Polyadenylated-RNA during the development of Ceratitis capitata

Insect Biochem. Vol. 14, No. 3, pp. 30%311, 1984 Printed in Great Britain. All rights reserved

0020-1790/84 $3.00 + 0.00 Copyright © 1984 Pergamon Press Ltd

P O L Y A D E N Y L A T E D - R N A D U R I N G THE DEVELOPMENT OF C E R A T I T I S C A P I T A T A JESOS M. FOMINAYA, JUAN M. GARCiA-SEGURA a n d Jos~ G. GAVILANES Departmento de Bioquimica, Facultad de Ciencias, Universidad Complutense, Madrid-3, Spain

(Received 24 June 1983) Abstract--Polyadenylated-RNA has been isolated at different stages of the life cycle of the insect Ceratitis capitata using chromatography on oligo-dT-cellulose. The molecular characterization of this RNA fraction has been carried out by melting, sedimentation, electrophoretic and CD studies. The distribution of this polynucleotide fraction during the development has been related to physiological changes of this holometabolous insect. The results obtained are discussed in terms of the presence of RNA-degrading activities previously reported for this insect.

Key Word Index: PolyA-RNA, insect development, RNase

INTRODUCTION

T h e m o r p h o l o g i c a l a n d physiological variations produced d u r i n g cell d e v e l o p m e n t are considered to be controlled by changes in the gene expression. These can be studied at various levels. Thus, changes in cell functions can be considered from the p o i n t o f view o f variations in the cellular protein population. Such variations should be magnified in h o l o m e t a b o l o u s insects. In fact, cell m e t a b o l i s m goes t h r o u g h dramatic modifications d u r i n g insect m e t a m o r p h o s i s . T h e larval stage is characterized by a rapid accumulation of c o m p o u n d s with c o n c o m i t a n t growth. Remodelling o f tissues occurs d u r i n g the p h a r a t e adult stage; the larval tissues are b r o k e n d o w n a n d characteristics o f the adult insect appear. Finally, stabilization of b o t h g r o w t h a n d differentiation occurs at the a d u l t stage. Obviously, all these modifications would be reflected in the protein p o p u l a t i o n a n d consequently in b o t h n u m b e r a n d c o n t e n t o f the m R N A species. A n y v a r i a t i o n in the m R N A p o p u lation could be related to the presence o f e n d o g e n o u s ribonuclease activities which could depend o n the stage o f d e v e l o p m e n t o f the insect. This p a p e r describes the isolation a n d molecular characterization as well as the study of the distribution o f p o l y a d e n y l a t e d - R N A d u r i n g the life cycle of Ceratitis capitata. In addition, the results o b t a i n e d are discussed considering the levels o f ribonuclease activities previously studied for this insect (GarciaSegura a n d Gavilanes, 1982). MATERIALS AND

METHODS

Chemicals Proteinase-K was purchased from Boehringer. Oligo-dTcellulose was obtained from Sigma. All other reagents were analytical grade and obtained from Merck or Sigma. Rearing of insects C. capitata (Wiedemann) was used at the egg, larval, pharate adult and adult stages of development. Diet, temperature and humidity conditions were carefully controlled as previously described (Fernandez-Sousa et al., 1971). Eggs, larvae, pharate adults and adults were reared at precise development times in synchronous culture.

Isolation of polyadenylated-RNA The different samples of biological material were disrupted in a Teflon-glass homogenizer in the presence of both 4vol of 25mM Tris buffer, pH 8.0, containing 25 mM EDTA, 75 mM NaCI and 0.5~ (w/v) SDS, and 4 vol of the same buffer saturated with phenol. After 30 min at 4°C, the homogenate was treated with 0.5vol of twice distilled chloroform. The obtained suspension was gently shaken for 3 min and the resulting emulsion centrifuged at 27,000g and 10°C for 30 min. The organic phase as well as the interphase were treated with 1 vol of Tris buffer, without phenol and re-extracted. Both aqueous phases were combined and made 0.2 M with respect to NaC1 and 2 vol of cold ethanol (-20°C) were added. The suspension was maintained for about 12 hr at -20°C. The precipitate was recovered by centrifugation at 27,000g and - 2 ° C for 15 min and dissolved in the minimum volume of 25 mM Tris buffer, pH 7.5, containing 25 mM EDTA, 75 mM NaCI and 0.5~ (w/v) SDS. This solution was treated with proteinaseK (0.1 mg/ml) for 18 hr at 37°C. The incubation mixture was then precipitated with cold ethanol as described above. The precipitate was recovered by centrifugation at 27,000g and - 2 ° C for 15 min and dissolved in the minimum volume of glass distilled water. Four volumes of 3.75 M sodium acetate, pH 5.0 were added and the suspension was maintained for 8 hr at -20°C. This treatment was repeated twice. The final precipitate was washed with ethanol and then dissolved in 10 mM Tris buffer, pH 7.5, containing 0.5 M NaC1, 0.2~ (w/v) SDS and 0.02~ (w/v) sodium azide. This sample was incubated at 65°C for 10 min and applied to an oligo-dTcellulose column (1.1 cm diameter x 4.0cm height) equilibrated with Tris buffer. The elution was performed with the same buffer until no absorbance at 260 nm could be detected in the eluate. Then, the elution buffer was substituted by the same one without NaCI. Fractions eluted at low ionic strength were collected and re-chromatographed. The material finally eluted at low ionic strength corresponds to the polyadenylated-RNA fraction. Molecular characterization RNA samples dissolved in 20 mM sodium acetate buffer, pH 5.8, containing 5 m M EDTA, were centrifuged in a linear gradient (5-20~, w/v sucrose) at 216,445 g (rotor SW 65) for 6 hr at 2°C in a Beckman L-4 centrifuge. Fractions containing 0.15ml were collected from the tubes (1.27 cm x 5.05 cm) and further diluted with 0.4 ml of the acetate buffer. Absorbance measurements were performed in a Cary 118 spectrophotometer, using 0.2 cm, optical-path cells. 307

JESIASM. FOMINAYAet al.

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The melting temperatures of the samples were obtained in a Beckman DU-8 spectrophotometer, using the "Compuset DU-8 TM" accessory (Beckman), by measuring the absorbance at 260nm; values were obtained in the range of temperature 20-100°C by increasing 0.5°C/min. Samples were dissolved in 50mM Tris-phosphate buffer pH 7.5, containing 0.1 M NaC1. Circular dichroism measurements were carried out in a Jobin Yvon Mark II dichrograph at 0.2 nm/sec scanning speed and using 0.1 cm optical-path cells. Electrophoretic characterization of the polyadenylatedRNA fractions was performed in 0.5~ agarose-2~ acrylamide slab gels. The gels were scanned in the Beckman DU-8, after methylene-blue staining (Peacock and Dingman, 1967) using the "Compuset DU-8 Gel Scan" accessory (Beckman) at 550 nm and 5 cm/min scanning speed.

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RESULTS

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Isolation o f the polyadenylated-RNA The isolation procedure for the polyadenylatedRNA has been developed after taking a number of findings into consideration. Thus, the phenol treatment dissociates protein-RNA complexes (Brawerman et al., 1972; Brawerman, 1974). A vigorous shaking during this treatment avoids the formation of protein-RNA aggregates (Palmiter, 1974). EDTA as well as SDS are used during the isolation in order to inactivate RNA-degrading activities as well as to avoid aggregate formation and to dissociate both ribosomes and messenger ribonucleoproteins (mRNP) (Wagner et al., 1967; Rosenfeld et al., 1972). Chloroform is employed in order to increase the recovery of the polyadenylated-RNA by preventing a partial distribution of the polynucleotide in the phenol phase (Penman, 1966; Perry et al., 1972; Palmiter, 1973). During the proteinase-K treatment, the presence of SDS increases the proteolytic effect (Hilz et al., 1975).

20 1,0

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36

52

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84

°C

Fig. 2. Melting profiles for the polyadenylated-RNA isolated from (A) 6-day-old larvae and (B) adult insect. (..... ) //26o, relative increase in A:~ and ( ) calculated first derivative. A typical profile for the affinity chromatography using an oligo-dT-cellulose column is given in Fig. 1. Concentration of the polynucleotide has been determined from the absorbance values at 260 nm with an E0.1~ 0.2 era, 260 nm of 4.0 (Chirgwin et al., 1979).

Thermal denaturation o f the polyadenylated-RNA The melting profiles for the polyadenylated-RNA isolated from 6- day-old larvae and adult are given in Fig. 2. Results obtained at other status of the insect development are summarized in Table 1. From these results, a broad thermal transition is observed for the polyadenylated-RNA fractions, melting interval of about 30°C, whereas for rRNA it is about 20°C (Table 1). These observations are in agreement with those reported for rRNA and mRNA (Holder and Lingrel, 1975), indicating a lower co-operativity for the melting of the mRNA than for that of rRNA. This fact would indicate that the average length of those regions showing an ordered structure in the

(B)

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Fig. 1. (A) Elution profile for the first chromatography on oligo-dT-celluloseof the total RNA fraction isolated from 6-day-old larvae. (B) Elution profile of the second chromatography on oligo-dT-cellulose of the RNA fraction eluted at low ionic strength. Arrows indicate the change to the low ionic strength buffer (see Methods). Absorbance was determined in 0.2 cm optical path cells. Fractions of 1ml were collected.

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Fig. 3. Result of centrifuging the polyadenylatcd-RNA fraction isolated from ( ..... ) egg and ( ) 4-day-old pharate adult.

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PolyA-RNA from C. capitata Table 1. Summary of the thermal den•tur•tion studies for polyadenylated-RNA fractions rRNAt mRNA mRNA mRNA mRNA mRNA

E L-6 P-5 P-8 A-2

H260"

ATe:

Tm§

~ Ordered structurell

30.2 + 1.2 18.2+0.7 18.0 _ 0.8 6.4 _ 0.5 20.4+1.1 20.2 + 0.9

20.0 + 1.1 31.0+1.8 35.0 _ 1.8 14.0 _ 0.6 12.0 _ 0.4 25.0+1.5 11.0+0.3 29.0 _ 1.7 10.0 _ 0.3

56.0 + 1.7 47.0+1.5 47.0 _ 1.4 41.0 _ 1.1 86.0 _ 2.3 47.0+1.5 86.0+2.2 47.0 4- 1.7 85.0 4- 2.5

71.1 42.8 42.4 15.1 48.0 47.5

(9.2) (42.1) (41.6)

E, egg; L-6, 6-day-old larvae; P-5, 5-day old pharate •dult; P-8, 8-day-old pharate •dult; A-2, 2-day-old adult insect. *Relative increase in absorbance at 260 n m ( + SD). tThis sample corresponds to the R N A fraction after removing polyadenylated-RNA, which is m•inly rRNA, from the insect eggs. :~Values corresponding to the melting interval. Two values are indicated when two different thermal tr•nsitions are observed ( _+ SD). §Melting points in °C. Two values are indicated when two thermal transitions are observed ( + SD). IIPercentage of estimated ordered structure. Values are determined according to Holder and Lingrel (1975). V•lues in parenthesis are those corrected for the biochrome considering that its contribution to the H l°°°c value is 2.5.

insect polyadenylated-RNA is lower than that observed for rRNA. Moreover, the content of ordered structure for polyadenylated-RNA (see Table 1) is also lower than for rRNA, as the//26o values indicate. The melting temperature values Tm agree also with these conclusions. In fact, the midpoint of the thermal transition for r R N A occurs at higher temperature values than for m R N A in agreement with both the higher content in ordered structure and the greater average length of the segments in such ordered conformation. The polyadenylated-RNA fractions from pharate-adult and adult insect exhibit two different thermal transitions (Fig. 2, Table 1). The one shown at a high temperature has been related to the presence of endogenous biochromes. This conclusion results from the existence of a shoulder at 275 nm in the spectra of the polyadenylated-RNA fractions from the pharate adult and adult stages. Exhaustive treatment of these samples with the C. capitalta alkaline ribonuclease and further ion exchange chromatography reveals the existence of a compound exhibiting an absorbance maximum at 275 nm, which is related to the above considered biochrome (Fominaya J. M., Garcia-Segura J. M. and Gavilanes J. G. unpublished results). The values in Table 1 are corrected for this contribution. Apart from the specificity of the mRNA-biochrome interaction, the strength of the binding would be considerable as the interaction remains during the selective purification procedure.

The Tm values as well as the broad interval of temperatures for the thermal transitions are characteristics of m R N A populations.

Other molecular characterizations for adenylated-RNA

the poly-

Table 2 summarizes the results obtained from the CD studies for polyadenylated-RNA from different stages of development of C. capitata. The ellipticity values obtained are in agreement with the existence of the polyA tail as it is derived from the CD spectrum of the adenine nucleotide. The different fractions of polyadenylated-RNA constitute heterogeneous populations exhibiting sedimentation coefficients around 10-16 S (Fig. 3), indicating that the average length would be around 2000-3000 nucleotides. Maximum heterogeneity is observed for samples taken at the middle of the pupal stage (Fig. 3). This result would indicate an important contribution of hnRNA to the total polyadenylated-RNA fraction. Such an hypothesis will be discussed below. The results obtained from the sedimentation studies are in agreement with the

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T•ble 2. Circular dichroism res,.:lts for polyadenylated-RNA from

C. capitata rRNAII mRNA mRNA mRNA mRNA

E L-6 P-5 A-3

k*

Ot

022o~

0/02:o§

264 265 266 267 268

25400 _ 700 18600+500 9600 _ 350 10300 + 400 12000 + 400

3000 + 100 6 0 0 0 + 180 6500 _ 190 5700 4- 180 2800 4- 80

8.47 _ 0.52 3.10+0.18 1.48 4- 0.10 1.81 + 0.13 4.29 _ 0.27

E, egg; L-6, 6-day-old larvae; P-5, 5-day-old pharate adult; A-3, 3-day-old •dult insect. *Wavelength corresponding to the maximum of ellipticity. "['Ellipticity values, in units of degrees x cm 2 x dmol - 1 of nucleotide, corresponding to the indicated wavelengths ( 4- SD). :[:Ellipticity values at 220 um ( + SD). §Maximum eUiptieity to 0220 ratio ( + SD). IIThis sample corresponds to the R N A fr•ction after removing polyadenylated-RNA, which is mainly r R N A , from the insect eggs.

8

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9 t l 13 ~5 17 19 Days Ap

P

A

Fig. 4. R N A c o n t e n t d u r i n g the d e v e l o p m e n t o f the insect

C. capitata: (

) polyadenylated-RNA and (..... ) total RNA. Values are expressed in mg of polynucleotide per g of tissue. Days corresponding to each stage (E, egg; L, larva; Ap, apolysis; P, pharate adult; A, adult) of the development are indicated, considering the egg-eclosion as zero time. This Figure represents the average of the two obtained from two different batches of biological materials; the difference was less than 5% at each point of development.

310

JESUS M. FOMINAYAet al.

observed patterns in the agarose-acrylamide gel electrophoresis, Distribution of polyadenylated-RNA during the development of C. capitata

The results obtained for the determination of the RNA content during the development of the insect are given in Fig. 4. In this Figure, both patterns of distribution for total RNA and polyadenylated-RNA are considered. All the values are referred to the starting amount of biological material which are determined from the absorbance at 260 nm. Eggs are obtained in aqueous suspension. Thus, the values for this stage of development are referred to the weight of the lyophylized material. At the pharate adult and adult stages, values are corrected for the weight of the non-solubilized material (Fominaya, unpublished observations). The highest content for total R N A is found at the egg-stage. As this stage corresponds to an embryonic phase, this result is in agreement with a high rate of cellular differentiation. DISCUSSION The polyadenylated-RNA, related to the m R N A content, represents 0.3-2.0~o weight of the total RNA. This is in the range normally found for the mRNA/total RNA ratio for preparations obtained from whole cells (Lewin, 1981). The highest content for the m R N A fraction is observed for the egg; however, a maximum is observed at the late larval stage. The emergence of the adult insect is accompanied by an increase in the polyadenylated-RNA content. The embryonic phase is characterized by fast growth and cellular differentiation which would be related to the high rate of protein biosynthesis and to the high content of mRNA, in agreement with the results obtained for the polyadenylated-RNA fraction at this stage of development. The maximum observed at the end of the larval stage would be explained as due to the preparation for the metamorphosis which requires specific molecules to control the mechanism for the formation of pupa and metamorphosis. The end of the pupal stage coincides with the synthesis of specific structures for the adult insect, which would require the corresponding mRNA. This would represent the observed progressive increase of the m R N A levels which would be enhanced at adult eclosion. This fact is explained by the appearance of the functionality of the adult structures previously formed at the end of the pupal stage. The content at the beginning of the pupal stage is in agreement with the tissue lysis that takes place. Another aspect to consider is the fact that the principal decreases are found at the stage-changes: embryo-larva, larvapharate adult. At these points the stock of m R N A for specific molecules from the former stage is no longer required and so it is degraded. The polyadenylated-RNA population in the pharate adult stage, which shows the lowest levels during the insect development, presents a particular characteristic. Thus, all the physico-chemical parameters obtained from this fraction exhibit clear differences in comparison to the normal behaviour related to the

fractions from other stages (see Table 1 and Fig. 3). This can be explained by a considerable increase of the ratio h n R N A / m R N A for this stage because such special properties agree with ones exhibited by the hnRNA (Lewin, 1981). m R N A would be present at low concentration because of the high RNAdegrading activity in the cytoplasm, while the hnRNA, confined to the nucleus would remain unchanged. These results are in agreement with the reported distribution for the RNA-degrading activities during the development of the insect (Garcia-Segura and Gavilanes, 1982). After the apolysis, acid ribonuclease from lysosomes is released as is expected when there is modification and re-absorption of tissues. High levels of this acid RNase are maintained during the pupal stage in agreement with the low levels of polyadenylated-RNA observed. The minimum in the content for both alkaline and acid ribonuclease activities at the egg stage (Garcia-Segura and Gavilanes, 1982) is also in agreement with the distribution in Fig. 4. Likewise, at the beginning of the larval stage there is a minimum in the level of polyadenylated-RNA, which coincides with the maximum of the alkaline RNase found in the insect. According to these results, a clear correlation may be established between m R N A levels, ribonuclease activities and development of this insect. This provides additional proof for control of protein synthesis at the level of R N A degradation. Such control would be exerted by the regulation of RNase activity. The study of this aspect is being pursued at the moment. REFERENCES

Brawerman G., Mendecki J. and Lee S. Y. (1972) Isolation of mammalian messenger ribonucleic acid. Biochemistry 11, 637-641. Brawerman G. (1974) Eukaryotic messenger RNA. A. Rev. Biochem. 44, 621-642. Chirgwin J. M., Przybyla A, E., MacDonald R. J. and Rutter W. J. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18, 5294-5299. Fernandez-Sousa J. M., Municio A. M. and Ribera A. (1971) Biochemistry of the development of the insect C. capitata. Changes in the positional distribution of fatty acids in diacylethanolamine phosphoglycerides. Biochim. biophys. Acta 231, 527-534. Garcia-Segura J. M. and Gavilanes J. G. (1982) Study of the RNA-degradating activities during the development of the insect C. capitata. Comp. Biochem. Physiol. 73B, 835-838. Hilz H., Wiegers H. and Adamietz P. (1975) Stimulation of proteinase-K action by denaturing agents. Application to the isolation of nucleic acids and the degradation of "masked" proteins. Eur. J. Biochem. 56, 103-108. Holder J. W. and Lingrel S. B. (1975) Determination of secondary structure in rabbit messenger RNA by thermal denaturation. Biochemistry 14, 4209-4215. Lewin B. (1981) Gene Expression, Vol. 2. Wiley, London. Palmiter R. D. (1973) Ovalbumin messenger ribonucleic acid translation. Comparable rates of polypeptide initiation and elongation on ovalbumin and globin messenger ribonucleic acid in a rabbit reticulocyte lysate. J. bioL Chem. 248, 2095-2106. Palmiter R. D. (1974) Magnesium precipitation of ribonucleoprotein complexes. Expedient techniques for the isolation of undegraded polysomes and messenger ribonucleic acid. Biochemistry 13, 3606-3614. Peacock A. C. and Dingman C. W. (1967) Resolution of

PolyA-RNA from C. capitata multiple ribonucleic acid species by polyacrylamide gel electrophoresis. Biochemistry 6, 1818-1827. Penman S. (1966) RNA metabolism in the HeLa cell nucleus. J. molec. Biol. 17, 117-130. Perry R. P., La Torre J., Kelley D. E. and Greenbnrg J. R. (1972) Lability of poly(A) sequences during extraction of messenger RNA from poly-ribosomes. Biochim. biophys. Acta 262, 220-226.

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Rosenfeld M. G., Abrass I. B. and Perkins L. A. (1972) Cleavage of the polyadenylate-rich region of polyadenylate-rich RNA. Biochem. biophys. Res. Commun. 49, 230-238. Wagner F. K., Katz L. and Penman S. (1967) The possibility of aggregation of ribosomal RNA during hot phenol-SDS deprotcinization. Biochem. biophys. Res. Commun. 28, 151-159.