Decay of transfer and total RNA in maturing reticulocytes

439

BIOCHIMICA ET BIOPHYSICA ACTA 13BA 96829

DECAY OF T R A N S F E R AND TOTAL RNA IN MATURING RETICULOCYTES T. Z E H A V I - W l L L N E R AND D. DANON

Section o] Biological Ultrastructure, The Weizmann Institute of Science, Rehovot (Israel) (Received January 5th, 1971)

SUMMARY

A gradual decrease in ribosomes and tRNA occurs during maturation of reticulocytes. The rate of decrease of ribosomes is higher than that of tRNA, so that there is a continuous increase in the proportion of tRNA to total RNA. No significant changes in the accepting activity of tRNA to a mixture of 16 amino acids could be observed during the maturation process. The decreased activity in protein synthesis of a cell-free system containing a soluble fraction of old reticulocytes could not be compensated by the addition of tRNA isolated from young reticulocytes. It is concluded that tRNA is not responsible for the diminution in the rate of protein synthesis in the maturing reticulocyte.

INTRODUCTION

As the reticulocyte matures into an erythrocyte, there is a gradual decrease in its capacity for protein synthesis as well as a decrease in its nucleic acid content. Some investigators 2-4 have studied the breakdown of the total RNA during reticulocyte maturation. Other studies have been restricted to the disappearance of the ribosomes 5-9, which are associated with the bulk of the RNA. The decline in ribosome content is closely correlated with the steady decline in protein synthesis 1A,z°. Since it was found that during the maturation process the decline in protein synthesis takes place faster than the breakdown of ribosomes ~, the decay or reduced activity of factors other than ribosomes involved in protein synthesis was studied 11, in order to determine what are the limiting factors which are responsible for the decay of protein synthesis before the ribosomes disappear. In the present study, the possibility that tRNA might be a rate limiting factor in protein synthesis in maturing reticulocytes was investigated. MATERIALS AND METHODS

Reticulocytes Reticulocyte-rich blood was obtained by bleeding common stock rabbits e. ~2P-labeled reticulocytes were prepared by injecting intravenously anemic rabbits with 5 mC [32P~phosphate /purchased from the Nuclear Research Center, Negev, Beer Sheva, Israel). Blood was collected 38-42 h after the administration of the radioactive phosphate. Biochim. Biophys. Aeta, 238 1197T) 439-446

440

T. ZEHAVI-WILLNER, D. DANON

Systems ]or studying decay o[ tRNA and rRNA in maturing reticulocytes in vivo (I) Translusion. 3~p-labeled reticulocytes were transfused into normal rabbits and the proportion of 3zP-labeled t R N A to 32p in ribosomes was determined at different time intervals. Transfusion was carried out essentially as described by CIVIDALLI AND DANON12. 25 ml of an 80 % suspension of a2p-labeled reticulocytes was transfused into a normal rabbit. I h after transfusion, 15 ml blood was collected from the marginal vein of the ear. This blood was considered to be the "zero time" sample. After 5 h, 30 ml of blood was collected and after I I h 60-80 ml of blood was taken from the animal's heart. In some experiments the blood samples were taken at zero, 16 and 24 h. Red blood cells were separated from the plasma and buffy coat by centrifugation at 800 × g for IO min, washed twice, lysed and fractionated as described by WARNER et al. 18. The ribosomes were purified on P-2oo Biogel columns; t R N A was prepared from the $1oo fractions. The proportion of *2P-labeled t R N A to total 32p_ labeled RNA was calculated from the data obtained for the ribosomal and t R N A fractions. Since the amount of t R N A bound to the ribosomes was less than I °/o of the rRNA it was considered not worthwhile to extract the ribosomal RNA and separate the ~RNA from the other RNA's. (2) Actinomycin D treatment. Actinomycin D was injected into rabbits (15o/~g/ kg body weight) to prevent further release of reticulocytes from the bone marrow into the circulation 4. The first blood sample (considered as zero time) was taken 28-30 h after actinomycin D administration, since it was found in preliminary experiments that release of reticulocytes from the bone marrow continues for 28-30 h after administration of the drug. Further procedures were as described in the transfusion experiments.

Puri]ication o/ ribosomes The ribosomal pellet obtained by differential centrifugation of the postmitochondrial supernatant at lO5 ooo x g for 2 h was resuspended in the following buffer: o.oi M Tris, pH 7.4, o.oo15 M MgC12, 0.05 M KC1 (RBC buffer). The hemoglobin-free ribosomes were prepared using a Biogel column ~4. The ribosomal suspension was applied to a P-2oo Biogel column (2.3 c m x 35 cm) and eluted with the RBC buffer. The amount of ribosomes in 2.4-ml fractions which were collected was estimated b y measuring the absorbance at 260 mk~ and the radioactivity by counting o.2-ml aliquots in IO ml of Bray's scintillation solution in a Packard Tri-carb liquid scintillation counter.

tRNA preparation The t R N A was extracted by phenol from the Slo0 fraction prepared from reticulocytes at different stages of maturation. The aqueous phase was precipitated with 2 vol. of ethanol containing I °/o magnesium acetate. When t R N A was extracted from $100 fractions prepared from transfused animals, 4 A2e0 nm units of unlabeled t R N A were added to the aqueous phase as carrier. The ethanol-precipitated RNA was purified as t R N A by means of Sephadex G-ioo chromatography 15. Fractions of 2.4 ml were collected and the absorbance at 260 m/~ and radioactivity of the t R N A determined. The phenol-extracted RNA from $100fractions prepared from reticulocytes of rabbits injected with actinomycin D were dialyzed for 5-1o h against RBC buffer before chromatography on a Sephadex G-Ioo column. Biochim. Biophys. Acta, 238 ti97 I) 439-446

RNA DECAY IN MATURING RETICULOCYTES

441

Gel electrophoresis 3~P-labeled tRNA samples isolated from $100 fractions of reticulocytes at different stages of maturation and purified on a Sephadex G-ioo column were combined with carrier reticulocyte tRNA and separated on polyacrylamide gels as outlined by LOENING1~. The tRNA samples were placed on gels containing 6 % (w/v) recrystallized acrylamide and o.II % (w/v) methylene bisacrylamide. Scanning of the gels was performed using an attachment to a Shimatzu QV 50 spectrophotometer, mounted between the sample chamber and the photomultiplier housing 17. The slicing and counting of the gels was carried out according to the procedure described by GRESSEL AND WOLOWELSKY18.

Methylated albumin kieselguhr chromatography This was performed as described by COMB AND ZEHAVI-WILLNER19.

Aminoacyl-tRNA synthesis assay The reaction mixture (total volume IOO/,1) contained 7.5/*moles KC1, I/*mole Tris-HC1, pH 7.4, I/,mole dithiotreitol; 0. 7/,mole MgC12; o.I/,mole ATP; 0.5/,mole phosphoenol-pyruvate, 2/,g pyruvate kinase; 20/,1 S10o fraction and one of the following labeled amino acids: 0.25 pmole Ix4C]phenylalanine (lO6 mC/mmole); o.I pmole E14C]leucine (312 mC/mmole); 0.05 pmole of a mixture of 16 [x4C]labeled amino acids (51 mC/milfiatom) (Radiochemical Centre, Amersham) and IO/,g of the test tRNA. After incubation for 30 rain at 37 °, samples were cooled to o ° and 0.2/,mole of an unlabeled amino acid as used in the incubation mixture was added to each tube. IOO-/,1portions of the incubated, cooled, mixture were pipetted on to Whatman 3MM filter discs. The discs were washed once in cold IO % trichloroacetic acid solution and 3 times in cold 5 % trichloroacetic acid solution and finally once in ethanol ether (i :2, v/v). The discs were dried and their radioactivity counted.

Assay/or amino acid incorporation Washed reticulocytes were lyzed and ribosomes and $10o soluble tractions prepared as described by WARNER et al. 13. The ribosomes were resuspended in their own $10o fraction or in $100 fraction prepared from old reticulocytes (48 h after actinomycin D administration). The incubation mixture consisted of 50 #1 of a solution containing salts, amino acids, E14C]leucine and an energetic system as described by ADAMSON et al. 2°, tRNA isolated from young reticulocytes (2.5/*g) and IOO/*1 of ribosomes suspended in either $10o fraction. After incubation at 37 ° for 15 min, the samples were cooled to o ° and 0.2/,mole of unlabeled leucine was added to each tube. IOO/,1 portions were pipetted on to Whatman 3MM filter discs for the determination of radioactivity in the hot trichloroacetic acid-insoluble protein fraction.

RESULTS

Relative decay o/tRNA in maturing reticulocytes In repeated experiments we have found that, at zero time, the tRNA in the reticulocyte Sxoo fraction represented 12-16 °/o of the total RNA. During maturation of reticulocytes, the absolute amount of tRNA and ribosomal RNA decrease at dif-

Biochim. Biophys, Acta, 238 (I97 I) 439-446

442

T. ZEHAVI-WILLNER, D. DANON

ferent rates; the relative amount of t R N A to total RNA of maturation there is a decrease of over 50 % in the ticulocytes and about 30 % in the t R N A content. After remain, whereas the $io0 fraction retains about 20 % (Table I).

increases. After the first 12 h ribosomal content of the re24 h practically no ribosomes of its initial t R N A content

TABLE I T H E R E L A T I V E D E C A Y OF

tRNA

Expt.

tRNA/total RNA

Time"

(h)

I

*

rRNA

IN MATURING RETICULOCYTES

tRNA decay

(%)

O

II

TO

rRNA decay

(%)

(%)

11.8

5

15.5

16

36

12

21. 3

31

62

65 84

78 92

o

15.5

16 24

24.o 29.2

F o r definition of "zero t i m e " see MATERIALS AND METHODS.

Homogeneity o/tRNA isolated ]rom reticulocytes at late stages o/maturation To determine whether t R N A isolated from reticulocytes at late stages of maturation and purified on Sephadex G-Ioo is contaminated with breakdown products of rRNA, the t R N A was subjected to different chromatographic procedures, i.e. gel electrophoresis, which would separate the RNA molecules mainly according to their molecular weight ~x and methylated albumin kieselguhr chromatography in which the secondary structure affects the separation ~2,~3. When 3~P-labeled t R N A isolated from reticulocytes at different stages of maturation was mixed with unlabelled carrier t R N A and reseparated b y the two methods mentioned above, no heterogeneity could be observed (Figs. I and 2). ~

,

r

i

,

i

F

,

i

i

--~

I

i

I

I

Oh

i

i

,

i i 1 , 1 1 1

T

[

24h

16h

400

02

300

02

200

0

4

6

8

0

4 Distance

migrated

6

8

0

2

4

6

8

(cm)

Fig. I. Polyacrylamide gel electrophoretic p a t t e r n s of t R N A from reticulocytes isolated at different stages of m a t u r a t i o n , t R N A samples extracted from 3~P-labeled reticulocytes at different stages of m a t u r a t i o n (o, i6 a n d 24 h) and purified on S e p h a d e x G - l o o columns were mixed w i t h o.6 A u0 mu unit of carrier t R N A and applied to gels. The average specific activity of t h e " P - l a b e l e d t R N A ' s was 28 ooo c o u n t s / m i n per A260 mr* unit. o h, I25 o c o u n t s / m i n ; i6 h, lO5O c o u n t s / m i n ; 24 h, 65o counts/min. - - - - , A260 m~; O - - - O , sap radioactivity.

Biochim. Biophys. Acta, 238 (1971) 439-446

RNA

DECAY IN MATURING RETICULOCYTES

o]

I

I

!

I

Oh

I

I

I

i

I

,6h

t

443 I

I |

24h

11400

o

t '°°° 600.~

° o

o.o i]' 20

,, 40

20 40 Fraction No.

2O

40

Fig. 2. Methylated a l b u m i n c o l u m n c h r o m a t o g r a m s of 82P-labeled t R N A isolated from reticulocytes at different stages of m a t u r a t i o n . 0.2 A2~0m~ unit of t R N A (approx. 4ooo c o u n t s / m i n ) f r o m reticulocytes at different stages of m a t u r a t i o n were mixed with 3 A 260m~ units of carrier t R N A a n d c h r o m a t o g r a p h e d on m e t h y l a t e d a l b u m i n kieselguhr columns as described in MATERIALS AND METHODS. O- - -O, A~eom#; 0 - 0 , 82p radioactivity.

Base analysis 32p-labeled tRNA isolated at different stages of reticulocyte maturation was hydrolyzed in 0.3 lV[ KOH for 14 h at 37 ° and subjected to electrophoresis, together with a mixture of four unlabeled major nucleotides. The electrophoresis was carried out at pH 3.5 at 3000 V for 9 ° min. The base composition was determined by identifying the nucleotide spots by ultraviolet light and counting their relative radioactivities. The base compositions of tRNA samples taken from different age groups of reticulocytes were very similar (Table II), indicating that the tRNA was not contaminated. TABLE II BASE COMPOSITIONOF $~P-LABELED t R N A FROM RETICULOCYTES AT DIFFERENT STAGES OF MATURATION The three samples were dissolved in 0. 3 M K O H contained in io pl capillaries in an oven at 37 °. The radioactivity of each sample was a b o u t 4ooo counts/min. Before electrophoresis the samples were diluted with IO pl of a solution of t h e four s t a n d a r d nucleotides (o.o25 M each).

Timeo[ isolation (h)

CMP (%)

o 16 24

29.7±o.3 3o.o~o.2 29.6~o.3

AMP (%) 16.3±o. 4 16.2±o.3 15.8±o.5

GMP (%)

UMP (%)

33.o±o.2 33.6±o.1 33.5io.2

2i.o~o. 4 2o.2~o.5 2I.I~O.5

Ratio o] tRNA to total RNA in maturing reticulocytes ]rom actinomycin D-treated rabbits and the accepting activity o/the tRNA The tRNA isolated from transfused 32p-labeled reticulocytes at different stages of maturation could not be compared on the basis of their biological activity because only the 32p-labeled RNA could be correlated to the degree of maturation of the reticulocytes. The actinomycin D prevents the release of new reticulocytes Biochim. Biophys. Aaa, 238 ~i97 x) 439-446

444

T. Z E H A V I - W I L L N E R ,

D. D A N O N

into the peripheral blood for a short peliod. This experimental system enables us to follow the tRNA to rRNA ratio and study the accepting activity of tRNA isolated at different stages of maturation. A rabbit with about 4 ° °/o reticulocytes was injected with 5 mC of Es*Plphosphate. After an interval of 5 h actinomycin D was administered, and 30 h thereafter a blood sample was taken. Taking of further blood samples and other procedures were then carried out as in the transfusion experiments. The results were essentially the same as those obtained in the transfusion experiments, namely, the ratio of tRNA to total RNA increased with maturation (Table III). TABLE

III

THE RELATIVE DECAY OF TOTAL I~NIA TO t R N A D-TREATED RABBITS

Time"

Ribosomal R N A ""

t R N A **

~sp

~sp

A SSOmu

(counts/rain) o 5 12

IN MATURING RETICULOCYTES IN ACTINOMYCIN

81 5 0 0 49 2oo 17 9 0 0

% tRNA A s6om/t

(counts / m i~ ) 4.12 2.35 1.47

723 ° 68oo 431o

32p

A 2e0m/~

(counts/rain) o.4o8 o.375 o.329

8.9 13.8 24.0

9.9 16.oo 22.4

" F o r d e f i n i t i o n of " z e r o t i m e " see MATERIALS AND METHODS. ** T h e a m o u n t of R N A i n t h e d i f f e r e n t f r a c t i o n s is c a l c u l a t e d p e r m l p a c k e d cells. T h e a c t u a l a m o u n t of cells u s e d w a s : z e r o t i m e , 35 m l ; 5 h, 6. 7 m l ; 12 h, i 2 . 8 - m l p a c k e d cells.

The tRNA fractions isolated from pooled anemic blood of two actinomycin Dtreated rabbits at different stages of maturation were tested for their total accepting activity and of phenylalanine and leucine separately. In four out of six experiments, the total accepting activity of the tRNA extracted from reticulocytes at different stages of maturation was practically identical, nor were there significant changes in the accepting activity of the tRNAph e and tRNALe u. In two experiments, a decrease of about 30 % from the original level in the accepting activity of the tRNAL~u was observed (see Table IV). TABLE

IV

THE AMINO ACID ACCEPTING ACTIVITY OF t R N A MATURATION

Expt.

Maturation time o[ reticulocytes

(h)

FROM RETICULOCYTES AT DIFFERENT STAGES OF

[a4C]Aminoacyl-tRNA (r6 amino acids)

[14C]Leu-tRNA

[14C!Phe-tRNA

Counts[rain per A zeo mu unit t R N A

II

o 5 12

4000 4155 431o

1483 ° 129oo 986o

5300 5275 489 o

o 12 18

5365 546o 482o

9700 1o310 8835

459 ° 4680 461o

Biochim. Biophys. Acta, 2 3 8 ( I 9 7 I) 4 3 9 - 4 4 6

RNA

DECAY IN MATURING RETICULOCYTES

445

The e]]ect o] tRNA isolated ]rom young reticulocytes on protein synthesis in a cell-]ree system containing $1oo ]raction ]rom young or mature reticulocytes ROWLEY AND MORRIS2a reported that the activity of $10o fraction in supporting protein synthesis decreases during maturation of reticulocytes. Our experiment described in the previous sections showed that the relative amount of t R N A to ribosomal RNA increases and no major differences in the total accepting activity of the t R N A could be detected. In order to exclude the possibility that one or more of the tRNA's was initially present in only a limited amount, and during maturation a stage was reached in which this t R N A was entirely absent, the following experiment was carried out. The t R N A from a whole population of reticulocytes was added to a cell-free system containing $1oo from mature reticulocytes. If the above mentioned hypothesis was correct, an increase in the protein-synthesizing capacity of the system would be observed. Ribosomes were prepared from total populations of reticulocytes. A portion of the ribosomes was resuspended in a Sx0o fraction from relatively young reticulocytes taken from a rabbit 3o h after injection of actinomycin D. Another portion of the ribosomes was resuspended in S~0o from relatively mature cells (taken 4 8 h after actinomycin D injection). Purified t R N A obtained from a total population of reticulocytes was added to each of the two systems. It can be seen from Table V that tRNA from a total population of reticulocytes did not enhance amino acid incorporation in either system, indicating that the decreased activity of the S100 fractions in maturing reticulocytes is not due to limited amounts of one t R N A species. TABLE

V

THE EFFECT OF t R N A ON PROTEIN SYNTHESIS IN CELL-FREE SYSTEMS CONTAINING YOUNG AND OLD RETICULOCYTE SlOo FRACTIONS

$1oo ]faction

tRNA (/*g)

[x4C]Leucine incorporation (counts/rain)

Young Young Old Old

-2.5 -2. 5

946 965 820 812

DISCUSSION

The data obtained in the present study indicate that during reticulocyte maturation there is a relative decrease in the amount of rRNA as compared to tRNA. The isolated t R N A fractions from reticulocytes at late stages of maturation have been shown not to be contaminated b y rRNA breakdown products. This was concluded from the results of chromatography, base analysis and the fact that no differences could be observed in the capacity of t R N A isolated from reticulocytes at different stages of maturation to form the different aminoacyl-tRNA's. In their search for an explanation of the more rapid decline in protein synthesis as compared to the decline in the ribosomal fraction, ROWLEY AND i~ORRIS24 found that during maturation of reticulocytes there is a decreased activity of both the ribosomal and soluble fractions. HERZBERG et al. n reported that ribosomes isolated from relatively Biochim. Biophys. Acta,

238 ¢i971) 4 3 9 - 4 4 6

446

T. ZEHAVI-WlLLNER, D. DANON

old reticulocytes were deficient in their capacity to initiate protein synthesis. Adding initiating factors from total reticulocyte population to a cell-free system isolated from old reticulocytes increased the activity to some extent, but not to the level of the young reticulocyte system. Further enhancement of protein synthesis could be obtained by the soluble fraction from a young cell population, indicating that in this fraction derived from old cells, there are additional limiting factors which are responsible for the decrease in protein synthesis. Since we have shown that during maturation of reticulocytes the relative amount of tRNA to ribosomes or ribosomal RNA increases, the possibility that tRNA is the limiting substance in the soluble fraction in mature reticulocytes is practically ruled out. It could be conceived, however, that a more rapid decrease of some species of tRNA, either because of more rapid degradation or because they were initially present only in minor amounts, might play a role in regulating the rate of protein synthesis. The experiment in which no enhancement of protein synthesis could be detected when tRNA extracted from a total population of reticulocytes was added to a cell-free system containing $100from mature reticulocytes negates this hypothesis. Thus the decreased activity of the soluble fraction from mature reticulocytes in supporting protein synthesis cannot be attributed to limited quantities of tRNA in general or of a specific tRNA. The possibility still exists that in the supernatant of the mature reticulocyte there are limiting amounts of aminoacyl synthetases or transfer enzymes that participate in the process of protein biosynthesis. This is a subject for further study. ACKNOWLEDGEMENTS

We thank Miss 0fra Stanger and Mrs. Miriam Shamir for excellent technical assistance. REFERENCES I 2 3 4 5 6 7 8 9 IO 11 12 13 14 15 16 17 18 19 20 21 22 23

24

L. M. LOWENSTEIN, Intern. Rev. Cytol., 8 {I959) 135. H. G. SCHWEIGER, S. M. RAPOPORT AND E. SCHOLZEL, Nature, 178 (1956) 141. J. F. BERTLES AND W. S. BECK, J. Biol. Chem., 237 (1962) 377 o. T. ZEHAVI-WILLNER, G. IZAK AND J. MAGER, Blood, 27 (1966) 319. P. A. MARKS, R. A. RIFKIND AND D. DANON, Proc. Natl. Acad. Sci. U.S., 50 (1963) 336. D. DANON, T. ZEHAVI~WILLNER AND Cr. R. BERMAN, Proc. Natl. Acad. Sci. U.S., 54 (1965) 873. E. GLOWACKI AND R. L. MILLETTE, J. Mol. Biol., i i (1965) 116. D. DANON AND L. CIVIDALLI, Biochem. Biophys. Res. Commun., 3o (1968) 717 . R. M. DE BELLIS, Biochemistry, 8 (1969) 3451 . B. W. HOLLOWAY AND S. H. RIPLEY, J. Biol. Chem., 196 (1952) 695. M. HERZBERG, M. REVEL AND D. DANON, European J. Biochem., i i (1969) 198. L. CIVIDALLI AND D. DANON, J. Haematol., 19 (197 o) 243. J. R. WARNER, P. M. KNOP AND i . RICH, Proc. Nail. Acad. Sci. U,S., 49 (1963) 122. M. HERZBERG, Compt. Rend., 268 (1969) 2530. T. SCHLEICH AND J. GOLDSTEIN, ]. Mol. Biol., 15 (1966) 136. U. E. LOENING, Biochem. J., 113 (1969) 131. J. GRESSEL AND J. WOLOWELSKY, Anal. Biochem., 24 (1968) 157. J. GRESSEL AND J. WOLOWELSKY, Anal. Biochem., 22 (1968) 352. D . G. COMB AND T. ZEI-IAVI-WILLNER, J. Mol. Biol., 23 ti967) 441. S. D. ADAMSON, E. HERBERT AND W. GOODCHIAUX, I I I , Arch. Biochem. Biophys., 125 (1968) 671. U. E. LOENING, J. Mo1. Biol., 38 (1968) 355U. Z. LITTAUER AND R. STERN, in L. O. FROHOLM AND S. G. I.ALAND, Structure and Function o/ Transfer R N A and 5 S - R N A , Academic Press, London, 1967, p. 93. N . SARKAR AND D. C-, COMB, J. Mol. Biol., 39 (1969) 31. P. T. ROWLEY AND J. A. MORRIS, J. Biol. Chem., 242 (1967) 1533.

Biochim. Biophys. Acta, 238 (1971) 439-446