The relationship between polyribosomal and latent membrane-bound messenger ribonucleoprotein particles in artemia embryos

Int. J. Biochm?. Vol. 16. No. 9, pp. 1015-1022, Printed m Great Brttam All rights reserved

THE

1984 Copyright

RELATIONSHIP BETWEEN POLYRIBOSOMAL LATENT MEMBRANE-BOUND MESSENGER RIBONUCLEOPROTEIN PARTICLES IN ARTEMIA EMBRYOS MATS

Department

of Cell Physiology, The Wenner-Gren 16. S-I 13 45. Stockholm. (Receiwd

0020-7 I I X/H4 s3 00 + 0.00 (‘, 1984 Pergamon Pre\s Ltd

AND

LAKE*

Institute, University of Stockholm, Sweden [Tel. 08-I 3-8000]

13 Junuq

Norrtullsgatan

1984)

Polysomal messenger ribonucleoprotein (mRNP) particles from developing Arremiu cysts were isolated, characterized and compared with latent membrane-bound mRNP particles isolated from dormant cysts. 2. The polyribosomal mRNP particles sedimented between 25-35 S in a sucrose gradient and had a buoyant density of 1.33 g/cm) in Cs$o,. 3. Latent particles had a higher sedimentation coefficient and lower buoyant density. 4. The poly(A) + RNA in the two kinds of particles was comparable in size, l&20 S. 5. The protein composition of the particles, as determined by electrophoresis, was different. 6. Polyribosomal particles contained 9 major and 6 minor proteins; a 72 k poly(A)-associated protein was present. 7. Latent particles were characterized by a complex protein pattern ranging in apparent mol. wt between 14,000-140,000. 8. Some proteins with similar molecular weight and isoelectric point were probably common to both kinds of particles. Abstract-l.

INTRODUCTION

Messenger ribonucleoprotein (mRNP) particles occur in the cytoplasm either in a translatable form in polyribosomes or as non-translated “free” particles. It has been discussed, to what extent the free mRNP particles are associated with other cell structures, primarily endoplasmic membranes (Endo and Natori, 1979; Lake and Hultin, 1980). It is currently held that the free particles are attached to components of the cytoskeleton, which in turn also serves as a framework of the endoplasmic reticulum (Lenk et ul., 1977; Cervera et al., 1981). The mRNP particles of resting embryonic cells are of special physiological interest. In the encysted gastrulae of Artemia the dormancy is exceptionally deep. The embryos are devoid of polyribosomes, and the latent mRNA is preserved as free mRNP particles, in the main associated with membrane structures (Nilsson and Hultin, 1974). When development is resumed, the sequestered mRNA is utilized for the formation of active polyribosomes (Golub and Clegg, 1968). It has been shown by in vitro translation that the latent and polyribosomal mRNAs code for essentially the same proteins (Hultin et al., 1977). Thus, dormant and developing Artemiu cysts provide unique opportunities for comparing closely related mRNP particles in very different states of activity. In a previous study membrane-associated mRNP particles were isolated from dormant Artemia cysts

and characterized in some detail (Lake and Hultin, 1980). The aim of the present investigation was to compare these latent particles with active mRNP particles derived from polyribosomes. Apart from dissimilarities in the physical properties of the particles, it was found that their patterns were in part comparable, as determined by gel electrophoresis and electrofocusing. MATERIALS

Latent membranes-bound mRNP particles were prepared from dormant Arterniu embryos as described (Lake and H&in, 1980). Polyribosomal mRNP particles were obtained from developing cysts at the stage of half emergence and were ground in a porcelain mortar in ice-cold homogenization buffer (75 mM KCI, 5 mM MgCl?, 35 mM Tris-HCl, pH 7.6). The homogenate was filtered through i; porous glass filter funnel and centrifuged for IOmin at 15,OOOg at O‘C. Sodium deoxychelate (DOC) was added to the supernatant at a final concentration of 1%. For the isolation of polysomes, the DOC-treated supernatant was layered over a two-step gradient of 0.5 and 1.8 M sucrose in homogenization buffer and centrifuged for 150 min at 250,OOOg at 0 C (Wettstein et al., 1963). mRNP particles were released from the polyribosomal pellet by treatment with 5 mM EDTA in Mg’+-free homogenization buffer. The suspension was centrifuged for 16 hr at 175.000a at 0 C in a l(f3OY.. (w/v) sucrose gradient containing 75 mM KCI ” and 35 mM Tris-HC1 fuH 7.6). The eradients were fractionated (Lake ef al., 1982i andmonitored using an u.v.-spectrophotometer. Pre-ribosomal fractions containing poly(A) + mRNP particles were concentrated by precip.I

*Present address: Sweden.

KabiGen

AB,

S-l 12 87

Stockholm,

AND METHODS

Prepuration of mRNP parrides

MATS LAKE

1016 itation with 2.5 vol of ethanol( precipitates at -20 C.

-20

C) and stored as ethanol

mRNP particles were iixed with 2”, glutaraldehyde. neutralized with NaCo, and dialysed against 75 mM KCI, 5 mM MgCl, and IOmM triethanolamine (pH 7.4). The suspension was adjusted with solid Cs$O, to a density ol I. I g/ml and combined in a gradient former with an equal volume of Cs&, solution with a density of 1.8 g/ml. Gradients were centrifuged for 17.5 hr at I lO.OOOg at 4 C.

RNA (1973). 0.15-0.46 acetate. Tris-HCI 0 C. The according

was extracted accordmg to Holmes and Bonner Sedimentation analysis was performed in M sucrose gradients. containing 20 mM sodium 5 mM EDTA. O.l”,, (w/v) SDS and 40mM (pH 7.7). centrifuged for 3.5 hr at 190,OOOg at poly(A) content of RNA fractions was determined to Sullivan and Roberts (1973).

5 Effluent

Fractions were analysed in 7-l5”,, (w/v) polyacrylamide gradient gels using the SDS system of Laemmli (1970). 2-D gel electrophoresis was performed according to O’Farrel (1975) using isoelectric focusing in the first dimension. Briefly, samples were focused overnight at 400 V in a 4.5O, polyacrylamide gel containing 8.5 M urea, 2.5”” NP 40 and 2”,, ampholine pH 3.5-9.0. The pH gradient was determined after equilibration of gel slices with I ml water. Gels were stained with 0. IO’,, Coomassie Brilliant Blue in 50”,, methanol and 7”,, acetic acid and a strip was cut out and equilibrated with SDS gel sample buffer for 15 min and electrophoresed in the SDS gel system of Laemmli (I 970).

mRNP particles were digested with RNase A and TI and chromatographed on an oligo d(T) cellulose column according to Vincent rr ul. (1981) Alternatively, the digests were centrifuged for 4 hr at 24O.OOOg at 0 C, in a SW 50 rotor usmg 5-20”” (w/v) sucrose gradients containing 50mm NaCl, 5 mM EDTA and IO mM triethanolamine (pH 7.4). Gradients were fractionated (Lake ef al.. 1982) and the protein content of consecutive fractions analysed by SDS gel electrophoresis; poly(A) was extracted and analysed as described above.

RESULTS

Charucterizution

of’polyrihosomul

I shows a typical

mRNP

particles

profile of the polyribosomes used in these experiments. A marked peak is observed at the size of approx. 8 ribosomes per mRNA. In the dimer and trimer regions of absorbance was relatively low. indicating that the mRNA strands were largely intact. After EDTA treatment the mRNP particles appeared as a small but distinct elevation in the sedimentation profile within the range of 25-35 S (Fig. 2A). The main part of the poly(A) + RNA was found within this region. The corresponding fractions were collected for further investigation. Glutaraldehyde-fixed polyribosomal mRNP particles had a buoyant density in Cs$O, gradients of 1.33 g/cm’ (Fig. 3). According to the formula developed by Preobrazhensky and Spirin (1978) this would correspond to a protein content of 87%. Without fixation the particles were disintegrated during gradient centrifugation (not illustrated). This is in marked contrast to the membrane-associated mRNP Figure

sedimentation

10 ml

Fig. I. Analysis of polyribosomes from developing cysts. The polysomal pellet was suspended in homogenization buffer, layered onto a 15-50”~~~sucrose gradient in homogenization buffer and centrifuged in a Beckman SW 41 rotor. for I IO min at 200,000 g at 0 C. The gradient was monitored at 260nm.

particles from dormant cysts (Lake and H&in, 1980). The poly(A) + RNA extracted from the polyribosomal mRNP particles sedimented within the range of 10-20s (Fig. 4). The sedimentation curve to that previously observed for was similar poly(A) + RNA from the membrane-associated, latent mRNP particles (Lake and H&in, 1980). Protein

composition

of mRNP

particles

Figure 2B shows the protein patterns of consecutive gradient fractions of EDTA-treated polyribosomes. Within the sedimentation range of 25-35s (fractions 4 and 5) the SDS gel electrophoresis revealed a set of proteins with apparent mol. wt of 14,00~140,000. The protein pattern in fractions 4 and 5 was different from the pattern in later fractions containing ribosomal subunits. The subunit containing fractions had most of their proteins below mol. wt = 35,000 which is the normal range for ribosomal proteins. poly(A) + RNA-containing fractions on the other hand had an even distribution of proteins over the whole molecular weight range. In Fig. 5 the electrophoretic pattern of the polyribosomal mRNP particles is compared with that of the membrane-associated latent mRNP particles from dormant cysts. The polyribosomal mRNP particles contained 9 major and 6 minor proteins. The most abundant protein had a mol. wt of 34,000. The protein pattern of the membrane-bound particles was strikingly more complex. About 30 polypeptide bands could be distinguished without much difference in staining intensity. In the two preparations 12 polypeptides had similar mobilities as measured in the SDS system (approx. mol. wts: 89,000, 72,000. 64,000, 55,000, 51,000,41.000. 34,000.24,000, 20,000, 16,000, 14,000 and 12,000). The two types of particles were further analysed by a 2-D gel procedure using isoelectric focusing in the

40

s

60 S

Effluent

ml (A)

94,000 68,000

43,000

30,000

20,100 14,400

123

4

5678

9

Fraction number

10 11

12

13

14

Markers

(W Fig. 2. Isolation of polyribosomal mRNP particles by sedimentation of a sucrose gradient and analysis of the poly(A) contents and protein patterns in consecutive fractions. PoIy~bosomal pellets were suspended in Mg’+-free homogeni~tjon buffer and EDTA was added to a concentration of 5 mM. The dissociated polysomes were layered on a lO-30% sucrose gradient and centrifuged in a Beckman SW 27 rotor for 17 hr at 95,OOOgat 0°C (A). Gradients were monitored at 260 nm (-f, fractions were collected and precipitated with ethanol. RNA was extracted and the poly(A) content (0-O) analysed by [3H]poly(U) hybridization (B). The protein composition of each fraction was analysed by SDS gel electrophoresis.

1017

145,000

-

105,000 - -i** 94,000h8,OOO~

:x:

1 '64,C - 55,c -51,c

i - 94,000

-94,000

-68,000

-68,000

60,000

53,000 44,000

43,000-

: ~~y~~~ 30,000--

c

-43,000

-30,000

29:ooo

r26,OOO -23,000 -22,000 20,100-

-20,100

14,400-

--14,400

Markers

Polyribosomal

Membrane-bound

mRBJPproteins

mR.W proteins

Markers

Fig. 5. Comparison of the polypeptide compositions of polyribosomal and latent, membrane-bound mRNP particles. Proteins from polyribosomal (A) and latent membrane-bound (B) mRNP particles were analysed by SDS gel electrophoresis. Protein markers: lysozyme, soybean trypsin inhibitor, carbonic anhydrase, ovalbumin, bovine serum albumin and phosphorylase b.

Polysomal

and latent

mRNP

particles

1021

focused proteins were probably washed away during equilibration in SDS sample buffer; and thirdly, basic proteins (PI> 8) could not be analysed in the system used. Protein composition fragments go! N <

Ld

Fig. 3. Cs$O, density gradient centrifugation of polysomal mRNP particlesAfter fixation with glutaraldehyde the mRNP particles were centrifuged in a preformed Cs$O, gradient in a Beckman SW 60 rotor for 17 hr at 115,OOOg at 0 C. Gradients were monitored at 260 nm and the density of consecutive fractions determined. first and SDS electrophoresis in the second dimension (Fig. 6). For both particle preparations the pI range of the proteins was on the acid side of neutrality. Consistent with the greater complexity of the latent particles their proteins covered a wider pI range. The 2-D method was of particular interest in assessing the possible identity of the proteins co-migrating in the SDS system. Evidence for identity was provided for the 89,000, 64,000, 55,000, 34,000 and 24,000 mol. wt proteins. Detection of proteins after 2-D electrophoresis could be limited for three reasons. Firstly, some proteins were precipitated and failed to migrate in the second dimension; secondly, a portion of the

2 Effluent

4

ml

Fig. 4. Sedimentation analysis of polysomal poly(A) RNA from developing cysts. RNA was extracted from polyribosomes and centrifuged in a 5%IT”,‘, sucrose gradient. Fractions were analysed for poly(A) content by hybridization with [‘H]poly(LJ). Arfemiu, 17s and 25s rRNA (arrows) were centrifuged in a parallel gradient as markers.

qf’ poly (A )-containing

pc~rticie

The polyribosomal and membrane-associated mRNP particles were degraded by incubation with ribonucleases A and Tl and fractionated by affinity chromatography on oligo d(T) cellulose. In the case mRNP particles of the polyribosomal the ribonuclease-resistant poly(A) sequences were associated with 4 major and 2 minor polypeptides (Fig. 7A). The major components had mol. wts of 72,000, 55,000, 28,000 and 24,000. In contrast, RNase-resistant fragments of membrane-associated mRNP particles, eluted from oligo d(T) cellulose, still showed a complex protein pattern. To elucidate the relationship of this complex material to poly(A) sequences the digest was analysed by sucrose gradient centrifugation. The poly(A) content of consecutive fractions was determined by extraction and [‘H]poly(U) hybridization. The proteins were analysed in parallel by SDS gel electrophoresis and the poly(A)-rich fractions contained major polypeptides with mol. wts of 44.000. 30.000, 26.000, 24,000 and 21,000 (Fig. 7B). DlSCUSSlON

The latent and polyribosomal mRNA particles described here contained poly(A) + RNA molecules within the same size range (IO-20 S), in accordance with the previously observed similarity between the translation products in vitro of comparable preparations (Hultin et cd., 1977). Despite the essential homology of the RNA constituents, the two mRNP preparations were markedly different with regard to sedimentation coefficient (40 and 25-35 S, respectively). stability and buoyant density in Cs$O, (1.27 and 1.33g/cm’, respectively) and in ultrastructural compactness (Lake and Hultin, 1980; Lake et al., 1983). The lower density of the membranebound, latent particles is probably explained by their higher protein content in combination with the presence of some firmly bound lipid (Lake and Hultin, 1980). A high protein content has been reported for non-polyribosomal mRNP particles of different origin (Huynh-van-Tan and Schapira, 1978; Northernan et cd., 1980; Moon et al., 1980: Vincent r/ al., 1980). Regardless of the great complexity of the proteins from the membrane-bound particles. the experiments suggest that some proteins were shared by the two particle populations. Thus, by combining electrofocusing with SDS electrophoresis. evidence was obtained in favour of the identity of 6 distinct proteins from the two preparations. Since all proteins were not accounted for by the 2-D method, this represents a minimum number. The proteins in common may represent primary RNA-binding proteins. Some of these also seem to be present in latent mRNP particles prepared from a postmicrosomal fraction of Artemia cysts (Slegers et al.. 1981). Digestion and Tl ha:

of mRNP particles with ribonucleases A been used in the identification of proteins

1022

MATS LAKE

bound to poly(A) sequences (Vincent et al., 1981). A protein with an approx. mol. wt of 24,000 was found in the poly(A)-containing fractions of both mRNP preparations. A poly(A)-binding protein with a mol. wt of 23,500 has been previously described (Slegers car al., 1981). The polyribosomal poly(A)-containing complexes contained a protein with an approx. mol. wt of 72,000, probably analogous to a previously described poly(A)-associated protein of comparable size (Preobrazhensky and Spirin. 1978; Moon et trl., 1980; Standart C? (II., 1981). This protein was not found in the poly(A)-containing fractions of digests of latent mRNP particles. However, a polypeptide of the same size was observed in the SDS electrophoretic patterns of whole latent particles. Unfortunately, the identity of the two proteins could not be clarified by electrofocusing within the available pl range. Fol poly(A)-binding proteins pl values above 8 have been reported (Standart et ul., 198 I). The present experiments together with earlier ultrastructural data suggest the following model of the relationship between the two kinds of mRNP particles. The mRNA strand together with a limited number of RNA-binding proteins constitute a primary core structure. In the latent particles a number of other proteins are added to produce the condensed appearance and spherical shape characteristic of these particles. The presence of lipoproteins facilitates the membrane attachment. In the case of the active particles a different and more restricted complement of proteins is added to the core structure, including a 72,000 mol. wt poly(A)-binding protein. The activation procedure involves a conformational change from a condensed to a relaxed structure mediated through an extensive modification of the protein composition. Acknor~lrd~en~rn~s-I wish to thank Professor Tore Hulun for valuable discussions and Miss Marianne Magnusson for typing the manuscript. The work was supported by a grant from the Swedish Natural Research Council to Tore Hultin. REFERENCES Cervera M., Dreyfuss G. and Penman S. (1981) Messenger RNA is translated when associated with the cytoskeletal framework in normal and USV-infected HeLa cells. Cell 23, 113-120. Endo Y. and Natori Y. (1979) Direct association of messenger RNA-containing ribonucleoprotein particles with membranes of the endoplasmic reticulum in ethioninetreated rat liver. Biochim. hionhvs. Actu 562, 28 I-291. Golub A. and Clegg J. S. (1968) Protein synthesis in Artemiu .sa/inu embryos--l Studies on polyribosomes. &I>/ Bid. 17, 644-656. Holmes D. S. and Bonner _I. (1973) Preparatton, molecular weight, base composition, and secondary structure of giant nuclear ribonucleic acid. Biodwmi.~tr~ 12. 233&233X. Hultin T., Kallin B., Lake M. and Nilsson M. 0. (1977) Membrane-associated messenger RNA in Arrrmiu sulinu.

In Trunslution of’ Naturul und Synrhrtic Pol~nuclrotides (Edited by Legochi A. B.), pp. 2388243. Agricultural Univ.. Poznan. Poland. Huyuh-van-Tan and Schapira G. (1978) Isolation and characterization of free cytoplasmtc messenger ribonucleoproteins from rabbit reticulocytes. Eur. J. Bio&n~. 85, 271 -281. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nuturc 227, 680-685. Lake M. and Hultin T. (1980) Characterization of a poly(A)+ RNA-containing structural component dtrectly associated with cytoplasmic membranes in dormant .4r/rtmu cysts. Biochim. hiophvs. Acta 609, 286-295. Lake M.. Nygdrd 0. and Naslund P. H. (1982) A new cap device for sample recovery from density gradients. Am[vt. hioc/Kv?I.

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IO.

Lake M., Namomork E. and Johansen B. V. (1983) A structural comparison of latent membrane-bound and polysomal messenger ribonucleoprotein particles in Artemiu embryos: a dark field electron microscopical study. J. suhmicrosc. Cytol. 16, 2533259. Lenk R.. ransom L., Kaufman Y. and Penman S. (1977) A cytoskeletal structure with associated polyribosomes obtamed from HeLa cells. Cdl 10, 67-78. Moon R. T., Moe K. D. and Hille M. B. (1980) Polypeptides of nonribosomal messenger ribonucleoprotein complexes of sea urchin eggs. b’iochrmi.t/r~~ 19, 272332730. Nilsson M. 0. and Hultin T. (1974) Characteristics and intracellular distributton of messenger like RNA in encysted embryos of Artenziu sdinu. Ded Biol. 38, 138149. Northemann W.. Schmelzer E. and Heinrich P. C. (1980) Characterization of 20-S and 40-S nonpolysomal cytoplasmic messenger ribonuclcoprotein particles from rat liver. Eur. J. Biochcm. 112, 451-459. O’Farrel P. H. (1975) High resolution two-dimensional electrophoresis of proteins. J. hiol. Chenz. 250,4407-4021. Preobrazhensky A. A. and Spirin A. S. (1978) Informosomes and their protein components: the present state of knowledge. Proc. nucleic~ Ad Rm. molrc. Bid. 21, I-3X. Slegers H., De Herdt E. and Kondo M. (1981) Nonpoly(A)-containing messenger ribopolysomal nucleoproteins of cryptobiotic gastrulae of Arrernicr sulinu. Eur. J. Biochmz. 117, I I I- 120. Standart N.. Vincent A. and Scherrer K. (1981) The polyribosomal poly(A)-binding protein is highly conserved in vertebrate species. FEBS Lett. 135. 5&--60. Sullivan N. and Roberts W. K. (1973) Characterization and poly(adenylic acid) content of Erlich ascites cell ribonucleic acids fractionated on in modiiied cellulose columns. Biochen7i.srr~~ 12, 239552403. Vincent A.. Ctvelli 0.. Maundrell K. and Scherrer K. (1980) Identification and characterization of the translationally messenger-ribonucleorepressed cytoplasmic globin protem particles from duck erythroblasts. Eur. J. Biochrm. 112, 617-633. Vincent A., Goldenburg S. and Scherrer K. (1981) Comparisons of proteins associated with duck-globin mRNA and its Polyadenylated segment in polyribosomal and repressed free messenger ribonucleoprotein complexes. Eur. J. Biochem. 114, 179-193. Wettstein F. O., Stdehlin T. and Nell H. (1963) Robosomal aggregate engaged in protein synthesis: characterisation of the ergosome. Nature 197, 43S435.