Synthesis and transport of various RNA species in developing embryos of Xenopus laevis

DEVELOPMENTAL

Synthesis

BIOLOGY

and Transport

K. SHIOKAWA, Department Kyushu

503-514

68,

Y. MISUMI,

(1979)

of Various RNA Species in Developing of Xenopus laevis Y. YASUDA, Y. NISHIO, K. YAMANA

S. KURATA,*

of Biology, Faculty of Science 33, and *Department of Agronomy, University, Fukuoka, 812 Japan; and TDepartment of Ophthalmology, Kagoshima University, Kagoshima, 890 Japan Received

March

2, 1978; accepted

in revised

form

September

Embryos

M. SAMEsnIMA,t

AND

Faculty of Agriculture Faculty of Medicine,

46,

7, 1978

Embryonic cells of Xenopus laevis were labeled for varying lengths of time, and their nuclear and cytoplasmic RNAs were analyzed, with the following results. (1) The synthesis of small nuclear RNAs (snRNAs) is detected from blastula stage on. (2) The initiation of 4 S and 5 S RNA syntheses occurs at blastula stage. However, while the former is transported into the cytoplasm immediately after its synthesis, the latter remains within the nucleus, until its transport starts later, concomitantly with that of 28 S rRNA. (3) As soon as “blastula” cells start to synthesize 40 S rRNA precursor at 5th hr of cultivation, 18 S rRNA is transported fist; the transport of 28 S rRNA begins 2 hr later. (4) On a per-cell basis, poly(A)-RNA is synthesized in blastula stage at a much higher rate than in the later stages. About one-third of the total blastula poly(A)-RNA, and about one-fifth in the case of tailbud cells, is transported quickly into the cytoplasm. Then, it appears that the RNAs which are synthesized at early embryonic stages are transported to the cytoplasm without delays, except for 5 S RNA and snRNAs. INTRODUCTION

During amphibian embryogenesis, the protein synthetic machinery and messages which are present in egg cytoplasm, and hence maternal in origin, have to be replaced eventually by new machinery and messageswhich are produced de nouo during embryogenesis. In order that new protein synthetic machinery and messages might be produced, the genes concerned must first be transcribed. It is reasonable, then, that most of the previous papers on embryonic RNA metabolism have dealt with mainly transcriptional activity of various kinds of genes during development. At present, we know fairly well when, where, and what kinds of RNAs are synthesized in the embryo in the course of development (Gurdon, 1974). However, it may be pointed out here that there is still little information currently available concerning when and how these newly transcribed RNA molecules are uti-

lized, or in other words, assembled into a functional protein synthetic machinery and set into operation. The present series of studies aims at the description of not only the transcription of embryonic RNAs, but also the process of transport and utilization or recruitment of the newly synthesized RNAs in the course of the early embryonic development. For this purpose, we have already started to explore methods of isolating a reasonably pure nuclear preparation from Xenopus neurula cells (Yasuda et al., 1977) and have recently studied some aspects of the transport of RNAs from the nucleus to the cytoplasm using the isolated neurula cells (Shiokawa et al., 1977b). In the present paper, we further extended our work along this line, and here much more attention was paid to clarifying RNA transport at blastula stage. Thus, nuclear and cytoplasmic RNAs from Xenopus blastulae, as well as from embryos of other stages, were obtained and analyzed, with a

503 OO12-1606/79/020503-12$02.00/O Copyright All rights

0 1979 by Academic Press, of reproduction in any form

Inc. reserved.

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DEVELOPMENTAL BIOLOGY

special reference to the time course of the accumulation of all the major RNA classes. The results obtained reveal that all the newly synthesized RNA species except 5 S RNA and snRNA are quickly transported into the cytoplasm without delay after their transcription and maturation. Parts of the present work have been presented at the Molecular Biology Meeting of Japan (Nishio et al., 1977), 8th International Congress of ISDB in Tokyo (Shiokawa et al., 1977a), and 11th Annual Meeting of Developmental Biologists of Japan (Shiokawa et al., 1978).

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used as the soluble cytoplasmic fraction, which contained only less than 1% of the total cellular DNA both in blastula and tailbud cells. It is possible to obtain purified nuclei from the centrifugal pellet (Yasuda et al., 1977). However, in the present experiments, the crude pellet was used as the nuclear fraction without any further purification, since the extensive purification of the nuclear pellet greatly reduced radioactive 40 S rRNA precursor as well as snRNAs, and furthermore, the pattern of nuclear RNAs obtained did not change greatly depending on the extent of nuclear purification. MATERIALS AND METHODS The nuclear fraction contained at most Preparation and isotopic labeling of em- 30% of the cytoplasmic materials as evibryonic cells. Embryos of Xenopus laevis denced by the presence of nonradioactive were obtained, dejellied, and then disso- rRNA and 4 S RNA. The nuclear pellets, ciated into isolated cells at the blastula fixed with glutaraldehyde and osmium te[stage 8 (Nieuwkoop and Faber, 1956)] and troxide, were examined under a light microtailbud (stage 23) stages. In some experi- scope. No intact cells were left in the numents, cells were obtained from the cleav- clear pellets of both blastula and tailbud age (stage 6), gastrula (stage ll), neural cells. Instead, yolk platelets, pigment granplate (stage 13), and neural fold (stage 17) ules, and other cytoplasmic structures were stages. Care was taken not to disrupt large observed in addition to nuclei. Electron miblastomeres, especially when cleavage and croscopic examination revealed that blasblastula stage embryos were cultured. The tula and tailbud nuclei were rather normal cells obtained were labeled in complete in their appearance and retained apparStearns’ solution at 21°C with either [8,3- ently intact nuclear envelopes (electron mi3H]adenosine (31 Ci/mmole), [5-3H]uridine crographs omitted). Although the quanti(25 Ci/mmole), [8-3H]guanosine (6 tative recovery of total DNA in the nuclear Ci/mmole), or [methyZ-3H]methionine (4.7 pellet alone does not necessarily imply the Ci/mmole), as has been previously de- preservation of intact nuclear organization scribed (Shiokawa.and Yamana, 1967). The (cf. Price et al., 1974), our electron microlabeled cells were washed once with fresh scopic examination, together with DNA reStearns’ solution by low-speed centrifugacovery, strongly suggests that our nuclei tion, and either used immediately for cel- are not greatly disrupted during the procelular fractionation or stored in a deep freeze dure. for later use. RNA extraction and poly(U)-Sepharose Fractionation of cells into nuclear and column chromatography. Cells and nuclear cytoplasmic fractions. Cells from 30-50 em- pellet were homogenized in 0.1 M sodium bryos were homogenized with a Dounce acetate buffer, pH 5.0, containing 0.5% SDS homogenizer in 0.5 ml of 0.25 M sucrose (sodium dodecyl sulfate) and 1 mg/ml of which contained 5 m&f MgCL, 1 mM bentonite, and then treated with phenol at spermine, and 5 mM N-ethylmaleimide a slightly elevated temperature (10°C) (Yasuda et al., 1977; Shiokawa et al., 1977). (Knowland, 1970). Soluble, cytoplasmic The homogenate was centrifuged for 5 min fraction was made up in 0.1 M sodium at 1800 rpm. The supernatant obtained was acetate (pH 5.0)-0.5% SDS-l mg/ml of

SHIOKAWA

ET AL.

RNA

Transport

bentonite, and then treated with phenol as above. All RNAs were precipitated twice with 0.1 A4 NaCl and 70% ethanol, and then fractionated into poly(A)-lacking, or poly(U)-Sepharose-void, RNA and poly(A)-containing RNA using poly(U)Sepharose 4B (Pharmacia) columns as described previously (Sagata et al., 1976; Shiokawa et al., 1977b). As has previously been suggested by Knowland (1970)) the phenol extraction at a slightly elevated temperature is efficient in deproteinization, and the contaminating protein content was significantly smaller by the present method than by the usual phenol method at 4°C or the chloroform-phenol method. The efficiency of the recovery of labeled nuclear RNAs by the present method was found to be comparable to ihat by the hot phenol method used previously (Shiokawa et al., 1977). The labeled poly(A)-RNA obtained by the present method amounted to around 5-10s of the total labeled RNA, depending on the length of the labeling. These values are similar to those obtained previously by the chloroform-phenol method (Sagata et al., 1976), and better than those by the hot phenol method (Shiokawa et al., 1977b). Gel electrophoresis of RNAs. For fractionation of poly(U)-Sepharose-void RNAs, composite-type 0.5% agarose-2.2% polyacrylamide or SDS-B% polyacrylamide gels were used as in previous studies (Shiokawa et al., 1977; Yamana and Shiokawa, 1975). Poly(A)-RNA was fractionated following a modified method of Staynov et al. (1972). Thus, poly(A)-RNA was first treated with 80% formamide solution at 60°C for 5 min and then subjected to electrophoresis on 3.8% polyacrylamide gels (0.45 X 10 cm) at 3 mA per tube for 3.5 hr at room temperature in 80% formamide solution, containing 20 m.M Tris, 2.5 m.M EDTA (ethylenediaminetetraacetic acid), 5 mM NaCl, and 20 mM boric acid. All gels were sliced and hydrolyzed in 3 N ammonia solution, and then radioactivity was counted in Triton X-toluene-DPO in

in Xenopus

a scintillation al., 1977b).

505

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spectrometer

(Shiokawa

et

RESULTS

Poly(A)-lacking RNAs, or Poly(U)-Sepharose-void RNAs Poly(U)-Sepharose-void RNAs isolated from [3H]uridine-labeled blastula cells were analyzed on agarose-polyacrylamide gels (Fig. 1). In the nuclear RNA labeled for 1 hr, so-called heterogeneous RNA, which distributed throughout rRNA region, small molecular weight RNA, migrating slower than 4S RNA, and 4S RNA were detected (Fig. 1A). The heterogeneous nuclear RNA species, quite actively synthesized in these cells, are assumed not to contain poly(A) sequences, since it was not retained on a poly(U)-Sepharose column. The occurrence of such nonpolyadenylated heterogeneous nuclear RNA has already been reported in other systems (Nemer et al., 1974; Levis and Penman, 1977). The small peak, which was specific to nuclei and therefore may be designated as snRNAs, migrated slower than 4 S RNA. In the cytoplasm, on the other hand, very little label was incorporated into heterogeneous RNA around the rRNA region (Fig. 1B). However, one large peak, the main component of which was 4 S RNA, appeared in the cytoplasm, indicating very rapid transport of this RNA (Fig. 1B). Labeling of blastula cells for another 2 hr did not change the labeling patterns of both nuclear and cytoplasmic poly(U)-Sepharose-void RNAs (data omitted). However, after 5 hr of labeling, a relatively large amount of 40 S rRNA precursor appeared for the first time in the nuclear fraction (Fig. 1C). The exclusively nuclear occurrence of 40 S rRNA precursor was also observed when nonradioactive RNA was analyzed at late gastrula and tailbud stages. The nuclear RNA contained very small peaks of 28 S and 18 S rRNAs, probably indicating the presence of rRNA shortly after maturation (Fig. 1C). At this time, a considerably large amount of 18 S rRNA

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DEVELOPMENTAL BIOLOGY

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FIG. 1. Gel electrophoretic profiles of the nuclear and cytoplasmic ‘RNAs of blastula cells. The isolated cells from 30 blastulae were labeled with 100 pCi of [3H]uridine in 2 ml of the medium for 1 and 5 hr. They were then homogenized and fractionated into the nuclear and soluble cytoplasmic fractions. RNA was extracted from each fraction, and then applied onto the column of poly(U)-Sepharose 4B to eliminate poly(A)-RNA. A portion of the RNA obtained was fractionated on 0.5% agarose-2.2% polyacrylamide gel. (A) I-hr-labeled nuclear RNA, (B) l-hr-labeled cytoplasmic RNA, (C) 5-hr-labeled nuclear RNA, and (D) 5-hr-labeled cytoplasmic RNA.

but not of 28 S rRNA was found in the cytoplasm (Fig. 1D). It has long been observed that rRNA synthesis starts by the end of 5 hr of culture in an in vitro culture of blastula cells (Shiokawa and Yamana, 1967). However, the present experiments revealed that only 18 S rRNA had been transported to the cytoplasm. Electrophoresis of the RNA from blastula cells labeled for 6 hr with [methyl3H]methionine showed a quite discrete peak of 40 S rRNA precursor in the nuclear RNA (data not shown). In the cytoplasm, not only 18 S rRNA but also 28 S rRNA appeared, although the relative amount of the latter was much smaller. This delayed appearance of larger rRNA component is consistent with the previous observation by Shiokawa et al. (1977). The clear difference in the labeling profiles between nuclear and cytoplasmic RNAs implies that the two fractions obtained are in fact distinct from each other. When nuclear non-poly(A)-RNAs from [3H]uridine-labeled tailbud cells were ana-

lyzed on the same type of gels, an active labeling of 40 S rRNA precursor was found after the short labeling (1 hr) (Fig. 2A). In addition, several peaks of radioactivity appeared between the 40 S and 18 S rRNA regions, suggesting the existence of nonpoly(A)-heterogeneous RNA and some intermediate molecules of rRNA processing. The nuclear RNA also contained a small discrete peak of snRNA and 4 S RNA. In the cytoplasm labeled for 1 hr, discrete peaks of only 4 S RNA and 18 S rRNA were observed (Fig. 2B). The profiles of tailbud nuclear RNA did not change greatly as the labeling time became longer (data omitted). The time course of the change in the amounts of 18 S and 28 S rRNAs was the same as that described previously (Shiokawa et al., 1977): At 3 hr after labeling a sharp peak of 28 S rRNA appeared in addition to that of 18 S rRNA, and by 5th hr, the amount of cytoplasmic 28 S rRNA increased greatly and the ratio of 28 S rRNA:18 S rRNA became very close to the steady-state value (2:l).

SHIOKAWA

RNA

ET AL.

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2. Gel electrophoretic profiles of the nuclear and cytoplasmic RNAs of tailbud embryo cells. Cells from 30 tailbud embryos were labeled with 200 @i of [3H]uridine in 2 ml of the medium for 1 hr. They were homogenized and then fractionated into the nuclear and cytoplasmic fractions. RNA was extracted, and poly(U)-Sepharose-void RNAs were obtained and electrophoresed on 0.5% agarose-2.2% polyacrylamide gels as in Fig. 1. (A) Nuclear RNA and (B) cytoplasmic RNA. FIG.

Nuclear and cytoplasmic distribution of small molecular weight RNAs were then studied in more detail on 8% gels. When blastula nuclear RNA labeled with [3H]uridine for 3 hr was electrophoresed, five discrete radioactivity peaks appeared in the low molecular weight RNA region (Fig. 3A). The small RNAs other than 4 S RNA may correspond to the small RNA which migrated on agarose-polyacrylamide gel slower than 4 S RNA (cf. Fig. 1A). In Fig. 3A, two fast-moving peaks were identified, from right to left, as 4 S RNA and 5 S RNA, respectively, based on their relative abundance, molecular weight, and labeling with and [methyZ-3H]methionine (Yamana Shiokawa, 1975). The three slower components, designated in Fig. 3A from right to left as a, b and c, respectively, so far have not been described as nuclear components in amphibian embryonic cells, although similar components were shown in the

Transport

in Xenopus

Embryos

507

RNA profile of whole blastulae (Miller, 1974). According to the procedure of Loening (1969), their molecular weights were estimated approximately as 5.2, 6.0, and 7.3 x lo*, respectively. Further, these RNA species were labeled with [methyl-3H]methionine. Thus, the three components a, b, and c can tentatively be identified as U1, Up, and U3 RNAs, respectively, which have originally been described by Busch and his co-workers (Ro-Choi and Busch, 1974). Between 4 S and 5 S RNAs, there seems to be another component as indicated by the shoulder of the 4 S RNA peak. This may be 4.5 S tRNA precursor (L&m, 19771, although there is no direct evidence for it at present. In a sharp contrast to these, there was only one radioactivity peak of 4 S RNA in the blastula cytoplasmic fraction (Fig. 3B; see also Fig. 1B). The labeling pattern of nuclear and cytoplasmic small RNAs remained unchanged even after 5 hr of labeling. The present finding that not only 4 S but 5 S RNA is synthesized in blastula cells is consistent with that of Miller (1974). However, we now report, for the first time, the absence of newly synthesized 5 S RNA in the cytoplasm even after 5 hr of labeling. The profile of the blastula nuclear small RNAs remained unchanged up to 6 hr of labeling, except that the 4.5 S shoulder became much smaller (Fig. 3C). However, in the cytoplasm, not only 4 S RNA but also 5 S RNA was found to occur at 6 hr (Fig. 3D). The cytoplasm contained about 70% of the total cellular 4 S RNA, whereas cytoplasmic 5 S RNA amounted to about 40% of the total, indicating slower transport of 5 S RNA as compared with 4 S RNA. The pattern of 3-hi-labeled tailbud cell nuclear small RNAs was essentially the same as those of the blastula cells (cf. Fig. 3A). However, in the cytoplasm, not only 4 S RNA but also 5 S RNA was observed. This profile is essentially the same as that of blastula cells labeled for 6 hr (Figs. 3C and D), and are not reproduced here. When

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DEVELOPMENTAL BIOLOGY

VOLUME 68,1979

5s4 0

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FIG. 3. Gel electrophoretic profiles of small molecular weight RNAs from blastula cells. Cells from 30 blastulas were labeled for 4 or 6 hr with 100 &i of [3H]uridine in 2 ml of the medium, and poly(U)-Sepharosevoid was obtained from the nuclear and cytoplasmic fractions as in Fig. 1. RNA was electrophoresed on SDS-% polyacrylamide gel. For determination of the molecular weights of a, b, and c components in Fig. 3A, 4s RNA (MW, 27,000) and 5 S RNA (MW, 39,060) were used as standards, and the approximate molecular weight calculated was included in the profile. (A) 4-hr-labeled nuclear RNA, (B) 4-hr-labeled cytoplasmic RNA, (C) 6hr-labeled nuclear RNA, and (D) 6-hr-labeled cytoplasmic RNA.

the duration of the labeling was shortened to 1 hr, neither 28 S rRNA nor 5 S RNA appeared in the cytoplasm.

label (Sagata et al., 1978), these changes in the label incorporation can be assumed to reflect the change in the net poly(A)-RNA synthetic activity, since [3H]adenosine apPoly(A)-RNA pears to be freely permeable to the isolated Synthesis of poly(A)-RNA was studied cells, and the amount of acid-soluble ATP by labeling cells obtained from embryos at is reduced only twofold between blastula different stagesfor 2 hr each with [3H]aden- and the later stages (Lovtrup-Rein et al., osine. The poly(A)-RNA obtained migrated 1974). on formamide gels quite heterogeneously Figure 4 shows a set of formamide-gel throughout the gel, with the main compo- electrophoretic profiles of the blastula nunent at around the 40 S RNA region. In clear and cytoplasmic poly(A)-RNAs, agreement with our previous results (Sa- which were labeled with [3H]uridine for 2 gata et al., 1978), these RNAs contain hr. Switching the label from [3H]uridine to poly(A) sequences about 150 nucleotides [3H]adenosine did not alter the profile of long. On a per-cell basis, the amount of the poly(A)-RNA. The nudlear poly(A)-RNA label incorporation was almost negligible at distributed quite heterogeneously here cleavage stage, but suddenly attained the again in the rRNA region, with the main highest level at blastula stage and then was component between the 40 S and 28 S sharply reduced about five-fold in the later regions. The size of the main component stages (gastrula, early and mid neurula, and was estimated as about 2 x lo6 daltons. In tailbud stages). Consistent to the previous contrast, cytoplasmic poly(A)-RNA distribmeasurement made with [3H]uridine as a uted between the 18 S and 4 S regions, and

SHIOKAWA

ET AL.

RNA

Transport

I .5

I .o

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:

2 x 6 ,”

0 1.5

I.0

0.5

0

0

20

40 Distance

60 moved

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FIG. 4. Gel electrophoretic profiles of the nuclear and cytoplasmic poly(A)-RNA of blastula cells. Cells from 30 blastulas were labeled with 100 PCi of [3H]uridine in 2 ml of the medium for 2 hr. They were then homogenized and fractionated into the nuclear and cytoplasmic fractions. RNA was extracted from each fraction, and then poly(A)-RNA was collected using poly(U)-Sepharose 4B columns as in Fig. 1. A portion of the RNA obtained was electrophoresed on 3.5% polyacrylamide gels in the presence of 80% formamide. (A) Nuclear poly(A)-RNA and (B) cytoplasmic poly(A)-RNA.

the size of its main component was about 2 lo5 daltons on the basis of its mobility. These poly(A)-RNAs may not be of mitochondrial origin, since even high dose of ethidium bromide (15 pg/ml), enough to completely inhibit mitochondrial transcription (Zylber et al., 1969; Craig and Piatigorsky, 1971), did not alter the amount of both nuclear and cytoplasmic poly(A)-RNA isolated. The overall profiles of both nuclear and cytoplasmic poly(A)-RNAs were indifferent to varying lengths of labeling (l-6 hr). Nuclear poly(A)-RNA from tailbud cells, labeled for 1, 3, and 5 hr, showed the size distribution which was essentially similar to that from blastula cells (data omitted). On the other hand, the cytoplasmic X

in Xenopus

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509

poly(A)-RNA from tailbud cells was found to be slightly larger in size and a little more heterogeneous as compared with that of blastula cells. This suggests that the poly(A)-RNA molecules synthesized by tailbud cells are slightly larger in size, and more complex in their composition. On sucrose density gradients, both blastula and tailbud nuclear poly(A)-RNA distributed mainly between the 28 S and 18 S regions as described previously (Sagata et al., 1976), while the cytoplasmic RNA appeared between the 18 S and 4 S RNA, with the main component at around the 10 S region. It appears, then, that nuclear poly(A)-RNA has some secondary structure, thereby giving rise to an apparently larger size distribution on the formamide gels than on the sucrose density gradients (cf. Kung, 1974). The size of the present nuclear poly(A)RNA is similar to those of neurula cells (Shiokawa et al., 1977a), kidney cells (Misumi et al., 1977), and liver cells (Ryffel, 1976) of Xenopus laevis. In contrast, the cytoplasmic poly(A)-RNA is apparently smaller than previously reported (Ryffel, 1976; Shiokawa et al., 1977), although comparable to messenger RNAs of a relatively small molecular weight proteins such as globin (Battaglia and Melli, 1977) and histone (Jacob et al., 1976; Levenson and Marcu, 1976) of Xenopus laevis. The accumulation of label was measured for nuclear and soluble cytoplasmic poly(A)-RNAs for blastula and tailbud cells during 6 hr of labeling (Figs. 5A and B). It is clearly shown that both cytoplasmic and nuclear poly(A)-RNAs of blastula cells accumulate exponentially throughout the period examined. The amount of the cytoplasmic RNA was about one-half of that of the nuclear RNA at all the points determined. In contrast, the accumulation of tailbud nuclear poly(A)-RNA reached a plateau after about 4 hr of labeling, although that of cytoplasmic poly(A)-RNA proceeded almost linearly for 6 hr. The

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5. Kinetics of the accumulation of poly(A)RNA in the nuclear and cytoplasmic fractions in blastula and tailbud cells. Data were collected from the experiments of the type shown in Fig. 4. (A) Blastula and (B) tailbud cells. Closed and open circles are for nuclear and cytoplasmic poly(A)-RNA, respectively.

amount of the cytoplasmic RNA accounted for one-seventh up to one-fifth of the total nuclear poly(A)-RNA, depending on the length of labeling. Thus, this is another support to the generally accepted view that only a part of the nuclear poly(A)-RNA is transported to the cytoplasm (Shiokawa and Pogo, 1974; Wu and Wilt, 1974).

Transport

of Various RNAs

Based on the results shown above, kinetics of the appearance of various RNA species in blastula cytoplasm may be summarized as in Fig. 6. The results with tailbud cells are also reproduced in an inset for a comparison, although they are largely as has been previously described (Shiokawa et al., 1977). In blastula cells, 4 S RNA, and poly(A)-RNA appear in the cytoplasm shortly after their labeling. At this time, 5 S RNA is transcribed but remains untransported within the nucleus. Only after 4-5 hr of labeling does the synthesis of 40 S rRNA

3

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6. Kinetics of the appearance of various RNA species in the cytoplasmic fraction in blastula and tailbud cells. Data were collected from the experiments of the type shown in Figs. l-4. Squares, filled circles, open circles, open triangles, and filled triangles are for 4 S RNA, poly(A)-RNA, 18 S rRNA, 28 S rRNA, and 5 S RNA, respectively. The time at which the synthesis of 40 S rRNA precursor starts is indicated by an arrow. In the inset is shown the kinetics of the appearance of various RNAs in the cytoplasmic fraction of tailbud embryo cells. FIG.

begin in the nuclei. This is the time when the sibling whole embryos reach the gastrula stage and commence rRNA synthesis. Immediately after that, 18 S rRNA is transported to the cytoplasm. After about 6 hr of cultivation, 28 S rRNA and 5 S RNA start to appear in the cytoplasm simultaneously. In tailbud cells, quite actively synthesizing all the RNAs, 4 S RNA, 18 S rRNA and poly(A)-RNA are all transported to the cytoplasm from the very beginning of labeling. After about 2 hr of labeling, 28 S rRNA and 5 S RNA emerge in the cytoplasm. Then, by about 5 hr, the relative amounts of 28 S and 18 S rRNAs reach a ratio of

SHIOKAWA

2:1, indicating molar amount

ET AL.

the accumulation in the cytoplasm.

RNA

of equi-

DISCUSSION

In the present

study,

embryonic cells of into the nuclear and soluble cytoplasmic fractions. Concerning the purity of these fractions, it could be argued that there might be some substantial contamination of the nuclear fraction with cytoplasmic materials, since about 30% of the total cellular nonradioactive rRNA occurred in the nuclear fraction. Contamination of the nuclei with a small amount of ribosomes attached to the outer nuclear membrane is a rather general phenomenon (Whittle et al., 1968; Smith et al., 1969). In the present experiments, however, the contamination seems to be relatively large, This may be due to the fact that the nuclear fraction used here was not the purified one and contained many cytoplasmic particulate materials such as yolk platelets, pigment granules, and others. However, it should be also pointed out that both 40 S rRNA precursor and snRNAs, whether radioactive or nonradioactive, were always confined to the nuclear fraction, with the majority of radioactive 4 S RNA and 18 S and 28 S rRNAs in the cytoplasmic fraction. In this connection, electron microscopy revealed that most nuclei remained intact. Therefore, it may be safe to conclude here that the present procedure provides a satisfactory method for studying at least the transport of newly synthesized RNAs into the cytoplasm. In the present study, snRNAs have been shown to be synthesized as early as in blastula cells. In A6 cells of adult Xenopus kidney, the synthesis of several snRNAs has previously been detected by Rein and Penman (1969). Furthermore, in studying metabolism of 5 S RNA, Miller (1974) obtained a similar pattern of small molecular weight RNAs, although no attention was paid to their nuclear localization. In the present work, these snRNAs have tenta-

Xenopus laevis were separated

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tively been identified as U1, U2, and U3 RNAs (Ro-Choi and Busch, 1974). The observation that the synthesis of snRNAs begins prior to gastrulation is interesting, since these RNAs have been suggested to play some roles in the regulation of gene expression (Goldstein, 1976; Rao et al., 1977). The time course of the appearance of rRNA, tRNA, and poly(A)-RNA in the tailbud cell cytoplasm was mostly in accord with our previous results obtained with neurula cells (Shiokawa et al., 1977). However, immediate transport of both 4 S RNA and poly(A)-RNA after their synthesis in blastula cells may be of special interest. It has previously been claimed in cultured cells that nucleolus is essential for the transport of these RNAs (Harris et al., 1969; Desk, 1973). The present results, however, do not support this, since blastula cells lack typical nucleoli (Nakahashi and Yamana, 1976). Another interesting point is that 5 S RNA remained confined within the nucleus after its synthesis at the blastula stage, until its transport commenced together with that of 28 S rRNA. In this connection, Miller (1974) showed that 5 S RNA starts to be synthesized at blastula stage, but does not replace 5 S RNA present in maternal ribosomes. However, our paper is the first one which discriminates between nuclear and cytoplasmic 5 S RNA and shows that transport of newly synthesized blastula 5 S RNA starts only when the embryo has reached the gastrula stage. This conclusion appears to be somewhat inconsistent with the previous reports suggesting the transport of 5 S RNA without concomitant transport of 28 S rRNA in early oocytes (Mairy and Denis, 1971) and in anucleolate mutants of Xenopus (Miller, 1973). In the systems other than Xenopus embryos, it is well known that 5 S RNA first becomes integrated with 28 S rRNA into a 60 S ribosomal subparticle (Warner and Soeiro, 1967; Edstrom and Lonn, 1976). Thus, it

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may be assumed here that 5 S RNA and 28 S rRNA comigrate into the cytoplasm in the form of the 60 S RNP particle. Concerning poly(A)-RNA transport, it has become clear that at the blastula stage newly synthesized poly(A)-RNA may be transported rapidly into the cytoplasm. In the nuclear fraction, we found a relatively large amount of heterogeneous nuclear RNA which may lack poly(A) sequences. However, in the cytoplasm, there was little such labeled RNA which could be assumed to be poly(A)-lacking mRNA, although Nemer et al. (1974) claimed that a large amount of presumptive mRNA lacks poly(A) sequences in sea urchin pregastrula embryos. At this point, the possibility may be ruled out that the cytoplasmic poly(A)-RNA detected here reflects the so-called cytoplasmic polyadenylation of the previously produced mRNA stock (Slater et al., 1973; Wilt, 1973). This is because the label used was not [3H]adenosine but [3H]uridine. Finally, relatively large differences have been observed between blastula and tailbud cells in the kinetics of poly(A)-RNA accumulation in the nuclei: While the accumulation curve shows a plateau in tailbud nuclei, it continues to increase for as long as 6 hr in blastula nuclei. It is now well known that nuclear poly(A)-RNA, as well as heterogeneous nuclear RNA, is metabolically unstable (Wu and Wilt, 1974; Levis and Penman, 1977). Thus, the plateau observed can be explained as a metabolic turnover of the RNA within the nucleus. The almost linear accumulation of poly(A)-RNA in tailbud cell cytoplasm may indicate the stable nature of this RNA (cf. Murphy and Attardi, 1973). The cbntinuous increase in the blastula nuclear poly(A) -RNA, on the other hand, may need special explanations. One of them is a more rapidly increasing number of nuclei in blastulas which commence poly(A)-RNA synthesis at this stage. Thus, our kinetic study of blastula nuclear poly(A)-RNA accumulation is also in favor

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