Polyadenylated RNA in diapausing and developing embryos of Bombyx mori

Insect Biochem. Vol. 9, pp. 125 to 128. © Pergamon Press Ltd. 1979. Printed in Great Britain

0020-1700/79/0301-0125 $02.00/0

P O L Y A D E N Y L A T E D R N A IN D I A P A U S I N G A N D D E V E L O P I N G EMBRYOS OF B O M B Y X M O R I KRYSTYNA GRZELAK, EL2;BIETA SZCZI~SNA a n d ZOFIA LASSOTA Institute of Biochemistry and Biophysics of Polish Academy of Sciences, Warsaw, Poland (Received 10 March 1978) Abstract--The polysome fraction from diapausing embryos of Bombyx mori contains polyadenylated RNA which does not show messenger activity in vitro. The enhanced synthesis and polyadenylation of RNA during development of the embryo results in the appearance of active mRNA in polysomes. Key Word Index: Polyadenylated RNA, polysomes, Bombyx mori embryo, diapause

storage overnight at - 18°C it yielded a precipitate denoted as fraction 105s. Homogenization and centrifugation were performed at 4°C. DIAPAUSING insects represent a convenient model for Polysomes were isolated in the presen,e of i/.o (w/v) the study of the biochemical events associated with the arrest a n d resumption o f gene expression. In deoxycholate and purified in a 12-36% linear sucrose density gradient according to WAStLEWSKAand CHERRY (1974) with L e p i d o p t e r a it has been f o u n d recently t h a t minor modifications described previously (SzYSZKO and diapausing e m b r y o s o f B o m b y x mori, in which protein LASSOTA, 1977). The only change introduced was, that before synthesis is quiescent, c o n t a i n polysomes (SzvszKo layering on the sucrose gradient, the material sedimenting a n d LASSOTA, 1977). T h e o b s e r v a t i o n was puzzling as within 10 min at lO,O00g was removed from the polysomal metabolic animal e m b r y o s such as the encysted fraction. Artemia salina, have been reported to be devoid o f RNA was isolated from the subcellular fractions and from polysomes a n d to c o n t a i n m R N A stored in the polysomes by the procedure of HASTINGSand KIRBY(1966) at cytoplasm as 40 S r i b o n u c l e o p r o t e i n complexes pH 7.2, applied as described previously (SzYSZKO and (GROSFELD a n d LITTAUER, 1975). Therefore we have LASSOTA, 1972) with 1.6% (w/v) SDS used instead of 3% (w/v) p-aminosalicylate, or by the procedure of BRAWERMA~(1974) examined the distribution o f polyadenylated R N A in 0.1 M Tris-HCl buffer, pH 9.0, containing 0.005 M EDTA a m o n g the subcellular fractions o f diapausing a n d ' and 0.5% (w/v) SDS as in the modification of this procedure developing silkworm e m b r y o s a n d have tested the by SIERRAet al. (1976). Digestion with DNAse (RNAse-free) messenger activity o f polyadenylated R N A found in was performed according to KNOWLAND(1970). The details polysomal structures. of the procedure and removal of enzyme protein are The occurrence in diapausing silkworm e m b r y o s o f described elsewhere (OLSZANSKA et al., 1974). Affinity 'resting' polysomes with polyadenylated R N A which chromatography on oligo (dT) cellulose of isolated RNA was is n o t translatable indicates t h a t different molecular performed according to EDMONDSand CARAMELA(1969) as m e c h a n i s m s are involved in the regulation o f gene modified by WASmEWSKA and KLECZKOWSKI (1974) or according to Avw and LEDER (1972). expression in resting animals. The binding capacity of the columns was calibrated with poly (A) standard solution and the nonspecific binding was MATERIALS AND METHODS tested with ribosomal RNA from Bombyx mori eggs separated on methylated albumin column prepared The silkworm eggs of the monovoltine white Japan hybrid according to MANDELL and HERSHEY (1960). Enzymatic strain of Bombyx mori (L.) were obtained from a commercial deproteinisation of subeellular fractions as recommended by culture in Milan6wek. The eggs were kept in cold storage WIEGERS and HILZ (1972), was performed together with the from December until May. Transfer of the hibernating eggs procedure of Avlv and LEDER(1972). Pronase was suspended to 25°C resulted in hatching of larvae after 14 days. The eggs in the application buffer containing 0.5% (w/v) SDS and was were investigated during diapause and on the eighth day of self-digested for 90 min at 37°C. This pronase solution was postdiapausal incubation. added to the subeellular fractions, suspended in the same Subeellular fractions were obtained after a mechanical buffer, to the final concentration of 10 mg enzyme per ml, and homogenization of eggs in 0.05 M Tris-HCl buffer, pH 7.8, the digestion of proteins was carried out at room temperature containing 0.25 M sucrose, 0.01 M MgCI2, 0.015 M KC1, for 90 min. Fractions 15p, 105p and polysomes were treated 0.005 M fl-mereaptoethanol, 3 ml of this solution being used after they had been put on the oligo (dT) cellulose column, per gram of eggs. From the resulting homogenate the nuclei while nuclei and fraction 105s were digested once before were spun for 10 min at 800g The postnuclear supernatant putting on the column and then again, with a new portion of was filtered through sterile cotton-wool and the filtrate was pronase, on the oligo (dT) cellulose column. The digestion centrifuged for I 0 min at 15,000g. The material sedimented products were eluted with the application buffer containing during ccntrifugation constituted the 15p fraction. The 0.5% (w/v) SDS and then SDS was removed by treatment supernatant was then centrifuged at 105,000g for 4 hr. The with the same buffer without SDS. After washing the column resulting sediment constituted the 105p fraction. The with the elution buffer, the retained material was removed remaining supernatant was treated immediately with 2 voi of and in the case of 15p fraction it was supplemented with NaC1 absolute enthanoi in the presence of 0.5 M NaCI. After (final concentration 0.5 M) and rechromatographed to 125 INTRODUCTION

126

KRYSTYNA GRZELAK, ELZBIETA SZCZ~SNA AND ZOFIA LASSOTA

remove the coloured contaminant. In the case of nuclei the material was digested with DNAse according to KNOWLAND (1970), then treated once more with pronase, as indicated above and finally after addition of NaCI to 0.5 M it was rechromatographed on the oligo (dT) cellulose column. Translation assay was carried out in a wheat germ cell-free system as described by ZAN-KOWALCZEWSKAet al. (1977). Wheat germ S 23 extract was prepared by the method of ROBERTS and PATERSON (1973) in the modification of Zagorski, quoted by ZAN-KOWALCZEWSKA et al. (1977). Incorporation of amino acids into protein was measured as the radioactivity remaining insoluble in hot TCA by the Whatman 3MM paper disc method of MANS and NOVELLI (1961).

o

4.6. c:3 4.2 ¢2) 0.90.4-

t

42

RESULTS D i a p a u s i n g eggs of B o m b y x mori contain small a m o u n t s of R N A binding to oligo (dT). The distribution o f this R N A a m o n g the subcellular fractions is presented in Table 1. The R N A o f the nuclear fraction has the highest relative content of polyadenylated R N A a m o u n t i n g to 1~o of the total. The bulk ( a b o u t 50~o) o f polyadenylated R N A o f the cell is present in fraction 105p which represents mainly the polysomes. F r a c t i o n 15p, with a b u n d a n t m e m b r a n e o u s structures is relatively p o o r in polyadenylated R N A . R i b o n u c l e o p r o t e i n s o b t a i n e d from diapausing eggs by the procedure used for isolation o f polysomes a n d sedimenting in sucrose gradient like polysomes (Fig. 1) were f o u n d to c o n t a i n as m u c h as 10/~g o f polyadenylated R N A per 10 g of eggs, more t h a n 90~o of this R N A being present in ribonucleoprotein particles heavier t h a n monosomes. D u r i n g postdiapausal d e v e l o p m e n t o f the e m b r y o the R N A c o n t e n t o f the egg increases (Table 2). After 8 days of post diapausal i n c u b a t i o n (6 days before the expected h a t c h i n g of larvae) the a m o u n t of total R N A in the egg doubles, this increase is u n i f o r m in the subcellular fractions, except nuclei where a six-fold a u g u m e n t a t i o n occurs. The c o n t e n t o f polyadenylated R N A also increases d u r i n g post diapausal embryogenesis o f B o m b y x mori. The changes found in the subcellular fractions after 8 days of post diapausal i n c u b a t i o n are presented in Table 3. The five-fold increase which occurs in the Table 1. Polyadenylated RNA in subcellular fractions of diapausing eggs of Bombyx mori Fraction Nuclei Membranes (15p) Ribosomes (105p) Postribosomal supernatant (105s)

1.1 0.2 0.2

1.7_+0.16 (n=3)

0.3

The subcellular fractions were separated as described in Materials and Methods. RNA was isolated by the procedure Of HASTINGS and KIRBY (1966) and that of BRAWERMAN" (1974). Polyadenylated RNA was bound on oligo (dT) cellulose according to EDMONDS and CARAMELA(1969) as modified by WASILEWSKA and KLECZKOWSKI (1974). The values are means (_+ S.E.M.) from at least three experiments performed by each of the RNA-isolation procedures mentioned above.

co~cen~*va'Hon % 36

Fig. 1. Sedimentation pattern ofpolysomes from diapausing eggs of Bombyx mori in a 12-36~o linear sucrose density gradient. Polysomes were isolated and tested according to WASlLEWSKA and CHERRY (1974).

polyadenylated R N A in the nuclear fraction and the two-fold increase in fraction 15p c6rrespond to the respective increases observed in total RNA content of these fractions. However, no such correlation was f o u n d for the fractions 105p a n d 105s in which the a m o u n t of polyadenylated R N A increased ten-fold a n d nine-fold, respectively, m a r k e d l y exceeding the increases noted in total R N A o f these fractions. Table 2. RNA content of the subcellular fractions from diapausing and developing embryos of Bombyx mori

Fraction Nuclei •15p 105p 105s Total RNA

RNA content mg/10g of eggs On eighth day of* At diapause* postdiapausal Increase development 0.11 _ + 0 . 0 7 0.33 _+0.02 1.47_+ 0.03 0.51 _ + 0 . 0 2 2.42_+0.14

0.72_+0.05 0.85 _+0.05 2.93 _+0.02 1.70_+0.03 6.20_+0.15

6x 2x 2x 3x 2x

Subcellular fractions were separated as described in Materials and Methods. RNA was isolated by the procedure of HASTINGSand KIRBY (1966). * ±S.E.M. (n=3). Table 3. Polyadenylated RNA in the subcellular fractions from diapausing and developing embryos of Bombyx mori

Polyadenylated RNA #g/10g of eggs ~ of total RNA _+S.E.M. of the fraction 1.3 _+0.26 (n = 4) 0.7_+0.17 (n=4) 3.5_+0.17 (n=4)

1 ~ucvo~e

Fraction Nuclei 15p 105p 105s

Polyadenylated RNA On eighth day of postAt diapause dispausal development % of % of /~g/10g of total RNA #g/10g of total RNA eggs* of embryo eggs* of embryo 0.9q-0.1 2.3+0.30 4.7+0.03 2.2_+0.16

0.04 0.09 0.19 0.08

4.8+0.05 5.3___0.25 41.7+0.1 19.3+0.15

0.07 0.08 0.67 0.31

Subcellular fractions were separated as described in Materials and Methods. RNA was deproteinized enzymatically after WIEGERS and Hmz (1972). Polyadenylated RNA was bound on oligo (dT) cellulose according to Avlv and LEDER (1972). * +S.E.M. (n=3).

Polyadenylated RNA in Bombyx embryos

127

with oligo (dT) (Table 1) indicate the presence of polyadenylated RNA in the 'resting polysomes' from diapausing eggs. The fact that, polyadenylated RN.A from the polysomal fraction of diapausing eggs of Polyadenylated RNA [14C]-Leucine Bombyx mori is not translated in a system in which added incorporated analogous RNA from developing eggs shows (#g) (pmole) Stage messenger activity, points to the defective structure of the presumably 'resting mRNA'. The possibility that 1.3 0 Diapause capping of the 5~ end is a requisite of translation of 2.6 0 'resting m R N A ' is not unlikely. PERRY (1976) 5.2 0 suggested that succession of capping and Eighth day of 1.2 7.2 polyadenylation may vary for various m R N A which postdiapausal 2.4 11.2 belong to the classes of message undergoing both development 4.8 9.6 modifications. The presence of polysomes containing the bulk of The value for control experiment without polyadenylated polyadenylated R N A of the cell and the fact that this RNA (4-5 pmole of L-[1-14C]-leucine was subtracted; RNA is not translatable, distinguish the diapausing counting efficiency was 60% (1 pmole = 78 counts/min); the results of representative experiments are shown; the embryo of Bombyx mori from the resting embryo of repetition yielded values differing by less than 15%. Artemia salin. In the latter no polysomal structures Polysomal fraction 105p was separated as described in were observed (GRoSFELD and LITTAUER, 1975) and Materials and Methods. RNA was deproteinized the translatable polyadenylated RNA was present in enzymatically according to WIENERS and HILZ (1972). the membraneous 15p fraction (NILSSON and Polyadenylated RNA was bound on oligo (~tT) cellulose HULTIN, 1974, 1975; SIERRA et al., 1976). Since a columns after AvIv and LEDER(1972). Translation assay in wheat germ cell-free system was perfdrmed as described by period of rest interrupts embryogenesis of both ZAN-KOWALCZEWSKAet aL 0977) with S-23 wheat germ Bombyx (CoULON, 1966) and Artemia (DUTRIEU, 1960) at an analogous stage of development (late extract prepared after ROBERTSand PATERSON(1973) in the modification of ZAGORSKIquoted by ZAN-KOWALCZEWSKA blastula--early gastrula) it seems that different molecular mechanisms operate in the regulation of et al. (1977). physiologically similar phenomena in these The assays in the wheat-germ, cell-free system invertebrates. presented in Table 4 revealed that the polyadenylated During postdiapausal development of Bombyx mori R N A of fraction 105p from diapausing eggs was not polyadenylated RNA bound in polysomes increases translatable, while the analogous R N A from markedly and becomes translatable. The increase of developing eggs stimulated the incorporation of polyadenylated m R N A in polysomes was also amino acids into proteins in this system. observed during the embryonal development of Artemia salina (SIERRA et al., 1976) and sea urchin (FROMSON and DUCHASTEL, 1975) as well as in DISCUSSION regenerating rat liver (GREENE and FAUSTO, 1974). The augumentation of polyadenylated RNA The small quantities ofpolyadenylated R N A found in diapausing eggs o f B o m b y x mori raised a problem in amount in the postribosomal supernatant (fraction the evaluation of preliminary results. Thus beside the 105s) occuring during the development of the procedure of HASanNGS and KIRBY (1966) applied as silkworm embryo may reflect enhanced translation if, described previously (SzYszro and LASSOrA, 1977) as postulated by MARBAIX et al. (1976) the bulk of two alternative procedures were also used for isolation nonpolysomal polyadenylated RNA of cytoplasm is of RNA. The first was the procedure of BRAWERMAN 'older' than the polyadenylated RNA in the (1974) and it was used because it has been polysomes. recommended as releasing effectively the polyJORNSON et al. (1976) suggested that the level of adenylated R N A from complexes with proteins. The mRNA in the cytoplasm is regulated by the efficiency second procedure was that of enzymatic of post-transcriptional modifications of pre-mRNA deproteinisation according to WIEGERS and HILZ rather than by the rate o f its transcription. Thus (1972) and this was used because, when applied to the polyadenylation of m R N A in the silkworm embryo, material already put on oligo (dT) cellulose column, it being probably at diapause a factor stabilizing the was expected to minimize the losses of hybrydizing "resting m R N A ' (HUEz et al., 1974), becomes during R N A particles. The results obtained with these development one of the modifications facilitating methods were found to be in reasonable agreement maturation, transport and binding of m R N A (Tables l and 3) except the values for polyadenylated molecules to ribosomes. R N A content of fraction 15p. Contaminations present Acknowledgements--We are grateful to Mrs. K. GOCMAN in this fraction resist the pronase treatment and the for her skilled .technical assistance. We also wish to thank repeated chromatography on oligo (dT) cellulose, Mrs. K. N AL~CZwho participated in part of the experiments resulting in an atypical absorption spectrum and as a postgraduate student. increased OD at 260 nm of final RNA solution (Table 3). Since no measurable binding of Bombyx mori REFERENCES embryonic m R N A to oligo (dT) cellulose was noted, the significant and reproducible values obtained for Avlv H. and LEDER P. 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Table 4. Translation in vitro of polyadenylated RNA from polysomal material of diapausing and developing embryo.of Bombyx mori

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KRYSTYNA GRZELAK, ELZBIETASZCZI~SNAAND ZOFIA LASSOTA

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