The dynamics of maternal poly(A)-containing mRNA in fertilized sea urchin eggs

Cell, Vol. 11, 673-661,

July

1977, Copyright

0 1977 by MIT

The Dynamics of Maternal Poly(A)-Containing in Fertilized Sea Urchin Eggs Fred H. Wilt Department of Zoology University of California Berkeley, California 94720

Summary Cytoplasmic polyadenylated RNA with the characteristics of sequestered mRNA exists in the unfertilized sea urchin egg. Following egg activation, the amount of poly(A) doubles, but total RNA content stays constant. Chromatography of the RNA on poly(U)-Sepharose shows that the amount of RNA that bears a poly(A) tract increases slightly (approximately 20-30%) during the 2 hr after fertilization. When a cDNA transcript of the poly(A)+ mRNA from 2 hr zygotes is reacted against poly(A)+ RNA from either eggs or zygotes, the kinetics of reassociation of the two preparations seem identical; hence the RNA sequences bearing poly(A) are the same in eggs and zygotes. Measurement of the length of the poly(A) tract in eggs and zygotes shows an increase in number average length from about 45 bases to 60 bases. Measurement of tract length of poly(A) in two cell zygotes by adenosine/AMP ratios of radioactive RNA shows that the poly(A) tract of the zygote is solely accounted for by radioactive RNA, indicating extensive turnover of the poly(A). It in concluded that the poly(A) tract in these cells is subject to both lengthening and shortening, with the former predominating in this instance. The increase in poly(A) does not involve polyadenylation of different sequences, but is due to an increase in the number of polyadenylated sequences and the length of the poly(A) tracts that they bear. Introduction Soon after the rediscovery of poly(A) and its implication in nuclear poly(A) metabolism (reviewed by Brawerman, 1976), it was shown there is also cytoplasmic adenylation of mRNA following activation of sea urchin eggs (Wilt, 1973; Slater, Gillespie and Slater, 1973). Cytoplasmic adenylation and turnover of 3’ terminus of the poly(A) tract have also been observed in mammalian cells, and it is probably a widespread phenomenon (Diez and Brawerman, 1974). The biological role of poly(A) in mRNA metabolism is still unclear; the mechanism and role of cytoplasmic adenylation are virtually unexplored. The newly activated sea urchin egg is uniquely suited for investigation of cytoplasmic adenylation because nuclear contributions are almost nonex-

mRNA

istent until later in development (Wu and Wilt, 1974). It is also of interest that the sea urchin egg is the best studied example of stored mRNA, although the relationship between mRNA sequestration, its mobilization on polyribosomes after fertilization and cytoplasmic adenylation is obscure (Mescher and Humphreys, 1974). We describe experiments on the mechanism(s) of adenylation which demonstrate that the poly(A) tract is subject to extensive turnover. The same mRNA sequences that bear poly(A) in the egg are those that possess the poly(A) in the early zygote. Both the amount of RNA that bears poly(A) and the tract length of the poly(A) increase after fertilization, accounting for most of yhe increase in poly(A) that is observed in these cells following activation. Results The Amount of Poly(A)+ RNA There is a gradual doubling of the poly(A) content of Lytechinus eggs following fertilization, and by 90 min following fertilization, shortly before the first cell division, 0.08-0.09% of the RNA of the cell is poly(A) (Slater, Slater and Gillespie, 1972; Slater et al., 1973; Wilt, 1973). This amount remains constant throughout development to the larval stage (F. H. Wilt, unpublished data) and represents 1 .5 pg of poly(A) per embryo. The total amount of RNA per egg or embryo is constant at about 1800 pg per embryo (Ficq, Aiello and Scarano, 1963). It has been impossible to label RNA throughout oogenesis to high specific activity, and hence the determination of the amount of RNA that bears a poly(A) tract [(poly(A)+ RNA)] requires a method of efficiently separating poly(A)+ RNA from the remainder in amounts that can be determined by routine chemical methods. Does the increase in poly(A), which occurs almost entirely in the cytoplasm at these stages, represent an addition of poly(A) to previously adenylated RNA, is it turnover of poly(A) or is it due to adenylation of a subset of maternal RNA that previously bore little or no poly(A)? These are not mutually exclusive possibilities. Oligo(dT)-Cellulose Chromatography The use of oligo(dT)-cellulose to fractionate poly(A)+ RNA is popular, but the efficiency of fractionation of RNA, as evaluated by 3H-poly(U) hybridization, is rather poor. Table 1 shows some selected representative data. For the two-cell zygote, about 1% of the total RNA binds to oligo(dT)cellulose when fractionated at room temperature. Repassage of unbound material at room temperature leads to additional binding near the noise level of the column. Oligo(dT)-cellulose is even less capable of selecting polyadenylated molecules from

C@ll 674

Table

Source

1. Binding

of RNA

Unfertilized

Egg

% RNA Bound @ Am)

% Poly(A) Selected [“h Total 3H-Poly(U) Hybrid in Bound Fraction]

0.54 t 0.08 (5)”

26

Repass of unbound (room temperature)

0.21

Repass of unbound at 4”C, elute at 37°C

1.02

37

Cumulative bound

1.56

63

1.03 2 0.06 (7)a

41

Two-Cell

No of nucleotides

of RNA to Oligo(dT)-Cellulose

90 v

45 v

25 v

amount

Zygote

Repass of unbound (room temperature)

0.16

Repass of unbound at 4”C, elute at 37°C

1.04

46

Cumulative bound

2.07

67

amount

a Number of determinations. 50-100 AzeO units of RNA were fractionated on oligo(dT)-cellulose as specified in Experimental Procedures. Aliquots of the column fractions were assayed for their poly(A) content by hybridization to 3H-poiy(U). Recovery of total poly(U)-hybridizable material ranged from 93-102%.

the RNA of unfertilized eggs. About 0.5%‘of the RNA binds, but it contains 26% of the poly(A), and repassage of the unbound fraction is not particularly efficacious. By repassing unbound RNA over oligo(dT)-cellulose at 4°C and then eluting the bound material at 37°C in 10 mM Tris, an additional 1% of the RNA can be bound, using RNA from either eggs or zygotes. This results in a total of 1.5% of the RNA from unfertilized eggs being scored as poly(A)+ RNA, and about 2% of the RNA from zygotes being scored as poly(A)+ RNA; these fractions contain 63% and 87%, respectively, of the total poly(A) of the preparation. The sedimentation in aqueous sucrose density gradients shows that the sedimentation of poly(A)+ RNA retained by the column is identical to the sedimentation of that which is not retained (data not shown). Oligo(dT)-cellulose does not seem to fractionate poly(A)+ RNA by virtue of total RNA size. It is more probable that the poly(A) tract length affects the chromatography of RNA on oligo(dT)cellulose, and accordingly, the electrophoretic behavior of poly(A) tracts fractionated on oligo(dT)cellulose has been examined. Figure 1 shows the distribution of poly(A) tracts from unfertilized eggs, as measured by 3H-poly(U) hybridization to eluants of gel slices. The total poly(A), prepared by nu-

,

L

20 Slice Figure

1. Fractionation

of Poly(A)

no. Tracts

Total poly(A) tracts from unfertilized eggs were fractionated on oligo(dT)-cellulose, and aliquots of the total poly(A) and the material bound and not bound to oligo(dT)-cellulose were electrophoresed on 12% polyacrylamide gels. Other conditions were the same as for Figure 4. Gel slices were eluted, and the distribution of poly(A) was measured by hybridization to 3H-poly(U).

clease digestion, and the poly(A) binding, or not binding, to oligo(dT)-cellulose in 0.5 M KCI at room temperature are shown. Bound and unbound poly(A) overlap in their distributions, but clearly, poly(A) binding to oligo(dT)-cellulose contains a larger proportion of the longer poly(A) tracts. The differential selectivity of oligo(dT)-cellulose for the longer poly(A) tracts helps to explain why oligo(dT)-cellulose is limited for determination of the total amount of poly(A)+ RNA. If one considers that the oligo(dT)-cellulose columns do not fractionate poly(A) on the basis of total RNA size, and since the size of the poly(A)+ RNA in eggs and zygotes is similar (Slater et al,., 1974), the data of Table 1 suggest that the amount of poly(A)+ RNA of the two stages is similar and that the increase in poly(A) is due in part to lengthening of the poly(A) tracts. In Table 1, for instance, 1% of the zygote RNA contains 41% of the poly(A), and 0.5% of the unfertilized RNA has 26% of the poly(A) in that preparation. If one corrects the amount of poly(A)+ RNA obtained by the recovery of poly(A), it would imply that the egg possesses 2.09% and the zygote 2.44% of its total RNA in the polyadenylated form.

Poly(A) 675

of Maternal

mRNA

Poly(U)-Sepharose Chromatography RNA from eggs and zygotes may be fractionated by chromatography on poly(U)-Sepharose, a method which might have higher specificity because of the longer chain lengths of the hybrids formed on the column matrix. This expectation was sustained. Table 2 shows that 2.39% of the RNA from unfertilized eggs and 2.6% of the RNA from zygotes are scored as poly(A)+ RNA. If adenosine-labeled RNA from zygotes is used, 50% of the radioactivity is collected in the poly(A)+ RNA fraction. Examination of the distribution of poly(A) by 3H-poly(U) hybridization shows good retention of poly(A)+ RNA by the column. It is necessary to sediment the RNA bound to and eluted from the poly(U)-Sepharose through DMSO gradients, followed by concentration of the RNA by ethanol precipitation, to measure accurately the poly(A) content of poly(A)+ RNA by 3Hpoly(U) hybrid formation, presumably because small amounts of poly(U) are released from the Sepharose matrix during chromatography. Determination of the distribution of poly(A) in the different fractions from the column shows that after a single pass, 85% of the poly(A) from unfertilized eggs and 95% of the poly(A) from zygotes are found in the bound fraction The sedimentation of the bound material in sucrose-DMSO gradients is shown in Figure 2. It is clear the molecular weight is high and very heterogeneous; the broad peak sediments near the large ribosomal RNA marker, in close agreement with previous results using aqueous gradients (Slater et al., 1974). There are minor differences between Table

2. Binding

of RNA of Poly(U)-Sepharose

of RNA

% RNA Bound W Am or cpm)

Source

Unfertilized Repass

Egg of unbound

Cumulative bound Two-Ceil

3H-A-labeled twocell zygoteb 3H-A-labeled twocell zygote”

POlY(U)

% Poly(A) Selected [% Total 3H-Poly(U) Hybrid in Bound Fraction]

2.39 AZ60

65.1

0.27 AZ60

7.2

2.66 Aaso

92.3

2.60 A26,,

94.7

6

lo 3

amount

Zygote

Premixed

poly(A)+ RNA from eggs and zygotes, the latter having an apparently somewhat higher molecular weight distribution, a phenomenon noted previously (Slater et al., 1974). Not enough careful physical measurements have been made on these very heterogeneous RNA populations to evaluate properly the extent and meaning of these small differences. The RNA is large enough that it is probably relatively intact. Contamination by ribosomal RNA is not obvious in the bound material from zygotes (Figure 2), although the column noise level might be present without being detected by visual inspection. There is some contaminating ribosomal RNA in the poly(A)+ RNA from eggs, and by empirically plotting mixtures of pure poly(A)+ RNA like that found in zygotes with pure ribosomal RNA, the contamination of the poly(A)+ RNA of eggs is estimated at about 25-30%. The noise level of the poly(U)-Sepharose column is determined by prehybridizing the RNA with poly(U) prior to chromatography, which should eliminate most hybrid formation on the column matrix. The noise level is 0.14% of the At60 units passed over the column, which is about 5% the total bound (0.14%/2.6%). The greater contamination of poly(A)+ RNA from eggs with ribosomal RNA than that found in zygote poly(A)+ RNA has been observed repeatedly, although the chemical basis of the differential contamination is not understood. The data rule out the possibility that there is a doubling of the amount of RNA that bears a poly(A) tract; the doubling of poly(A) following fertilization cannot simply and solely be a recruitment of under (un) adenylated molecules into the poly(A)+ class. On the other hand, there is apparently some increase in poly(A)+ RNA. For instance, 2.66% of the

0.q 0.1

40

20 50.20

cpm

Fraction Figure

4.50 cpm

with 0.14 As60

a Average of three determinations. Determination of poly(A) in bound fractions was carried out after sedimentation of the RNA through dimethyl sulfoxide and concentration by ethanol precipitation. b Labeled with 5 @ml of 3H-adenosine for 100 min. c The same RNA specified in a was mixed with 1 AZBO unit of nonradioactive poly(U) before fractionation.

2. Sedimentation

of Poly(A)+

RNA in DMSO

RNA from unfertilized eggs and two-cell zygotes was fractionated on poly(U)-Sepharose, and the bound fraction was eluted by 70% formamide. The RNA was concentrated by ethanol precipitation and sedimented through 5-20% sucrose gradients in 99% dimethyl sulfoxide (Kung, 1974). 46 fractions of 16 drops were collected and diluted with water, and the A*,, was determined. The Azss of gradients devoid of RNA was also determined after dilution with water, and these values (0.09 A,,, units per fraction) were subtracted from the RNA data before plotting. Purified ribosomal RNA from Lytechinus gastrulae was run in a parallel gradient as a molecular weight marker.

Cell 676

RNA template prepared from zygotes have been determined. The poly(A)+ RNA used for synthesis of cDNA and for the hybridization reaction is isolated on poly(U)-Sepharose and purified on DMSO-sucrose gradients. Figure 3 presents data on the rate of reassociation of cDNA in the presence of poly(A)+ RNA from either eggs or zygotes and a computer fit to the data. The striking conclusion is that the pattern of reassociation is indistinguishable whether the excess “driver” RNA is prepared from eggs or zygotes. Thus within the limitations of this technique, the sequences present in poly(A)+ RNA from eggs and zygotes seem to be the same. The reassociation kinetics are very complicated, extending over a very large range of Rot values, although this is characteristic of reactions involving total poly(A)+ RNA from cells. There are several assumptions in the interpretation and analysis of these data which should be kept in mind: one assumes that the cDNA is a faithful reflection of the RNA population, that the effects of salt and RNA size on rate follow accepted rules for DNA-DNA hybridization, and that the use of cDNA probes that are only complementary to a portion of the templates (for example, see Weiss et al., 1976) does not introduce serious errors. It is also assumed that the standard used for complexity measurements is valid for other RNA molecules of different composition and size. Keeping these reservations in mind, it is of interest to compare the rate of reassociation observed here with that of a pure globin mRNA. The RotliZ (at 0.12 M phosphate buffer) for globin mRNA and its cDNA is 2.65 x 10m3 mol-set/l, determined as a part of these experiments, and this compares very closely to previously reported values (Bishop et al., 1974b; Rosen and Barker, 1976) when differences in salt concentration are taken into account. Table 4 presents an analysis of the reassociation data by the methods introduced by Bishop et al. (1974b). The best fit of the computer gives three transitions with RotI,, values of 2.5 x 10m3, 1 .4 and 102. The first transition will be neglected, since there is a paucity of data in this part of the curve, and the level of hybrid is close to the

RNA from unfertilized eggs bears 92% of the poly(A) tracts, but fully a quarter of this RNA is ribosomal RNA. Thus 92% of the poly(A) resides on 2% of the cellular RNA. 95% of the poly(A) tracts in the two-cell zygote reside on 2.6% of the cellular RNA; ribosomal contamination would not be likely to exceed 5% of this amount. There is quite probably, then, a 20-25% increase in the amount of RNA that bears a poly(A) tract following fertilization. The considerations above suggest there must be a net lengthening of the poly(A) tract following fertilization. The elution of the bound poly(A)+ RNA from poly(U)-Sepharose by formamide is consistent with this hypothesis. Table 3 shows the amount of bound RNA eluting at various formamide concentrations. Two thirds of the RNA from unfertilized eggs elutes without any formamide, while RNA from zygotes shows a distinctly different elution pattern, eluting at higher formamide concentrations. Radioactive RNA from zygotes shows an elution pattern requiring formamide for elution of much of the RNA. Since poly(A) of greater tract lengths requires higher formamide concentrations to be eluted from poly(U)-Sepharose (Firtel, Kindle and Huxley, 1976), the data are consistent with the idea that poly(A) tract lengths increase after fertilization. The Sequences in Poly(A)+ of Eggs and Zygotes Although the poly(A)+ RNA of zygotes seems only slightly augmented in terms of amounts and sedimentation compared to that from eggs, there is no knowledge of the nucleotide sequences in the two populations. If, as has been suggested above, there is a net lengthening of the poly(A) tract following fertilization, one should find that the sequences in poly(A)+ RNA of eggs and zygotes are about the same. The available data on the kinds of polypeptides synthesized in eggs and zygotes would suggest that the kinds of abundant mRNA being translated in the two states are the same (Brandhorst, 1976). The reassociation kinetics of poly(A)+ from the two stages with cDNA synthesized on a poly(A)+

Table

3. Elution

Unfertilized 2 Hr Zygotes 3H-A-Labeled (% cpm)

Eggs

of Poly(U)-Sepharose-Bound

(% A&

(% A&

RNA by Formamide

0% Formamide

23%

Formamide

66.0

27.3

(36)

(55)

47% Formamide

70%

Formamide

4.5 (6)

1.3 (1.7)

40.2

52.2

4.8

2.8

31 .o

49.0

10.8

6.9

2 Hr Zygotes

RNA was prepared from unfertilized eggs, two-cell zygotes or two-cell zygotes labeled for 100 min with 5 &ml of 3H-adenosine. After adsorption of the RNA to poly(U)-Sepharose and extensive washing, the bound fraction was eluted stepwise with increasing concentrations of formamide. The distribution of total recovered RNA in the various fractions is shown. Numbers in parentheses are a calculation of the amount of poly(A)+ RNA found in each fraction assuming that 25% of the bound unfertilized RNA is ribosomal RNA and that the ribosomal RNA is eluted entirely at 0% formamide.

Poly(A) 677

of Maternal

mRNA

blank values. Two other clear transitions are observed: one with a kinetic complexity of 3 x 10’ daltons and the second with a complexity of 1 x 1O’O daltons. The total complexity observed is very close to that reported by Anderson et al. (1976) using different methods. The complexity is sufficient to code for 15,000-30,000 different proteins. The analysis of Table 4 is subject to the limitations of the method mentioned previously; furthermore, there may be, in fact, a much more continuous range of abundance classes. Table 4 uses the total amount of hybrid found (64%) for the calculations; if one sets the observed maximum level of hybridization equal to lOO%, the complexities would be increased about 1.5 fold. The Length of the Poly(A) Tract I have examined several methods for determining poly(A) tract length and find that conventional polyacrylamide gel electrophoresis has greater resolving power that any other chromatographic methods that have been tried (for example, Stanley, 1967). Poly(A) migrates anomalously in polyacrylamide gels, even under denaturing conditions 707. 60

IO r

Figure

3. The Formation

of Sl Nuclease-Resistant

Hybrids

cDNA was prepared from poly(A)+ RNA of two-cell zygotes. Poly(A)+ RNA from unfertilized eggs (0) or two-cell zygotes (A) was reacted for 1, 20, 44 or 120 hr at 67°C with RNA concentrations varying from 0.1 Fg to 1 mg/ml. The concentration of RNA (Ro) and time is expressed in mole-se&l corrected to 0.18 M Na+ ion concentration at 62°C.

Table

4. Sequence

Complexity

of Poly(A)+

mRNA

Component

Observed

1

2.5 x IO-3

0.074

2

1.4

0.094

3

1.02

x 102

Rot,,,

(Burness, Pardoe and Goldstein, 1975), and standards are necessary. I have used standards of 90, 45, 25 and IO-12 (A) residues in length. There is a linear relationship between log molecular weight and migration through the gel. Transfer RNA behaves as if it were composed of 28 adenylate residues; 5.9 RNA migrates as though composed of 52 adenylate residues. The migration of the markers relative to tRNA and 55 RNA and bromphenol blue dye is very close to that observed by other investigators using similar conditions (Nokin et al., 1976; Morrison, Merkel and Lingrel, 1973). Figure 4 shows the electrophoretic migration of poly(A) prepared from unfertilized eggs and two cell zygotes, the distribution of poly(A) being followed by hybridization ‘of 3H-poly(U) to eluants of the gel slices. Figure 5 shows the mobility of radioactive poly(A) obtained from two-cell zygotes labeled continuously from fertilization to the two-cell stage under conditions where the ATP specific activity is constant. There is great heterogeneity, and considerable overlap, when RNA from eggs and zygotes is compared. Zygotes contain a greater proportion of the higher molecular weight poly(A) tracts. Table 5 presents calculations for the determination of number average molecular weights for the different preparations (Vournakis, Gelinas and Kafatos, 1974). Although the molecular weight averages will not be very accurate for such heterogeneous preparations, they do show that the poly(A) tracts of zygotes are longer than those in eggs. The most surprising finding is that when the tract length of the radioactive RNA is determined by A/ AMP ratios of the alkaline hydrolysates, the total length of the tracts is due to radioactive poly(A). The poly(A) tracts are metabolically labile and turn over completely, or nearly so, during the transition from egg to the two-cell stage. The observations have been repeated many times, both on alkaline hydrolysates of total RNA and on fractions eluted from gels, and the data are shown in Table 5. Even though there is a net lengthening of the poly(A) tract, it occurs by a dynamic turnover process and is not merely a chain elongation of preexisting poly(A) tracts.

Fraction

0.47

of RNA

Pure

0.128 48

Rot,,,

Number of Sequences (6 x 105)

50 18,000

The data of Figure 4 are analyzed for the apparent complexity of the observed major transitions by the method of Bishop et al. (1974b). It is assumed that a sequence with a complexity of 6 x lo5 daltons reacts under the specified conditions with a Rot,,* of 2.65 X 10eJ mole-see/l. Blank values (no RNA) of 8% S.1 nuclease resistance were subtracted prior to computing and plotting (Figure 3) the data.

Cell 676

Discussion Two principal conclusions emerge from the experiments. The observed increase in poly(A) following activation of sea urchin eggs is due to an increase in the number of RNA molecules that bear poly(A) and to an increase in the net length of the poly(A) tract. The sequences in poly(A)+ RNA of eggs and zygotes are the same. The reservation one must attach to the conclusion is a quantitative one about the relative importance of increase in poly(A)+ RNA and increase in tract length. Determination of the increase in the amount of poly(A)+ RNA is limited by estimations of the contamination of poly(A)+ RNA with ribosomal RNA, the degree of difference in the molecular weight of poly(A)+ RNA of eggs and zygotes, and the ability of poly(U)-Sepharose to retain a// the poly(A)+ RNA. I estimate that these errors could contribute an uncertainty of 20-30%. Nevertheless, the data adequately show that the increase in poly(A)+ RNA is much less than 2 fold, and an increase in poly(A)+ RNA cannot solely account for the increase in poly(A) content. Measurements of the tract lengths are more accurate, variaNo 90

of nucleotides 45

25 v

13 . IOOO-

tion from experiment to experiment not exceeding 20%. It is probable that the errors discussed above are the reason that only about two thirds of the poly(A) doubling is accounted for. The second conclusion is that lengthening of the poly(A) tracts occurs by a dynamic extension involving turnover of the poly(A). Dolecki, Duncan and Humphreys (1977) have also shown extensive turnover of the poly(A) tract in sea urchin embryos. This may have quite general significance for many eucaryotes. Limited turnover has been observed in mammalian cells, and gradual shortening of poly(A) tracts has been observed frequently (reviewed by Brawerman, 1976). I propose that the lengthening and shortening of poly(A) tracts occurring in different situations occur generally by extensive turnover, the amount of net lengthening or shortening determined by the relative activities of cytoplasmic adenylating and hydrolyzing enzymes. The hypothesis could be examined in cytochalasinenucleated mammalian cells. There are two additional consequences of the present observations that deserve comment. First, the determination of sequence complexity of the poly(A)+ RNA is similar to that determined by other methods using total mRNA from Arbacia (Anderson et al., 1976). Hence the poly(A)- RNA probably contributes very little to the total measured RNA se-

A

No. 90 .

of nucleotides

45 v

25 T

13 -

20,000

500 E %

p t i

40 Figure

20

Slice no. of Poly(A) Tracts

4. Electrophoresis

The electrophoretic migration in 10% polyacrylamide gels of total poly(A) tracts prepared from nonradioactive RNA of unfertilized eggs and two-cell zygotes is shown. Each fraction was composed of the eluant from two adjacent 1 mm slices of the gel. 3H-poly(U) was hybridized to an aliquot (0.2-0.6 ml) of the eluant. Markers of 90, 45, 25 poly(A), E. coli tRNA and 5s RNA, and bromophenol blue were run on parallel gels. Table

5. Size of Poly(A)

Source

I

60

Slice no. of Radioactive

5. Electrophoresis

Poly(A)

The electrophoretic migration of RNA from two-cell zygotes labeled with 2-3H-adenosine for 110 min is shown. Other conditions are the same as in Figure 4. The radioactive poly(A) was eluted from fractions lo-16 in several instances, and the ratio of A/AMP was determined afler alkaline hydrolysis, as shown in line 4 of Table 5

Method

of Determination

47, 43, 41

‘H-poly(U)

distribution

gels

57, 57. 64

‘H-poly(U)

distribution

of gels (see Figure

53, 67, 71

A/AMP

ratio of acid-precipitable

A/AMP

ratios

La

Eggs

Two-Cell

Zygotes

Two-Cell

Zygotes,

Fractions

10-16,

B Number b Zygotes

average molecular were labeled with

b 3H-Adenosine Figure

Figure

1

40

Tracts

of Poly(A)

Unfertilized

‘k.9

$0

Labeled

5

101,91,93 weights were =H-adenosine;

of.gel

eluants

(see Figure

poly(A) from

fractions

4) 4) tracts 10-16,

calculated from distributions in gels as outlined by Vournakis et al. (1974). this is the same RNA preparation used for electrophoresis in Figure 5.

Figure

5

Poly(A) 679

of Maternal

mRNA

and “C-poly(A) from Miles Laboratories; oligo(dT)-cellulose (T-3) from Collaborative Research; PEI-cellulose thin-layer plates from Baker Chemicals; Sepharose 4-B from Pharmacia; Si nuclease from Sigma; Ti and T2 ribonuclease from Calbiochem; pancreatic ribonuclease and RNAase-free DNAase from Worthington. Poly(A) molecular weight markers of 25, 45 and 90 nucleotides in length were a gift from Dr. Borek Janik (Miles Laboratories). Poly(A) lo12 nucleotides long was donated by Dr. Joan Egrie. Dr. Jerry Lingrel provided rabbit hemoglobin mRNA. Dr. Peter Duesberg supplied Rous virus-infected chick embryo cells. J

I

I

I

> a I

I

109

pM poly Figure

6. The

Formation

I I 545

I

218

U added

of Poly(U)-Poly(A)

Hybrids

Radioactive 3H-poly(U) (97 cpm/pM of uridylate) was added in increasing concentrations to 46 pM of nonradioactive poly(A), and the presence of RNase-resistant counts was determined as outlined in Experimental Procedures.

quence complexity. This could fact that the poly(A)sequences

be

due

either

to the

are different from the poly(A)+ sequences, and the sequences in the poly(A)RNA have a much lower total complexity than the poly(A)+ population, or to the fact that the poly(A)population contains the same sequences as the poly(A)+ RNA. Although it has been shown that poly(A)mRNA from sea urchin blastulae has sequences different from the poly(A)+ RNA of this stage (Nenier, Graham and Dubroff, 1974), there is no information on the complexity of the poly(A)RNA other than that it contains large amounts of histone mRNA (Lifton and Kedes, 1976). Second, the preponderance of poly(A)+ RNA in the two-cell zygote is found in the polyribosomal fraction (Slater et al., 1972; Wilt, 1973; Dolecki et al., 1977). 2% of the cellular RNA [poly(A)+ mRNA] should be sufficient to occupy fully all the ribosomes in the cell, yet only approximately 20% of the cellular ribosomes are in polyribosomes at the two cell stage (Humphreys, 1971), and the average size of the polyribosomes indicates some 4-9 ribosomes per mRNA. I therefore propose that during these early transitions from dormancy to active protein synthesis, very large mRNA molecules are severely underloaded with ribosomes, either because of small and restricted coding regions within the mRNA, or rather slow initiation of protein synthesis. This suggestion is amenable to experimental confirmation. Experimental

Procedures

Materials The following materials were obtained from the sources listed: ZH3-adenosine (20 Ci/mM), V-H-UDP (11 Ci/mM), V-H-dCTP (23 Ci/mM) and unlabeled UDP from Schwa&Mann; polynucleotide phosphorylase (type 15) from P-L Biochemicals; poly(U), poly(A)

Embryo Culture and RNA Extraction Embryos of Lytechinus pi&us (Pacific Biomarine) were cultured at concentrations of 1.2 x lo4 embryos per ml of sea water at 15°C and were labeled with nucleosides dissolved in sea water at l-40 &ml. When constant specific activity ATP pools were necessary, the 3H-adenosine was diluted by adding sea water to the cultures 10 min after isotope administration (Brandhorst and Humphreys, 1972). After labeling, embryos were washed several times in sterile 1.5 M dextrose, and the RNA was prepared by standard phenol-chloroform extraction methods after dispersion of cells in SDS-containing buffers (Hogan and Gross, 1972; Kung, 1974). All reagents used were autoclaved after addition of 0.1% diethyl pyrocarbonate, and all glassware was acid-washed. RNA preparations were always dissolved in 1 mM EDTA, heated at 65°C for 5 min, adjusted to contain either 2% K acetate (pH 5) or 0.2 M NaCI, and reprecipitated with 2 vol of ethanol at -20°C. This procedure was carried out 2 or 3 times. Sedimentation of RNA in aqueous or DMSO-sucrose gradients was carried out by conventional procedures (Kung, 1974). Isolation of Poly(A)+ RNA RNA was fractionated in oligo(dT)-cellulose in 0.5 M KCI. 0.01 M Tris buffer (pH 7.4), and bound material was eluted in 10 mM Tris buffer (pH 7.4) as previously described (Wilt, 1973). The temperature of adsorption and elution varied in different experiments, as described in the text. Poly(U)-Sepharose containing 5 As60 units of poly(U) per ml of Sepharose suspension was synthesized (Poonian, Schlabach and Weissbach, 1971). RNA in a concentration which never exceeded 50 A26,, units per 2 ml of poly(U)-Sepharose suspension was applied to the column in 0.36 M NaCI, 1 mM EDTA, 0.5% SDS, 1 mM Tris (pH 7.4), and the column was washed extensively with this same buffer. Bound RNA was eluted with formamide (deionized) water mixtures (O-90% formamide, v/v) containing 10 mM NaCI, 2 mM EDTA and 0.2% SDS. RNA was precipitated from fractions obtained from oligo(dT)-cellulose or poly(U)-Sepharose by adjusting the salt concentration to 0.2 M NaCl and adding 2 vol of ethanol. Several ethanol reprecipitations were necessary to free RNA from formamide contained in the buffers. Slight formamide contamination of an RNA sample may be detected by the absorbance ratios at 2401260 nm. The true absorbance at 260 nm of an RNA preparation containing traces of formamide (x) can be determined by application of the formula X=

14.5 Am - A,,, 13.925

This relationship was experimentally derived absorption spectra of pure RNA and formamide

from water

data on the mixtures.

Poly(A) Tract Analysis Poly(A) was obtained from RNA fractions by digestion with pancreatic (5 pg/ml) and Tl (10 units per ml) ribonuclease in 0.3 M NaCI, 5 mM Tris (pH 7.4), 0.001 M EDTA at 37°C for 30 min, followed by digestion with DNAase (20 pg/ml) for 30 min at 37°C after addition of Ms+~ to 5 mM. Digests were extracted twice with phenol-chloroform and ethanol-precipitated. Yeast RNA was added as a carrier when necessary. It is possible to collect as little as 4 pg of RNA from 10 ml of cold 67% ethanolic solutions by centrifugation at 100,000 x g for 30 min in polycarbonate centrifuge tubes.

Cell 660

Determination of the amount of poly(A) in nonradioactive RNA samples was determined by hybridization of samples to 3Hpoly(U). Radioactive poly(U) was synthesized as outlined by Bishop, Roshbash and Evans (1974a). The products, with a specific activity of 350 mCi/mM were purified on aqueous sucrose gradients to obtain material sedimenting faster than marker E. coli tRNA. After centrifugation, the 3H-poly(U) was precipitated with ethanol and dissolved in water. Hybridization of poly(U) to RNA was carried out by a modification of a previously published procedure (Wilt, 1973). There is some difference in the literature on the number of poly(U) strands bound per poly(A) tract (for example, see Slater et al., 1972; Bishop et al., 1974b), and I have encountered some variability in this respect. The method used is presented here in detail, which results in a stable hybrid of two strands of radioactive poly(U) per 1 strand of poly(A). RNA in water and 3H-poly(U) in water were mixed with concentrated buffer to obtain final concentrations of 0.3 M NaCI, 0.001 M EDTA, 0.01 M Tris buffer (pH 7.8). 200 ~1 reaction mixtures (this may be scaled up or down) were placed at 45°C for 30 min. 10 vol of 0.5 N NaCI, 0.002 M EDTA, 0.002 M Tris buffer (pH 7.8) containing 20 pglml of pancreatic ribonuclease were added, and the mixtures were incubated for 30 min at 37°C. Samples were then chilled on ice, and 1 A,,, unit of yeast tRNA and 0.3 A,,, units of poly(A) were added, after which cold TCA was added to attain a final concentration of lo%, and the mixture was collected on glass fiber filters within 15 min. A standard curve of mixtures of known amounts of poly(A) and 3H-poly(U) is shown in Figure 6. If a sufficient excess of poly(U) is added, a 2:l hybrid is formed. All experiments reported here were carried out with varying amounts of RNA to ensure that the poly(U) was in excess. Routinely 20,000 cpm of poly(U) were added to a standard reaction mixture, and blank values (no RNA) between 25 and 50 cpm were obtained. When sea urchin RNA is hybridized by the method described above, it results in twice the radioactivity as when the same RNA and poly(U) are used in the method used by Slater et al. (1972); this latter method results in a I:1 hybrid (D. W. Slater, personal communication). Application of this improved method to RNA of eggs and zygotes reconfirmed the doubling of poly(A) following fertilization. Electrophoresis of poly(A) obtained by nuclease digestion of total RNA was carried out with 10% or 12%, 0.7 x 10 cm cylindrical polyacrylamide gels in Loening’s E buffer (1968) containing 0.2% SDS. Gels were preelectrophoresed for 1 hr at 5 mA per gel and run at 7 mA for4-5 hr. Optical density scans at 260 nm of the gels were performed on a Gilford gel transport apparatus. The gel was mechanically. fractionated into 1 mm slices, and the slices were homogenized and eluted in 0.5 M NaCI, 0.002 M EDTA, 0.002 M Tris buffer (pH 7.8) for 18 hr at 37°C. Recovery of RNA from gel slices was routinely 290%. RNA was hyrolyzed in 0.2 N NaOH at 37°C for 18 hr and acidified with HC104. and adenosine and 2’-3’AMP were separated by chromatography on PEI-cellulose (Reyes, 1972). Calculation of molecular weight averages was by the methods of Vournakis et al. (1974). cDNA Preparation and Hybridization Reverse transcriptase was purified from Rous sarcoma virus (Wang and Duesberg, 1973). Poly(A)+ RNA prepared by poly(U)Sepharose chromatography was sedimented through DMSO-sucrose gradients, recovered from the gradient by ethanol precipitation and used as a template under the reaction conditions specified by Sullivan et al. (1973). The cDNA product was sometimes purified on hydroxylapatite (Sullivan et al., 1973) and always by alkaline sucrose density gradients (Harrison et al., 1972). cDNA prepared with a sea urchin RNA template gave a broad size distribution ranging from approximately 50 bases to 650 bases in length. Only fractions sedimenting faster than 250 bases were used, and the mean molecularsize was350 bases. cDNA prepared with rabbit Hb mRNA as a template had a sharp unimodal distribution in alkaline sucrose gradients corresponding to 470 nucleotides in length. cDNA was prepared using 3H-dCTP with a specific

activity of 23 CilmM. Assuming the product contained 20% dCMP, the calculated specific activity of the cDNA used here was between 4-5 CitmM. Hybridization of cDNA (1000 cpm) and RNA was carried out in 50 PI vol in plastic microcentrifuge tubes overlaid with mineral oil. Incubation was at 67°C (0.304 N Na+ ion). Reaction conditions and digestion with Sl nuclease were carried out precisely as outlined by McKnight and Schimke (1974). All calculations were carried out after converting experimental Rot values to those values equivalent too.12 M phosphate buffer (Britten and Smith, 1970). A best-fit plot of the data and calculation of Rotliz values of the plotted curve were carried out by the procedures described by Ryffel and McCarthy (1975). Acknowledgments I am very much indebted to Dr. Borek Janik, Dr. Lu-Hai Wang, Dr. Peter Duesberg, Dr. Jerry Lingrel, Dr. Richard Firtel, Dr. Brian McCarthy, Dr. Joan Egrie and Galvin Swift for their suggestions and gifts of valuable research materials. I appreciate the expert technical assistance of Mrs. Ellen E. Hyland. Research was supported by a grant from the NIH and a fellowship from the John Simon Guggenheim Memorial Foundation. Received

February

25, 1977;

revised

April

7, 1977

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