Differentiation 12, 91-97 (1978) Differentiation 0 Springer-Verlag 1978

Postfertilization Poly(A) Protein Complex Formation on Sea Urchin Maternal Messenger RNA CYNTHIA PETERS and WILLIAM R. JEFFERY*

Department of Biophysical Sciences, University of Houston, Houston, Texas 77004, USA * Department of Zoology, University of Texas, Austin, Texas 78712, USA

A two-fold increase in polyadenylate [poly(A)l content occurs between fertilization and the two-cell stage in sea urchin zygotes. In this report the role of this cytoplasmic polyadenylation process in the provision of binding sites for poly(A)-associated proteins during early development of Lyrechinus picrus is evaluated. Protein-associated poly(A) sequences, from ribonuclease-treated, post-mitochon- drial supernatants of various developmental stages, were collected by nitrocellulose filtration and quanwied by 3H-poly(U) complex formation. The proportion of protein-associated poly(A) rose from about 27% to about 60% of the total poly(A), on a nucleotide basis, during the period between fertilization and the eight-cell stage. However, the actual increase in number of poly(A) sequences associated with protein was more extensive, about 2.5-fold, since protein-associated poly(A) se- quences average about 45 nucleotides longer than free poly(A). The protein-associated poly(A) of eggs and zygotes is found in two types of protease-sensitive complexes which sediment at 8-12 S and 15-20 S. The 8-12 S complex appears to be selectively increased in amount following fertiliza- tion. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the poly(A) protein complex fraction indicates the presence of 87,000 and 130,000 molecular weight polypeptides in both eggs and zygotes. It is concluded that quantitative, but not qualitative, alterations in the proportion of protein-associated poly(A) accompanies post-fertilization cytoplasmic polyadenylation in sea urchin zygotes. The attachment of specific proteins to the 3’ terminus of maternal RNAs may be involved in their subsequent activities during early embryogenesis.

Introduction

The polyadenylate [poly(A)l content of sea urchin zy- gote RNA rises about two-fold during the period be- tween fertilization and the two-cell stage [l-41. This in- crease is thought to reflect both an elevation in the titer of poly(A)-containing RNA and in some cases a length- ening of poly(A) sequences already present in the unfer- tilized eggs [l, 51. Maternal RNA primers are utilized for this type of polyadenylation since it is resistant to actinomycin D [l, 21 and occurs in enucleate merogons [21. Despite considerable study [ 1- 101 the physiological significance of the cytoplasmic polyadenylation process in sea urchin zygotes, like the related but less extensive

Offprint requests should be addressed to: W. R. Jeffery

process discovered in cultured mammalian cells [ll-131, is currently unknown. Maternal mRNA acti- vation appears to be an unlikely role for this process since its suppression by high levels of cordycepin does not effect translation [41 and a cytoplasmic polyadeny- lation phenomenon does not accompany the enhance- ment of translation in several other types of eggs

The present investigation was undertaken in order to examine the possibility that the sea urchin cytoplasmic polyadenylation process may serve to provide more ef- fective binding sites for proteins which are associated with the 3’ terminus of poly(A)-containing mRNA mole- cules in eukaryotic cells [ 17-221. Our results, which are based on the separation of poly(A) - protein by its inter- action with nitrocellulose filters, indicate that both the

[ 14- 161.

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proportion and the number of poly(A) sequences asso- ciated with protein increase at the time of cytoplasmic polyadenylation. m e formation of a poly(A). protein tail following fertilization may be related to maternal RNA function during subsequent embryogenesis.

C. Peters and W. R. Jeffery: Sea Urchin Poly(A) . Protein Complex

acrylamide/bisacrylarnide gels (8 cm long) containing 375 mM Tris- HCI (PH 8-81 and 0.1% SDS after an initial separation at 1 mA/gel through 1 cm long acrylamide/bisawldde stacking gels Contain- ing 375 mM Tris-HC1 (PH 6.8) and 0.1% SDS [271.

Methods Results and Discussion

Lytechinus pictus (obtained from Pacific Biomarine Laboratories, Separation Of and '

Venice, California) eggs or zygotes cultured in Milliporefiltered sea water (MFSW) at 200 C were collected by low speed centrifugation, washed twice in MFSW, and twice in 50 mM Tris-HCl (PH 7-61!

attained by three passages of the final egg suspension through a 23

tion was prepared by two cycles of centrifugation at 9500g for 20 min. Total post-mitochondrial fractions used for further experi-

Infante and Nemer 1241 have shown that sea urchin ri- bonucleoprotein (RNP) is adsorbed to nitrocelldose fd-

400 mM NaC1* lo mM MgC1,, and '"ldrnl bentonite. LYsis was

gauge sterile hypodermic needle [23], The post-mitochondrial frat-

ters at low ionic strength whereas under these conditions free RNA is refractive and can be recovered in the fil- trate- PolY(A) * Protein Particles also bind to nitroceh- lose filters [ 171 but it is not currently known whether

mentation were brought to 50 mM Tris-HC1 (PH 7.6), 50 mM KC1, 1 mM MgCI, (TKM) by dialysis. The preparation and labeling of Ehrlich ascites tumor cell post-mitochondrial fractions and poly- somes was conducted as previously described [221.

Poly(A) containing components were prepared from the post- mitochondrial fraction by treatment with 1 pg/ml RNase A for 1 h at 37O C. Poly(A). protein was then separated from free poly(A) by filtration through nitrocellulose fdters (0.45 p; Millipore Corp., New Bedford, Mass.) in the presence of 10 mM Tris-HC1 (PH 7.6), 10 mM KCl, 1 mM MgCl, 1241. In a typical experiment about 10 ml (20 A, units/ml) of the digest was passed through a nitrocellulose filter at a flow rate of about 0.25 dmin . The initial fdtrate was collected and passed through two fresh fdters under the same condi- tions and the resulting filtrates were pooled. The fdtrate RNA was precipitated at -20" C after the addition of NaCl to a final concen- tration of 0.1 M and 2.5 volumes of cold 95% ethanol. The nitrocel- lulose fdters were also pooled and the bound material was eluted by incubation at 25O C for 1 h in a 1.5 ml suspension of 0.1 M Tris-HC1 (pH 7.6) and 0.5% sodium dodecyl sulfate (SDS). RNA was ex- tracted from the eluted material by the method of Brawerman et al. [251.

The poly(A) titer of the nitrocellulose-bound and refractive RNA was measured by a modified version of the [3H]-poly(U) binding method [261. A 50 p1 aliquot of each RNA preparation was added to the hybridization mixture which contained 200 mM NaCl, 10mM Tris-HCl (pH 7.6), 5 m M MgCl,, and 2pCi /d PHI- poly-uridylic acid [poly(U)] (4.65 Ci/mM; New England Nuclear Corp., Boston, Mass.). Annealing was performed at 300C for 30 min. The uncomplexed ['H]-poly(U) was removed by treatment with 5 pdml RNase A for 1 h at 300 C, and the RNase-resistant complexes were collected on glass fiber fdters (Whatman GF-A) after precipitation in the cold with 10% trichloroacetic acid (TCA). The fdters were dried and counted in a toluenebased cocktail using a scintillation spectrometer at 17% efficiency.

Poly(A). protein particles were isolated by oligo(dT)-cellulose chromatography from the post-mitochondrial fraction as described

this interaction is quantitative. Below we consider the eficiency of separation of free poly(A) from poly(A) - protein by nitrocellulose filtration. Poly(A) components prepared from Ehrlich ascites polysomes were utilized for these pilot studies since their physical characteristics are well known [22,281 and they can be labeled to high speciftc activities. Approximately 3% of the total radioactivity of phenol-extracted poly(A), ob- tained from [3H]-adenosine labeled Ehrlich ascites poly- somes, was retained by the nitrocellulose filters. In con- trast, RNase-digested polysomes or total post-mito- chondrial fractions, which contain poly(A) - protein, showed 94% and 75% poly(A) retention, respectively. In order to establish whether the nitrocellulose filters quantitatively retained poly(A) * protein, the sedimenta- tion behavior of the filtrate poly(A) was compared to that of the poly(A)-containing components of the origi- nal post-mitochondria1 digest. Unfortunately, the nitro- cellulose adsorbed poly(A) * protein cannot be analyzed directly since SDS treatment is required for its release from the filters. As shown in Fig. 1, the original post- mitochondrial digest contained a 10 S RNase-resistant RNP (Fig. lA), which has been previously identifed as a poly(A) - protein complex [221, and more slowly-sedi- menting material (4-6s) similar in characteristics to free poly(A) (Fig. 1C). Material sedimenting at 10 S was absent from the nitrocellulose fdtrate (Fig. 1B) suggest- ing quantitative retention of poly(A) - protein by the ni- trocellulose filter. The adsorption of poly(A) - protein complexes to nitrocellulose filters under conditions in

C. Peters and W. R. Jeffery: Sea Urchin Poly(A) Protein Complex 93

4s @I 4s + @ 4 - +

Fii. 1. Sedimentation analysis of poly(Ajcontaining fractions from the RNase A-digested post-mitochondrial supernatant of Ehrlich ascites cells. A Total post-mitochondrial supernatant. B Nitrocellulose fdtrate. C Phenol- extracted poly(A). Centrifugation was at 4" C for 20 h through 12 ml 10-30% linear sucrose gradients in TKM in a Spinco SW 41 rotor at 40,000 rpm

X

10 20 30 10 20 FRACTION NUMBER

0 12 2

X

8 - 9 N

4

10 20 30

Table 1. The proportion of protein-associated poly(A) on a nucleotide and a sequence number basis present in the post-mitochondrial fraction of developing sea urchin eggs

Developmental stage'

% Nitrocellulose bound % Nitrocellulose poly(A) on a nucleotide basisb

Normal Actinomycin D-treatedd

bound poly(A) on a sequence number basisC

Unfertilized egg One-cell zygote

26.7 f 7.3 (10)' - 19.2

58.5 f 10.3 (4) 55.8 f 3.8 (4) 48.2

Eight-cell embryo 61.2 f 14.8 (4) 50.9 f 9.6 (4) 50.2

Blastula 58.2 f 10.3 (4) 44.4 5 7.9 (3) 47.9

Samples for poly(A) titer determination were removed from cultures of developing embryos at 30 min (one-cell zygotes), 3 h (eight-cell

% Nitrocellulose bound poly(A) was calculated from the RNasbinsensitive [3H1-poly(U) radioactivity as follows; embryo), and 6 h (blastula) post-fertilization

. . . .

[3Hl-poly(U) CPM adsorbed [3Hl-poly(U) CPM adsorbed + [3Hl-poly0 CPM unbound

% poly(A) adsorbed = x 100

The sum of [3HI-poly(U) CPM adsorbed and ['H]-poly(U) CPM unbound was calculated to be equal to poly(A) contents of 0.39 f 0.07 ng/pg RNA (0.039% of the total RNA) of unfertilized eggs and 0.86 f 0.08 ng/pg RNA (0.086% of the total RNA) of eight-cell embryos

The % protein-associated poly(A) sequences was calculated from the data of this table and Fig. 3 by the following formula:

x 100 PHI-poly(U) CPM adsorbedmean poly(A) size adsorbed 13H1-wlv(U) CPM unbound

% nitrocellulose adsorbed sequences = PHl-~olvM CPM adsorbed mean poly(A) size adsorbed ' mean poly(A) size unbound

Unfertilized eggs were pre-treated with 20 pg/ml actinomycin D for 30 min prior to fertilization Data is expressed as the mean proportion of nitrocellulose-bound poly(A) f standard deviation. The numbers in parentheses represent the

number of different post-mitochondrial preparations utilized for each determination

Proportion of Poly(A) Associated with Protein in Sea Urchin Eggs and Zygotes

RNase-digested post-mitochondrial fractions from un- fertilized eggs, one-cell zygotes (30 min post-fertiliza- tion; streak stage), eight-cell embryos (3 h post-fertiliza- tion), and blastulae (6 h post-fertilization) were sub- jected to nitrocellulose filter fractionation. The poly(A) titer was then determined by application of the PHI-

p l y 0 assay to phenol-extracted aliquots of the ad- sorbed and refractive fractions. Sea urchin poly(A) - pro- tein, like the mammalian poly(A) - protein analyzed in the pilot experiments, was quantitatively-bound to nitro- cellulose filters since only free poly(A) could be recov- ered in the refractive material (data not shown). As sum- marized in Table 1, the post-mitochondrial digests of each developmental stage analyzed showed significant proportions of protein-associated poly(A). About 27%

94

of the poly(A) from the post-mitochondrial fraction of unfertilized eggs, on a nucleotide basis, interacted with nitrocellulose. This value was increased to almost 6096 at the streak stage and remained about the same in the eight-cell and blastula stages.

The increase in protein-associated poly(A) observed after fertilization may be based on the attachment of protein to the modified 3’ terminus of pre-formed RNA or the appearance of newly-synthesized transcripts con- taining poly(A) . protein tails. In order to resolve this issue post-mitochondrial digests from actinomycin D- treated zygotes were also subjected to nitrocellulose fractionation. The proportion of protein-associated poly(A) was not markedly altered from the normal lev- els in actinomycin D-treated streak and eight-cell stages (Table 1). However, by the blastula stage, when zygote- synthesized transcripts make up a considerable propor- tion of the poly(A)-containing RNA population [291, the proportion of protein-associated poly(A) was reduced from the control value.

The nitrocellulose filtration data suggest that the proportion of poly(A) associated with protein on mater- nal RNA molecules rises about 2.2-fold, on a nucleotide basis, during the period of cytoplasmic polyadenylation in the zygote. It is also implied that the poly(A) proteins added to the 3’ termini of these molecules are either stored in an uncomplexed form in the unfertilized egg or synthesized using pre-formed RNA immediately follow- ing fertilization.

C. Peters and W. R. Jeffery: Sea Urchin Poly(A). Protein Complex

Size of Free and Protein-Associated Poly(A)

Since some reports [l, 51 indicate that the poly(A) se- quences of sea urchin eggs increase in length during cy- toplasmic polyadenylation, the average size of free and protein-associated poly(A) tracts must be established in order to estimate the increase in number of poly(A) se- quences associated with protein. The electrophoretic mobilities of RNA extracted from the nitrocellulose fil- ter adsorbed and filtrate fractions are shown in Fig. 2. Two distinct size classes of fragments, detected by their ability to form RNase-resistant complexes with [’HI- poly(U), were observed in all the gels. A very rapidly- migrating class, possibly representing oligo(A) se- quences [30-321, was present in both the nitrocellulose bound and refractive fractions. In contrast, the more slowly migrating sequences, which exhibit the electro- phoretic mobilities expected of eukaryotic poly(A) [331, were fractionated by nitrocellulose into a long subclass, distinctive of the poly(A) - protein complex material, and

I 5 0

100 H a

50 n N W

. I

n

: I

U

4 150 =l b

100 -

50 - ; I

0 Q , , , i , I , , , , : I

10 20 10 20

SLICE NUMBER

mg. 2. Polyacrylamide gel electrophoresis of poly(A) sequences in the nitrocellulose adsorbed (A, C) and refractive (B, D) fractions of unfertilized eggs (A-B) and eight-cell embryos (C-D) of the sea urchin. The position of poly(A) migration was determined by PHI- poly(U) assay of RNA eluted from each gel slice. Vertical dashed line represents the position of the poly(A) radioactivity maximum in the refractive fraction and the vertical dotted line represents this posi- tion in the adsorbed fraction. Electrophoresis was carried out through 7 cm, 6.5% polyacrylamide gels at 7 mA/gel as described previously [341

a shorter subclass, primarily represented in the free poly(A) fraction. The occurrence of the longer se- quences in the poly(A) e protein complex suggests that the limiting factor in the formation of these associations in unfertilized eggs may be poly(A) size.

In order to determine the average sequence length represented in a heterogeneous population of poly(A) molecules it is necessary to convert the poly(A) electro- phoretic profiles illustrated in Fig. 2 into length fre- quency distributions. When this conversion was made (Fig. 3), average sequence lengths for protein-associated and free poly(A) of 130 and 85 nucleotide residues, re- spectively, were obtained. Taking these values into ac- count, the increase in proportion of poly(A) associated with protein can be expressed on a sequence number rather than a nucleotide basis (Table 1). We estimate that the proportion of poly(A) sequences present in the protein complex fraction rises 2.5-fold following fertili- zation. This finding is consistent with the possibility that poly(A) sequences elongated after fertilization also be- come associated with protein.

95 C. Peters and W. R. Jeffery: Sea Urchin Poly(A). Protein Complex

30 @ Unfertilized Bound 1 @ A Unfertilized Unbound I

-

2 W

I- z a 10

I I I I I I I I I I I

[ / f l I I I I

@ Fertilized Bound @ Fertilized Unbound

50 100 150 200 50 100 150 200 POLY(AI LENGTH (AMP RESIDUES)

Fig. 3. Frequency distribution of mean poly(A) sequence length in the nitrocellulose adsorbed (A, C) and refractive (B, D) fractions of unfertilized eggs (A-B) and eight-cell embryos (C-D) of the sea urchin calibrated in relation to the electrophoretic migration of poly(A) standards of known size [351. Radioactivity values from Fig. 2 were corrected so 88 to represent the relative number of poly(A) sequences in each size class. Vertical dashed lines represent the average sequence length of the adsorbed poly(A) fraction where as the vertical dotted lines represent the average sequence length of the refractive poly(A) fraction

Fig. 4. Sedimentation behavior of the poly(A) a protein complex from the post- mitochondrial supernatant of sea urchin eggs and embryos. A Unfertilized egg. B Unfertilized egg pretreated with proteinase K for 1 h at 37O C. C Eight-cell zygote. Centrifugation was at 4 O C for 15 h through 12 ml 10-3096 linear sucrose gradients in TKM in a Spinco SW 41 rotor at 40,000 rpm

5

4 D: 0 x 3

h " 2

I

Size and Polypeptide Composition of Sea Urchin Poly(A) * Protein Complexes

In order to examine the qualitative characteristics of poly(A) - protein complexes in unfertilized eggs and zy- gotes their sedimentation behavior was compared on su- crose density gradients. Poly(A) a protein complexes de- rived from RNase-treated digests of mammalian cells generally occupy the 8- 12 S region of sucrose gradients [17-22, 26, 28, 36, 371. As shown in Fig. 4A, PHI- poly(U) binding was observed primarily in the 15-20 S and the 4 S regions of gradients containing unfertilized egg material, although a minor peak can also be seen in the 8-12s region. The [3Hl-poly(U) binding compo- nents present in the 8-12 S and 15-20 S regions of the gradients are likely to represent poly(A) - protein com- plexes since they were sensitive to proteinaseK treat- ment (Fig. 4B).

In gradients containing the post-mitochondrial di- gest from eight-cell embryos the proportion of PHI- p o l y 0 binding activity in the 8- 12 S region of the gra- dient was markedly increased relative to that of 15-20 S material (Fig. 4C). These results suggest that the elevation in protein-associated poly(A) sequences at the time of cytoplasmic polyadenylation primarily en- tails the formation of 8-12 S poly(A) - protein com- plexes. At present the basis for the presence of what appears to be two size classes of poly(A) protein com- plexes in sea urchin eggs is obscure. The 15-20 S com- plex may be associated with a distinct class of abundant maternal mRNA or it may be an artifact due to dimeri- zation of 8-12 S complexes. Further studies will be ne- cessary to resolve this point.

Egg, proteinase K Q Zygote

0 10 20 0 10 20 0 10 20 30

FRACTION NUMBER

96 C. Peters and W. R. Jeffery: Sea Urchin Poly(A). Protein Complex

I .o 0.50 Fig. 5. SDS-polyacrylamide gel electrophoresis of polypeptides of the sea urchin poly(A) protein complex. A Total RNase A-treated post- mitochondrial supernatant from unfertilized eggs, E

approximately 20 pg of total protein was applied. B Oligo(dT)-cellulose eluted poly(A) - pro& UJ cu fraction from unfertilized eggs, the total oligo(dT>

0) u $ cellulose eluate from approximately 800 Ax0 units c 0 1.0 0.50 “N of post-mitochondrial supernatant was applied. e 0 C Total RNase A-treated post-mitochondrial

supernatant from fertilized eggs, approximately 20 pg total protein was applied. D OIigo(dQ n cellulose eluted poly(A). protein fraction from a 0.5 0.25 eight-cell zygotes, the total oligo(dT)-eluate prepared from approximately 400 A, units of post-mitochondrial fraction was applied

o.25 C 0 0.5 2 u 2 -

0 0 In 3

1 2 3 4 5 6 7 1 2 3 4 5 6 7

Distance M i g r a t e d ( c m )

The oligo(dT)-cellulose-eluted poly(A) . protein com- plex fraction from eggs and zygotes showed two major polypeptide bands, of molecular weight 87,000 and 130,000, and two minor bands, of molecular weight 76,000 and 67,000, when analyzed by SDS-polyacryl- amide electrophoresis (Fig. 5B and D). These polypep- tides were not major components of the total post-mito- chondrial supernatants (Fig. 5A and C). These data sug- gest that, although the proportion of protein-associated poly(A) sequences increase following fertilization, the polypeptide composition of the new poly(A). protein complexes formed remains unaltered. It is also of inter- est to note that in the poly(A). protein complex of sea urchin maternal RNA the major poly(A)-associated po- lypeptide of other eukaryotic systems, the 7 5 4 1 , 0 0 0 molecular weight protein 118-22, 36, 371, is not the dominant component. It will be important to determine whether this is a general feature of maternal RNA.

Concluding Remarks

The results of the present investigation provide the basis of a possible role for the phenomenon of post-fertiliza- tion cytoplasmic polyadenylation in sea urchin eggs. They suggest that the synthesis and possibly the length- ening of poly(A) tails on maternal RNA primers may facilitate the attachment of specific proteins to the 3‘ termini of these molecules. Although the function of poly(A)-associated proteins from eukaryotic cells is cur- rently unresolved, in the fertilized sea urchin egg they could be involved in activities related to the activation of the maternal mRNA, such as the regulation of meta-

bolic stability or promotion of RNA interaction with membranes and other sub-cellular structures. It is also possible, but cannot be decided by this study, that some of the polypeptides which are associated with the 3‘ terminus of sea urchin maternal RNA are poly(A) poly- merases responsible for, rather than the effect of, cyto- plasmic polyadenylation. Acknowledgements: This research was supported by grants from the National Institutes of Health (GM-25119-02) and the National Science Foundation (PCM-77-24767).

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C. Peters and W. R. Jeffery: Sea Urchin Poly(A) Protein Complex

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Received May 1978/Accepted October 1978