. laevis 28S rRNA probe and the results were then normalized to an equal amount of rRNA per sample. Control lanes containing increasing levels of unfertilized egg RNA were included to ensure t h a t the Xenopus probe bound proportionally to the amount of sea urchin RNA present (data not shown). The fraction of mRNA in polysomes was based on the quantification of probe binding to slot blots containing RNA isolated from 15 to 50% linear glycerol gradients with 80% glycerol pads used to fractionate postmitochondrial supern a t a n t s from different developmental stages (KelsoWinemiller et al., 1989). The 0D26o of the gradients was determined and used to isolate three fractions. The first two fractions contained n o n t r a n s l a t e d RNA, and the third fraction contained the RNA associated with polysomes. Similar fractions from control gradients containing EDTA were also probed to ensure t h a t the message in the third fraction of the gradient was released by EDTA and therefore polysomal. The fraction of mRNA in polysomes was then calculated as the a m o u n t of mRNA in polysomes divided by the total mRNA in the gradient and expressed as a percentage. A probe for ~486
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FIG. 1. Expression of EFI-a during development. A Northern blot containing 5.0 ttg of total RNA from various developmental stages was probed with labeled EFI-a cDNA. The stages represented in each lane are indicated as hours after fertilization. The major transcript at each stage is 2.7 kb.
tubulin (gift of P. H a r l o w a n d M. N e m e r ) w a s used in parallel e x p e r i m e n t s to c o m p a r e the t r a n s l a t i o n a l p a t t e r n of this p r o t e i n to E F I - a . M e a s u r e m e n t s of m R N A levels a n d the f r a c t i o n of m R N A in p o l y s o m e s w e r e rep e a t e d twice w i t h R N A isolated f r o m two different sets of e m b r y o s . RESULTS AND DISCUSSION
Identification of KL1 as sea urchin EFI-~. A c D N A clone encoding sea urchin E F I - ~ w a s isolated f r o m a b l a s t u l a - s t a g e c D N A l i b r a r y b a s e d on its ability to hybridize to a p r o b e m a d e f r o m Xenopus E F I - ~ . The plasm i d c o n t a i n i n g a 2.0-kb insert, r e f e r r e d to as KL1, w a s excised. I t s i d e n t i t y w a s verified by s e q u e n c i n g a n d comp a r i s o n of the sequence to t h a t of the Xenopus clone. The deduced a m i n o acid sequences f r o m the two species w e r e s t r i k i n g l y s i m i l a r , i n c l u d i n g a r e g i o n of 69 i d e n t i t i e s over a region of 75 a m i n o acids, c o r r e s p o n d i n g to a m i n o acids 5-82 of the p u b l i s h e d Xenopus sequence ( d a t a not shown). Expression of EFI-a during development. To d e t e r m i n e the p a t t e r n of e x p r e s s i o n of this clone d u r i n g sea urchin d e v e l o p m e n t , N o r t h e r n blots of t o t a l R N A isol a t e d f r o m different d e v e l o p m e n t a l s t a g e s w e r e p r o b e d w i t h 32p-labeled KL1. The p r o b e h y b r i d i z e s to a single 2.7-kb t r a n s c r i p t , as s h o w n in Fig. 1. T h e r e l a t i v e a m o u n t of E F I - a m R N A is s h o w n in Fig. 2. The level of E F I - a r e m a i n s r e l a t i v e l y c o n s t a n t t h r o u g h the first 12 h r of d e v e l o p m e n t , which c o r r e s p o n d s to the e a r l y blast u l a s t a g e in this species. By 18 h r of d e v e l o p m e n t , or l a t e b l a s t u l a , t h e levels of E F I - a b e g i n to i n c r e a s e , r e a c h i n g m a x i m u m levels a t 48 a n d 72 h r ( p r i s m a n d pluteus stages). T h e r e is a p p r o x i m a t e l y 10 t i m e s m o r e E F I - a m R N A p r e s e n t a t t h e s e s t a g e s t h a n in the first 12 hr. This p a t t e r n of a c c u m u l a t i o n is s i m i l a r to o t h e r sea u r c h i n m R N A s such as t u b u l i n ( H a r l o w a n d N e m e r , 1987).
i
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FIG. 2. Changes in EFI-a mRNA levels during development. An autoradiograph of a Northern blot containing RNA from the indicated developmental stages probed with the sea urchin EFI-a clone was scanned with a densitometer to determine the relative amounts of EFI-c~ mRNA at each stage.
Translation regulation of EFl-~. In Xenopus, the l a r g e i n c r e a s e in E F I - a m R N A a f t e r the m i d b l a s t u l a t r a n s i tion is a c c o m p a n i e d b y the a s s o c i a t i o n of the m R N A w i t h p o l y s o m e s to a l a r g e extent, i n d i c a t i n g t h a t the m R N A is b e i n g t r a n s l a t e d . In sea u r c h i n s the s i t u a t i o n a p p e a r s to be different (Fig. 3). The p e r c e n t a g e of E F I - a in p o l y s o m e s in the u n f e r t i l i z e d egg is low, as is expected because of the d e p r e s s e d r a t e of p r o t e i n s y n t h e s i s in t h e egg. Following fertilization, the p e r c e n t a g e of m R N A in p o l y s o m e s increases, r e a c h i n g a p e a k of 90% by 4 hr. This i n d i c a t e s t h a t s y n t h e s i s of E F I - a p r o t e i n occurs d u r i n g t h e cleavage stage, even t h o u g h the r e l a t i v e level of the m R N A is quite low. H o w e v e r , by 18 hr, the t i m e in d e v e l o p m e n t a t which the a b u n d a n c e of E F I - a m R N A is s t a r t i n g to increase, t h e r e is a decrease in the p e r c e n t 100" E o
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i
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FIG. 3. Fraction of mRNA associated with polysomes. Postmitochondrial supernatants from various developmental stages were fractionated on glycerol density gradients to separate polysomal from nontranslated mRNAs. RNA isolated from gradients was probed with 82p-labeled probes to both EFI-a and tubulin and the fraction of mRNA in polysomes was quantified.
488
DEVELOPMENTALBIOLOGY
age of m R N A in polysomes, so t h a t by 72 hr, only 30% of the m R N A is being actively t r a n s l a t e d . O t h e r m e s s a g e s w h i c h show s i m i l a r i n c r e a s e s in a b u n d a n c e do not und e r g o t h i s d e c r e a s e d level of a s s o c i a t i o n w i t h p o l y s o m e s . F o r e x a m p l e , ~ - t u b u l i n m R N A r e m a i n s a t its p e a k level of p o l y s o m e a s s o c i a t i o n d u r i n g l a t e r developm e n t , as shown in Fig. 3. The f~-tubulin p r o b e used recognizes several of t h e t u b u l i n t r a n s c r i p t s , which all act in a s i m i l a r m a n n e r in t e r m s of p o l y s o m e association. Actin also exhibits a s i m i l a r p a t t e r n of p o l y s o m e r e c r u i t m e n t to/~-tubulin ( d a t a not shown). We h a v e not found a n y o t h e r m R N A s in the sea u r c h i n which e x h i b i t the d e c r e a s e d level of p o l y s o m e a s s o c i a t i o n d e m o n s t r a t e d for E F I - a . Thus, t h e r e is only an a p p r o x i m a t e l y 3-fold i n c r e a s e in the r a t e of E F I - a p r o t e i n s y n t h e s i s d u r i n g d e v e l o p m e n t , despite the 10-fold i n c r e a s e in a b u n d a n c e in the m R N A encoding this protein. I t is unclear w h y an m R N A would exhibit a p p a r e n t l y c o u n t e r p r o d u c t i v e m o d e s of t r a n s c r i p t i o n a l a n d t r a n s l a t i o n a l control. A loss of t r a n s l a t i o n a l efficiency during d e v e l o p m e n t h a s been r e p o r t e d for o t h e r m R N A s , for example, for the m R N A s encoding r i b o s o m a l prot e i n s in d i f f e r e n t i a t i n g m o u s e m y o b l a s t s ( A g r a w a i a n d B o w m a n , 1987), but in t h e s e s y s t e m s , the loss of efficiency is a c c o m p a n i e d by a decrease in the level of m e s sage as well. Thus, it is not clear w h a t , if any, is the f u n c t i o n a l significance of t h e s e c h a n g e s in E F I - ~ m R N A levels a n d r e c r u i t m e n t into polysomes. One possible e x p l a n a t i o n is t h a t the t r a n s l a t i o n depression m i g h t be a control m e c h a n i s m s u p e r i m p o s e d over t r a n s c r i p tional control in o r d e r to reduce t r a n s l a t i o n of the E F 1 protein. I t will be of i n t e r e s t to m e a s u r e the levels of E F I - a p r o t e i n d u r i n g d e v e l o p m e n t to d e t e r m i n e h o w t h e s e levels c o r r e l a t e to t h e o b s e r v e d c h a n g e s in m R N A levels. A l t h o u g h we only o b s e r v e a single size t r a n s c r i p t during d e v e l o p m e n t , the o b s e r v e d t r a n s l a t i o n a l depression m a y be the r e s u l t of the t r a n s c r i p t i o n of a second population of E F I - a m e s s a g e s (possibly encoded by a s e p a r a t e gene) which a r e t u r n e d on a t the end of the b l a s t u l a stage. These m e s s a g e s m a y be less efficiently r e c r u i t e d into p o l y s o m e s t h a n E F I - a m R N A p r e s e n t e a r l y in dev e l o p m e n t due to c h a n g e s in s e q u e n c e or p r o c e s s i n g
VOLUME142, 1990
steps. A l t e r n a t i v e l y , t h e r e m a y be c h a n g e s or modifications of the e x i s t i n g p o p u l a t i o n of E F I - a m R N A a t the b l a s t u l a s t a g e which reduce t h e efficiency, of t h e i r rec r u i t m e n t into polysomes, for e x a m p l e , c h a n g e s in the proteins, or o t h e r f a c t o r s a s s o c i a t e d w i t h the m R N A which influence t r a n s l a t i o n . We hope t h a t f u t u r e experi m e n t s will clarify the level a t w h i c h t h i s u n u s u a l t r a n s lation r e g u l a t i o n is o p e r a t i n g . We gratefully acknowledge the generous gifts of Dr. Paul Kreig who donated the Xenopus clone and provided technical advice, Dr. William Lennarz who provided the cDNA library, and Drs. Patricia Harlow and Martin Nemer who supplied the fl-tubulin clone. This work was supported by an American Cancer Society Postdoctoral Fellowship to M.T.P. (Grant PF-3078) and by Public Health Service Grant (HD 17722-06) to M.M.W. from the National Institutes of Health. REFERENCES AGRAWAI, M. G., and BOWMAN,L. H. (1987). Transcriptional and translational regulation of ribosomal protein formation during mouse myoblast differentiation. J. BioL Chem. 262, 4868-4875. BRANDS,J. H. G. M., MAASSEN,J. A., VAN HEMERT,F. J., AMONS,R., and MOLLER,W. (1986). The primary structure of the subunit of human elongation factor 1. Eur. J. Biochem. 155, 167-171. HARLOW,P., and NEMER,i . (1987). Developmental and tissue-specific regulation of/~-tubulin gene expression in the embryo of the sea urchin Stongylocentrotus purpuratus. Genes Dev. 1, 147-160. HOVEMANN,B., RICHTER,S., WALLDORF,U., and CZIEPLUCH,C. (1988). Two genes encode related cytoplasmic elongation factors 1 (EFI-a) in Drosophila melanagaster with continuous and stage specific expression. Nucleic Acids Res. 16, 3175-3194. KELSO-WINEMILLER,L., DRAWBRIDGE,J., and WINKLER,M. M, (1989). A new ultracentifuge technique for analysis and isolation of polysomes. Nucleic Acids Res. 17, 4896. KELSO-WINEMILLER,L., and WINKLER,i . M. Identification of a novel class of "cleavage-stage specific" maternal mRNAs in sea urchins. Submitted for publication. KRIEG,P. A., VARNUM,S. M., WORMINGTON,W. M., and MELTON,D. A. (1989). The mRNA encoding elongation factor 1-(~is a major transcript at the midblastula transition in Xenopus. Dev. Biol. 133, 93100. NAGATA, S., NAGASHIMA,K., TSUNETSUGU-YOKOTA,Y., FUJIMURS,K., MIYAZAKE,M., and KAZIRO,Y. (1984). Polypeptide chain elongation factor 1 (EFI-a) from yeast; nucleotide sequence of one the two genes for EFI-~ from Saccharomyces cervisae. E M B O J 3, 18251830. NEMER, M., TRAVAGLINI,E. C., RONDINELLI,E., and D'ALoNZO, J. (1984). Developmental regulation, induction, and embryonic tissue specificity of sea urchin metallothionein gene expression. Dev. BioL 102, 471-482.
Counterproductive Transcriptional and Translational Regulation of Elongation Factor 1-c Synthesis during Early Development in Sea Urchins M A R G A R E T T R U S C H E L P E E L E R , 1'2 L E S L I E K E L S O - W I N E M I L L E R , M I N G - F A N W U , J A M E S K . S K I P P E R , AND M A T T H E W M. W I N K L E R
Center for Developmental Biology, Department of Zoology, University of Texas at Austin, Austin, Texas 78712 Accepted August 13, 1990 We have isolated a cDNA clone encoding elongation factor 1-a (EFI-a) and used probes prepared from this cDNA to measure levels of E F I - a transcripts during early development. We also determined the fraction of E F I - a transcripts in polysomes during this time period. Following the blastula stage there is a s h a r p increase in the amount of E F I - a mRNA. This pattern of accumulation is similar to other previously described sea urchin mRNAs. However, while the level of E F I - a mRNA is increasing, the fraction of E F I - a mRNA in polysomes decreases. Thus, there is an apparently counterproductive decrease in efficiency of recruitment into polysomes occurring concurrently with an increase in the overall amount of E F I - a mRNA. © 1990AcademicPress, Inc. INTRODUCTION
The a subunit of eukaryotic elongation factor i (EF1a) functions in the elongation step of protein synthesis, where it mediates the binding of a charged tRNA molecule into the P site of the ribosome. This protein has been cloned from several diverse species and exhibits a high degree of sequence conservation (Hovemann et al., 1988; Nagata et al., 1984; Brands et al., 1986). Recently, it has been shown t h a t in Xenopus, the mRNA encoding E F I - a is a major new transcription product after the midblastula transition, and t h a t the message partitions into polysomes to a high degree, indicating active synthesis of EFI-~ protein (Kreig et al., 1989). We have isolated a cDNA clone corresponding to E F I - a from the sea urchin Strongylocentrotus purpuratus. The mRNA for E F I - a undergoes a 10-fold increase in abundance of the message beginning at the late blastula state. However, this message appears to have an unusual p a t t e r n of translational regulation, such t h a t there is a decrease in the association of the message with polysomes accompanying the increase in message abundance. Thus, there is apparently only an estimated 3-fold increase in the synthesis of E F I - a protein in sea urchins despite a 10fold increase in mRNA abundance during development. MATERIALS AND METHODS
The E F I - a clone was isolated from a blastula stage S. purpuratus (Lambda Zap; Stratagene) cDNA library (a
1 To whom correspondence should be addressed. 2 Current address: D e p a r t m e n t of Biology, Susquehanna University, Selinsgrove, PA 17870. 0012-1606/90 $3.00 Copyright© 1990by AcademicPress, Inc. All rights of reproductionin any form reserved.
gift from Dr. William Lennarz). The library was screened with 32p-labeled Xenopus E F I - a cDNA (a gift from Dr. Paul Krieg). For Northern blots, 5.0 ~g of total RNA, isolated from postmitochondrial supernatants, was separated on agarose gels containing formaldehyde, blotted onto Hybond N nylon membranes, and hybridized as described previously (Kelso-Winemiller and Winkler, submitted for publication). The autoradiograms obtained from the blots were scanned using a densitometer and the area under the peaks was integrated. To correct for possible loading errors, the Northerns were stripped and reprobed with a probe made from a X. laevis 28S rRNA probe and the results were then normalized to an equal amount of rRNA per sample. Control lanes containing increasing levels of unfertilized egg RNA were included to ensure t h a t the Xenopus probe bound proportionally to the amount of sea urchin RNA present (data not shown). The fraction of mRNA in polysomes was based on the quantification of probe binding to slot blots containing RNA isolated from 15 to 50% linear glycerol gradients with 80% glycerol pads used to fractionate postmitochondrial supern a t a n t s from different developmental stages (KelsoWinemiller et al., 1989). The 0D26o of the gradients was determined and used to isolate three fractions. The first two fractions contained n o n t r a n s l a t e d RNA, and the third fraction contained the RNA associated with polysomes. Similar fractions from control gradients containing EDTA were also probed to ensure t h a t the message in the third fraction of the gradient was released by EDTA and therefore polysomal. The fraction of mRNA in polysomes was then calculated as the a m o u n t of mRNA in polysomes divided by the total mRNA in the gradient and expressed as a percentage. A probe for ~486
BRIEFNOTES 0
I 2
487
3 4 6 8 1012 18 2 4 3 6 4 8 ? 2
100" 80' 60"
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W
40, 20O
FIG. 1. Expression of EFI-a during development. A Northern blot containing 5.0 ttg of total RNA from various developmental stages was probed with labeled EFI-a cDNA. The stages represented in each lane are indicated as hours after fertilization. The major transcript at each stage is 2.7 kb.
tubulin (gift of P. H a r l o w a n d M. N e m e r ) w a s used in parallel e x p e r i m e n t s to c o m p a r e the t r a n s l a t i o n a l p a t t e r n of this p r o t e i n to E F I - a . M e a s u r e m e n t s of m R N A levels a n d the f r a c t i o n of m R N A in p o l y s o m e s w e r e rep e a t e d twice w i t h R N A isolated f r o m two different sets of e m b r y o s . RESULTS AND DISCUSSION
Identification of KL1 as sea urchin EFI-~. A c D N A clone encoding sea urchin E F I - ~ w a s isolated f r o m a b l a s t u l a - s t a g e c D N A l i b r a r y b a s e d on its ability to hybridize to a p r o b e m a d e f r o m Xenopus E F I - ~ . The plasm i d c o n t a i n i n g a 2.0-kb insert, r e f e r r e d to as KL1, w a s excised. I t s i d e n t i t y w a s verified by s e q u e n c i n g a n d comp a r i s o n of the sequence to t h a t of the Xenopus clone. The deduced a m i n o acid sequences f r o m the two species w e r e s t r i k i n g l y s i m i l a r , i n c l u d i n g a r e g i o n of 69 i d e n t i t i e s over a region of 75 a m i n o acids, c o r r e s p o n d i n g to a m i n o acids 5-82 of the p u b l i s h e d Xenopus sequence ( d a t a not shown). Expression of EFI-a during development. To d e t e r m i n e the p a t t e r n of e x p r e s s i o n of this clone d u r i n g sea urchin d e v e l o p m e n t , N o r t h e r n blots of t o t a l R N A isol a t e d f r o m different d e v e l o p m e n t a l s t a g e s w e r e p r o b e d w i t h 32p-labeled KL1. The p r o b e h y b r i d i z e s to a single 2.7-kb t r a n s c r i p t , as s h o w n in Fig. 1. T h e r e l a t i v e a m o u n t of E F I - a m R N A is s h o w n in Fig. 2. The level of E F I - a r e m a i n s r e l a t i v e l y c o n s t a n t t h r o u g h the first 12 h r of d e v e l o p m e n t , which c o r r e s p o n d s to the e a r l y blast u l a s t a g e in this species. By 18 h r of d e v e l o p m e n t , or l a t e b l a s t u l a , t h e levels of E F I - a b e g i n to i n c r e a s e , r e a c h i n g m a x i m u m levels a t 48 a n d 72 h r ( p r i s m a n d pluteus stages). T h e r e is a p p r o x i m a t e l y 10 t i m e s m o r e E F I - a m R N A p r e s e n t a t t h e s e s t a g e s t h a n in the first 12 hr. This p a t t e r n of a c c u m u l a t i o n is s i m i l a r to o t h e r sea u r c h i n m R N A s such as t u b u l i n ( H a r l o w a n d N e m e r , 1987).
i
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FIG. 2. Changes in EFI-a mRNA levels during development. An autoradiograph of a Northern blot containing RNA from the indicated developmental stages probed with the sea urchin EFI-a clone was scanned with a densitometer to determine the relative amounts of EFI-c~ mRNA at each stage.
Translation regulation of EFl-~. In Xenopus, the l a r g e i n c r e a s e in E F I - a m R N A a f t e r the m i d b l a s t u l a t r a n s i tion is a c c o m p a n i e d b y the a s s o c i a t i o n of the m R N A w i t h p o l y s o m e s to a l a r g e extent, i n d i c a t i n g t h a t the m R N A is b e i n g t r a n s l a t e d . In sea u r c h i n s the s i t u a t i o n a p p e a r s to be different (Fig. 3). The p e r c e n t a g e of E F I - a in p o l y s o m e s in the u n f e r t i l i z e d egg is low, as is expected because of the d e p r e s s e d r a t e of p r o t e i n s y n t h e s i s in t h e egg. Following fertilization, the p e r c e n t a g e of m R N A in p o l y s o m e s increases, r e a c h i n g a p e a k of 90% by 4 hr. This i n d i c a t e s t h a t s y n t h e s i s of E F I - a p r o t e i n occurs d u r i n g t h e cleavage stage, even t h o u g h the r e l a t i v e level of the m R N A is quite low. H o w e v e r , by 18 hr, the t i m e in d e v e l o p m e n t a t which the a b u n d a n c e of E F I - a m R N A is s t a r t i n g to increase, t h e r e is a decrease in the p e r c e n t 100" E o
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FIG. 3. Fraction of mRNA associated with polysomes. Postmitochondrial supernatants from various developmental stages were fractionated on glycerol density gradients to separate polysomal from nontranslated mRNAs. RNA isolated from gradients was probed with 82p-labeled probes to both EFI-a and tubulin and the fraction of mRNA in polysomes was quantified.
488
DEVELOPMENTALBIOLOGY
age of m R N A in polysomes, so t h a t by 72 hr, only 30% of the m R N A is being actively t r a n s l a t e d . O t h e r m e s s a g e s w h i c h show s i m i l a r i n c r e a s e s in a b u n d a n c e do not und e r g o t h i s d e c r e a s e d level of a s s o c i a t i o n w i t h p o l y s o m e s . F o r e x a m p l e , ~ - t u b u l i n m R N A r e m a i n s a t its p e a k level of p o l y s o m e a s s o c i a t i o n d u r i n g l a t e r developm e n t , as shown in Fig. 3. The f~-tubulin p r o b e used recognizes several of t h e t u b u l i n t r a n s c r i p t s , which all act in a s i m i l a r m a n n e r in t e r m s of p o l y s o m e association. Actin also exhibits a s i m i l a r p a t t e r n of p o l y s o m e r e c r u i t m e n t to/~-tubulin ( d a t a not shown). We h a v e not found a n y o t h e r m R N A s in the sea u r c h i n which e x h i b i t the d e c r e a s e d level of p o l y s o m e a s s o c i a t i o n d e m o n s t r a t e d for E F I - a . Thus, t h e r e is only an a p p r o x i m a t e l y 3-fold i n c r e a s e in the r a t e of E F I - a p r o t e i n s y n t h e s i s d u r i n g d e v e l o p m e n t , despite the 10-fold i n c r e a s e in a b u n d a n c e in the m R N A encoding this protein. I t is unclear w h y an m R N A would exhibit a p p a r e n t l y c o u n t e r p r o d u c t i v e m o d e s of t r a n s c r i p t i o n a l a n d t r a n s l a t i o n a l control. A loss of t r a n s l a t i o n a l efficiency during d e v e l o p m e n t h a s been r e p o r t e d for o t h e r m R N A s , for example, for the m R N A s encoding r i b o s o m a l prot e i n s in d i f f e r e n t i a t i n g m o u s e m y o b l a s t s ( A g r a w a i a n d B o w m a n , 1987), but in t h e s e s y s t e m s , the loss of efficiency is a c c o m p a n i e d by a decrease in the level of m e s sage as well. Thus, it is not clear w h a t , if any, is the f u n c t i o n a l significance of t h e s e c h a n g e s in E F I - ~ m R N A levels a n d r e c r u i t m e n t into polysomes. One possible e x p l a n a t i o n is t h a t the t r a n s l a t i o n depression m i g h t be a control m e c h a n i s m s u p e r i m p o s e d over t r a n s c r i p tional control in o r d e r to reduce t r a n s l a t i o n of the E F 1 protein. I t will be of i n t e r e s t to m e a s u r e the levels of E F I - a p r o t e i n d u r i n g d e v e l o p m e n t to d e t e r m i n e h o w t h e s e levels c o r r e l a t e to t h e o b s e r v e d c h a n g e s in m R N A levels. A l t h o u g h we only o b s e r v e a single size t r a n s c r i p t during d e v e l o p m e n t , the o b s e r v e d t r a n s l a t i o n a l depression m a y be the r e s u l t of the t r a n s c r i p t i o n of a second population of E F I - a m e s s a g e s (possibly encoded by a s e p a r a t e gene) which a r e t u r n e d on a t the end of the b l a s t u l a stage. These m e s s a g e s m a y be less efficiently r e c r u i t e d into p o l y s o m e s t h a n E F I - a m R N A p r e s e n t e a r l y in dev e l o p m e n t due to c h a n g e s in s e q u e n c e or p r o c e s s i n g
VOLUME142, 1990
steps. A l t e r n a t i v e l y , t h e r e m a y be c h a n g e s or modifications of the e x i s t i n g p o p u l a t i o n of E F I - a m R N A a t the b l a s t u l a s t a g e which reduce t h e efficiency, of t h e i r rec r u i t m e n t into polysomes, for e x a m p l e , c h a n g e s in the proteins, or o t h e r f a c t o r s a s s o c i a t e d w i t h the m R N A which influence t r a n s l a t i o n . We hope t h a t f u t u r e experi m e n t s will clarify the level a t w h i c h t h i s u n u s u a l t r a n s lation r e g u l a t i o n is o p e r a t i n g . We gratefully acknowledge the generous gifts of Dr. Paul Kreig who donated the Xenopus clone and provided technical advice, Dr. William Lennarz who provided the cDNA library, and Drs. Patricia Harlow and Martin Nemer who supplied the fl-tubulin clone. This work was supported by an American Cancer Society Postdoctoral Fellowship to M.T.P. (Grant PF-3078) and by Public Health Service Grant (HD 17722-06) to M.M.W. from the National Institutes of Health. REFERENCES AGRAWAI, M. G., and BOWMAN,L. H. (1987). Transcriptional and translational regulation of ribosomal protein formation during mouse myoblast differentiation. J. BioL Chem. 262, 4868-4875. BRANDS,J. H. G. M., MAASSEN,J. A., VAN HEMERT,F. J., AMONS,R., and MOLLER,W. (1986). The primary structure of the subunit of human elongation factor 1. Eur. J. Biochem. 155, 167-171. HARLOW,P., and NEMER,i . (1987). Developmental and tissue-specific regulation of/~-tubulin gene expression in the embryo of the sea urchin Stongylocentrotus purpuratus. Genes Dev. 1, 147-160. HOVEMANN,B., RICHTER,S., WALLDORF,U., and CZIEPLUCH,C. (1988). Two genes encode related cytoplasmic elongation factors 1 (EFI-a) in Drosophila melanagaster with continuous and stage specific expression. Nucleic Acids Res. 16, 3175-3194. KELSO-WINEMILLER,L., DRAWBRIDGE,J., and WINKLER,M. M, (1989). A new ultracentifuge technique for analysis and isolation of polysomes. Nucleic Acids Res. 17, 4896. KELSO-WINEMILLER,L., and WINKLER,i . M. Identification of a novel class of "cleavage-stage specific" maternal mRNAs in sea urchins. Submitted for publication. KRIEG,P. A., VARNUM,S. M., WORMINGTON,W. M., and MELTON,D. A. (1989). The mRNA encoding elongation factor 1-(~is a major transcript at the midblastula transition in Xenopus. Dev. Biol. 133, 93100. NAGATA, S., NAGASHIMA,K., TSUNETSUGU-YOKOTA,Y., FUJIMURS,K., MIYAZAKE,M., and KAZIRO,Y. (1984). Polypeptide chain elongation factor 1 (EFI-a) from yeast; nucleotide sequence of one the two genes for EFI-~ from Saccharomyces cervisae. E M B O J 3, 18251830. NEMER, M., TRAVAGLINI,E. C., RONDINELLI,E., and D'ALoNZO, J. (1984). Developmental regulation, induction, and embryonic tissue specificity of sea urchin metallothionein gene expression. Dev. BioL 102, 471-482.