Transfer RNA methylases during sea urchin embryogenesis

558

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 96927

T R A N S F E R RNA METHYLASES DURING SEA URCHIN EMBRYOGENESIS

OPENDRA

K. SHARMA,

LAWRENCE

A. LOEB*

AND ERNEST

BOREK

Department o/ Microbiology, University o/ Colorado Medical Center, Denver, Colo. 80220 and *The Institute /or Cancer Research, Fox Chase Philadelphia, Pa. I 9 r I I (U.S.A.) (Received March I5th, 1971)

SUMMARY

Methylation of tRNA in sea urchin Strongylocentrotus purpuratus, during embryogenesis was studied by labeling with EMe-3H]methionine. The changes in methylated bases in tRNA following fertilization were not uniform. The N2-mono methylguanine was reduced to half and methylated uridylic acids increased 3-fold. Extracts of eggs and of embryos of various stages after fertilization were active in incorporating methyl groups from S-adenosyl-L-EMe-14CJmethionine into Escherichia coli B tRNA.

INTRODUCTION

The tRNA methylases have been found to be invariably constant in stable biological systems. However, these enzymes show alterations in a variety of systems undergoing shifts in regulatory processes, such as insect metamorphosis 1, phage infection 2, embryonic and neonatal tissues 3, differentiating lens tissue a, slime mold morphogenesis 5, thyroxine induced morphogenesis in bull frog tadpole 8, mammary gland differentiation 7, ovariectomized uterus 8, and a variety of tumor tissues 9. The examples cited do not represent merely quantitative changes in the level of enzymes but also alterations in the specificity of a number of base specific enzymes. COMBTM observed the incorporation of the methyl group of methylmethionine into tRNA and DNA only after 14-15 h after fertilization (gastrulation) in JLytechinus variegatus. On the contrary, SCARANO et al. n reported that methylation of DNA took place at all stages of early development in Paracentrotus lividus and Sphaerechinus granularis. They did not study the methylation of RNA. Since the tRNA methylases modify a cardinal component of protein synthesis (tRNA), and undergo the alterations noted above in so many biological systems, it appeared anomalous that they should show no alteration during early embryogenesis; therefore, we have reinvestigated the methylation of tRNA in vitro and in vivo in Strongylocentrotus purpuratus. In this communication we report that tRNA methylases are present in unfertilized eggs and their activity is manifested following fertilization and that the enzymes and their product, the tRNA's, show qualitative alterations during early embryogenesis. Biochim. t3iophys. Acta, 240 (1971) 558-563

tRNA METHYLASES DURING EMBRYOGENESIS

559

MATERIALS AND METHODS

Eggs of the sea urchin, Strongylocentrotus purpuratus, were collected into artificial sea water b y injecting 0.5 M KC1 into mature adults. After washing 3 times b y sedimentation under gravity they were fertilized in artificial sea water and grown in presence of 200/,g/ml penicillin and 12o/,g/ml streptomycin at 17 ° to the stages indicated.

Preparation o/enzyme extracts The unfertilized eggs and embryos at various stages of development were washed twice b y low speed centrifugation with I M dextrose containing 0.02 M potassium phosphate (pH 7.4), o.oo4 M reduced glutathione and 0.0004 M EDTA. A cell free extract was prepared b y homogenizing with 5 vol. of 0.02 M potassium phosphate (pH 6.8), I M dextrose, 0.004 M fl-mercaptoethanol and 0.0004 M EDTA in a Sorval omnimixer, operated at full speed for 30 sec. The IOO ooo x g supernatant was used as the source for methylase enzymes.

Assay o/tRNA methylases The reaction mixture contained Tris-HC1, p H 8.2 (5o/zmoles), MgClz (5 /zmoles), fl-mercaptoethanol (5/,moles), Escherichia coli B t R N A (2o/,g, General Biochemicals Corporation), S-adenosyl-L-EMe-14C]methionine (o.2#C, specific activity 46.5 C/mole, International Chemical and Nuclear Corporation) and enzyme in a total volume of 0. 5 ml. The control tubes received no tRNA. After incubation for 30 min at 37 ° the reaction was terminated by adding 2 ml of 5 trichloroacetic acid. The precipitates were collected on W h a t m a n GF/A filters and were washed 3 times with 5 % trichloroacetic acid and once with 95 % ethanol. After drying, the filters were counted in a Nuclear Chicago liquid scintillation counter.

In vivo labeling with [Me-aH]methionine Portions containing 2 ml of washed unfertilized eggs or washed embryos were collected by low speed centrifugation and were suspended in 2o ml of artificial sea water (2oo#g/ml penicillin and I2o/~g/ml streptomycin) containing 0. 5 mC of [Me-3H]methionine (specific activity 5.4 C/mmole, Amersham/Searle) at 17 °. After 3 h of incubation the tubes were centrifuged at 300o × g for IO rain and the pellets were frozen and were used for the isolation of tRNA.

Preparation o/tRNA Transfer RNA was prepared b y homogenizing pellets with 5 vol. of I.O M NaCl-o.oo5 M EDTA in o.i M Tris-HC1 (pH 7.5) and an equal volume of watersaturated phenol for I rain TM. The aqueous layer was separated from the phenol layer by centrifugation, and was again extracted with an equal volume of phenol. The RNA was precipitated from the aqueous layer by the addition of 2. 5 vol. of 95 % ethanol containing 2 % potassium acetate. After standing at --2o ° overnight, the precipitate was collected b y centrifugation and was drained free of ethanol. The precipitated RNA was extracted with I M NaC1 in o.i M Tris-HC1 (pH 7.8) in the cold and was again precipitated with ethanol. It was dissolved in IO ml of o.oi M MgCI~, o.I M Tris-HC1 (pH 7.5) and 200 #g of ribonuclease-free deoxyribonuclease Biochim. Biophys. Acta, 240 (1971) 558-563

560

o . K . SHARMAet al.

( W o r t h i n g t o n ) was a d d e d . The solution was i n c u b a t e d at 37 ° for 3o rain followed b y i n c u b a t i o n w i t h 500 # g of pronase. The reaction m i x t u r e was d e p r o t e i n i z e d with an equal volume of w a t e r - s a t u r a t e d phenol a n d t R N A was p r e c i p i t a t e d from the aqueous l a y e r b y 3 vol. of ethanol. The s t r i p p i n g of endogenous a m i n o acid was done b y i n c u b a t i n g in IO ml 1.8 M Tris-HC1 (pH 8.0) at 37 ° for 9 ° rain. The A2eonm/A2son m r a t i o of isolated t R N A was 1. 9 a n d t h e counts in R N A were a l k a l i labile.

RESULTS

The m e t h y l a t i n g c a p a c i t y of e x t r a c t s of unfertilized eggs a n d of e m b r y o s a t various stages of d e v e l o p m e n t d e t e r m i n e d i n vitro on heterologous t R N A from E . coli is shown in T a b l e I. The e x t r a c t s of unfertilized eggs are active in t r a n s f e r r i n g m e t h y l groups from S-adenosyl-L-metliionine to t R N A . This is in c o n t r a s t to the n e g a t i v e findings in t h e literature. The source of this d i s c r e p a n c y m a y stem from an a t t r i b u t e of sea urchin eggs we have observed. If t h e eggs are w a s h e d with water, a p r o c e d u r e we h a d a t t e m p t e d to e m p l o y to remove ions, the t R N A m e t h y l a s e s are lost, b y leaching, into the wash water. I n order to p r e v e n t loss of the enzymes d u r i n g washing a modified washing with I M dextrose was e m p l o y e d . 15 rain after fertilization the e n z y m e e x t r a c t s showed a m a r k e d decrease in s a t u r a t i o n capacities. The decrease in i n vitro a c t i v i t y was not due to e n h a n c e d ribonuclease a c t i v i t y of the fertilized eggs, which could v i t i a t e results b y the h y d r o l y s i s of t R N A ; nor was it due to d e s t r u c t i o n of S-adenosyl-L-methionine. These two possibilities were r u l e d o u t b y i n c u b a t i n g 14Clabeled t R N A w i t h the e x t r a c t s in the absence of S-adenosyl-L-methionine a n d t h e r e c o v e r y of t h e labeled s u b s t r a t e a n d in the second instance b y the s e q u e n t i a l a d d i t i o n of S-adenosyl-L-[14CJmetliionine which y i e l d e d no increase in the t o t a l methylation.

TABLE I tRNA

METHYLASE

C A P A C I T Y OF E X T R A C T S

OF S E A U R C H I N

EGGS AND

EMBRYOS

The IOO ooo×g supernatant was used as the enzyme source and the enzyme capacity was determined in three different sets of experiments with increasing amounts of enzymes. The values reported below are the maximum levels of incorporation in vitro. Enzyme source

Eggs Fertilized eggs 15min 4h 24 h 48 h

Counts/rain per 20 fig E. coli B t R N A Expt. I

Expt. I I

Expt. 1II

23oo

2400

145o

84o 870 85o 750

96o 790 81o 74°

7o5 510 500 35°

The m e c h a n i s m of the decrease in e n z y m e c a p a c i t y in this s y s t e m is obscure at present. I n o t h e r biological systems, such as in an i n d u c e d lysogenic organism following u l t r a v i o l e t i r r a d i a t i o n , a n i n h i b i t o r of uracil m e t h y l a s e a p p e a r s 14 a n d inBiochim. Biophys. Acta, 240 (1971) 558-563

tRNA METHYLASES

561

DURING EMBRYOGENESIS

hibitors of methylases are present in mammalian tissues15 as well as in the colonized slime moldle. The changes observed in the enzyme capacity have been confirmed by the analysis of the tRNA methylated in vitro with enzyme extracts (Table II). The major bases methylated were N2-methylguanine and NZ-dimethylguanine, significant methylation of cytosine and uridine also occurred in later stages. T A B L E II

In vitro METHYLATION OF E. coli B t R N A BY SEA URCHIN EXTRACTS 2OO # g of E. eoli 13 t R N A w a s m e t h y l a t e d in vitro b y IOO ooo x g s u p e r n a t a n t of sea u r c h i n ext r a c t s a n d S-adenosyl-L- [Me-X*C]methionine as described earlier. T h e v o l u m e of i n c u b a t i o n m i x t u r e w a s increased io-fold. T h e t R N A was isolated b y p h e n o l e x t r a c t i o n a n d alcohol p r e c i p i t a t i o n a n d w a s h y d r o l y z e d in 5 °/zl of I IV[ HC1 in a sealed glass t u b e for 6o m i n at IOO°. T h e h y d r o l y s a t e was a n a l y z e d for m e t h y l a t e d c o n s t i t u e n t s according to B J O R K AND SVENSSON13. T h e v a l u e s h a v e been e x p r e s s e d as t h e p e r c e n t a g e of t o t a l r a d i o a c t i v i t y recovered a n d are t h e a v e r a g e of 3 different e x p e r i m e n t s .

Eggs

1V[ethylated cytidylic acids 1V[ethylated uridylic acids N2-Methylguanine N2-Dimethylguanine

Fertilized eggs, after 15 rain

Blastula (24 h)

Pluteus (48 h)

4.24-2

13.54-1. 7

6. 5 4-2

i o . i 4-0. 5

5.1 4- i 5o.o4-3.1 38.24-1.5

4.o4-2.1 36.14-1.9 44.34-1.1

13.1 4-1.3 38.74-2.7 40.04-2.9

17.o±4 29.14-2 42.14-1.3

The methylation of tRNA in vivo was studied by pulse labeling with [Me-all] methionine and by the analysis of the isolated tRNA for individual methylated bases. N*-Methylguanine was the major base methylated in addition to methylation of NZ-dimethylguanine, cytidylic and uridylic acids in unfertilized eggs. There occurred a 50 % decrease in N~-methylguanine and a 3-fold increase in methylated uridylic acids following fertilization (Table III). The incorporation of radioactivity in the purine TABLE III DISTRIBUTION OF METHYL&TED BASES IN SEA URCHIN E M B R Y 0 G E N E S I S

t R N A LABELLED i n v i v o WITH [ M e - 3 H ] M E T H I O N I N E DURING

T h e eggs before a n d a t v a r i o u s stages following fertilization were i n c u b a t e d w i t h [Me-3H]me t h i o n i n e as described u n d e r MATERIALS AND ~tETHODS. T h e isolated t R N A w a s a n a l y z e d for t h e d i s t r i b u t i o n of r a d i o a c t i v i t y in m e t h y l a t e d b a s e s according to BJORK AND SVENSSON18. T h e v a l u e s h a v e b e e n e x p r e s s e d as p e r c e n t a g e of r a d i o a c t i v i t y recovered a n d are t h e a v e r a g e of 3 determinations.

lV[ethylated cytidylic acids Methylated uridylic acids N2-Methylguanine N2-Dimethylguanine

Eggs

Fertilized e g g s after 15 rain

Blastula

Plutei

2o.o i 2

24.14-1.3

26.2 4- 2.9

29.7 4- I. 2

I I . i 4-1. 7 52.14-2 15.64-2.2

32.2£3 27.o4-2.1 I5.94-1.9

17.1 4-1. 4 26.74-2. 5 27.24- 4

18.7±3.1 19.74-2 29.7£3

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ring via the one carbon pool was unlikely because no radioactivity was detected in spots corresponding to other purines, i.e. adenine and guanine. This is to be expected since the 3H-atoms are exchanged with the aqueous pool during conversion to the formate moiety. The differences in the patterns of labeling in vitro and in vivo are worthy of comment. One contributing factor is, of course, the difference in the structures of the substrates: the first is from a heterologous source; the second is indigenoous. The tRNA methylases are known to have high specificity for nucleotide sequences 17. Therefore, the different patterns of methylation of the two substrates are not unexpected, those obtained by in vivo labeling mirroring with fidelity the reactions within the cell. However, in vitro patterns of labeling are not without significance for they represent changes in the enzyme systems visa vis the same substrate under identical conditions.

DISCUSSION

Sea urchin embryogenesis is a useful system for studying the nature of regulatory mechanisms involved during differentiation. It is known that when RNA synthesis after fertilization is prevented, the templates made and stored in the cytoplasm during oogenesis allow both nearly the normal rate of post-fertilization protein synthesis and morphological development to the swimming blastula stage TM. It has been suggested that translational control of the preformed messenger RNA is involved in regulating the protein synthesis during early stages of embryogenesis18,19. Following fertilization a substantial addition of pCpCpA to terminal sequences of tRNA was detected ~°,21. The significance of this modification to functional capacity is not clear at present. The tRNA's made immediately following fertilization are different from those present in unfertilized eggs in their endowment of methyl groups, and the new tRNA's formed presumably function differently in protein synthesis. Some of the functions of methylation are now clear. Methylation of tRNA in E. coli serves a cognitive role in the interaction with the charging enzyme 2~, its coding response *z and its ability to attach to ribosomes ~. What other functions of tRNA are served by methylation is obscure at present.

ACKNOWLEDGMENTS

This work was supported by a grant to E.B. from the American Cancer Society and by a contract from the Atomic Energy Commission, grants to L.A.L. from the National Institutes of Health (CA-o6927) and National Science Foundation (GB18419) and to the Institute for Cancer Research from the National Institutes of Health (RR-o5539) and from the Commonwealth of Pennsylvania. REFERENCES 1 2 3 4

B. S. BALIGA, P. R. SRINIVASAN AND E, BOREK, Nature, 208 (i965) 555E. WAINFAN, P. R. SRINIVASAN AND E. ]~OREK, Biochemistr% 4 (I965) 2845. R. L. HANCOCK, P. M~CFARLAND AND R. R. FOX, Experientia, 23 (1967) 806. S. J. [{ERR AND Z. DISCHE, Invest. Ophthalmol., 9 (197 o) 286.

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Commun., 31 (1968) 404 • 23 A. pETERKOFSKY, C. JESENSKY AND J. D. CAPRA, Cold spring Harbor Syrup. Quant. Biol., 31 (1966) 515 • 24 IV[. L. GEFTER AND R. L. RUSSELL, J. Mol. Biol., 39 (1969) 145.

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