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Flow of informational RNA from cytoplasmic poly(A)-containing particles to polyribosomes in Artemia salina cysts at early stages of development
DEVELOPMENTAL
BIOLOGY
59, 49-61
(1977)
Flow of Informational RNA from Cytoplasmic Poly(A)-Containing Particles to Polyribosomes in Artemia salina Cysts at Early Stages of Development PAOLA
PIERANDREI
National
Research Received
AMALDI, Council November
LUCIANO (C.N.R.J,
2.1976:
FELICETTI,
Laboratory nrcrpted
of Cell
in rwwpd
AND
NADIA
Biology,
00196
form
March
CAMPIONI Rome,
Italy
28, 1977
When Artemia salina cysts are incubated at 28°C in artificial seawater, the pool of cytoplasmic mRNA as measured by the [“H]poly(U) hybridization technique is depleted, and a parallel increase in the polysome bound mRNA is observed. The shift of informational RNA from subribosomal particles to polyribosomes occurs without a significant increase in the amount of poly(A)-containing RNA per embryo, at least during the first hours of development. In order to ascertain whether resumption of development is due mainly to utilization of preformed mRNA, the polypeptides synthesized in a wheat germ lysate under the direction of cyst cytoplasmic mRNA were compared with those obtained using polysomal mRNA from developed embryos (1 and 4 hr). The complex pattern of proteins synthesized in vitro was resolved into 70-80 radioactive spots by two-dimensional gel electrophoresis and was shown to be almost identical in unincubated and incubated cysts, except for a few novel spots. Our results do not exclude that early transcription of a few mRNA species could play a role in the activation of metabolic processes which occur when development is resumed. INTRODUCTION
et al., 1975; Sierra et al., 1976). In a previous paper (Felicetti et al., 19751, we have shown that poly(A)-containing particles with sedimentation values ranging from 4 to 80s are present in the cyst postmitochondrial supernatant. In addition, poly(A)-containing RNA can be fractionated by sucrose density gradient centrifugation, and RNA fractions of different S values can be translated in a wheat germ lysate, resulting in the formation of a characteristic pattern of proteins (BOOO-40,000 M. W.) The question arises as to whether the activation of protein synthesis in the early stages of development is mainly due to utilization of cytoplasmic preformed mRNA or whether an early transcriptional event is also required to cope with the growing needs of developing embryos. No direct evidence of RNA synthesis in early development can be achieved due to the fact that the inner cuticle layer of the cysts is extremely impermeable to amino
Artemia salina cysts contain primarily dessiccated embryos at the gastrula stage, and, under this form, they can survive for long periods of time in unfavorable environments. In the extracts of dry cysts or cysts hydrated in seawater at O”C, the level of protein synthesis is very low, the polyribosomes are almost absent, and the ribosomal population is almost entirely composed of monosomes. If the cysts are hydrated in seawater and incubated at 3O”C, relatively few polysomes are detected within 5 min (Finamore and Clegg, 1969). The temperature-dependent activation of protein synthesis in the early stages of development can be exploited as a model system to study regulatory mechanisms at the translational level, since preformed mRNA not associated with ribosomes has been detected in subcellular fractions of the cyst homogenate (Nilsson and Hultin, 1974; Grosfeld and Littauer, 1975; Felicetti 49 Copyright All
rights
0 1977 by Academic of reproduction in
Press, any form
Inc. reserved.
ISSN
0012-1606
50
DEVELOPMENTAL
BIOLOGY
acids, nucleic acid precursors, and antibiotics (Finamore and Clegg, 1969). We have tried to circumvent the failure of in vivo labeling with two experimental approaches: (a) measurement of the amount of poly(A)-containing RNA extracted from the postmitochondrial supernatant of undeveloped and developed cysts; (b) comparison of translational products at early stages of development with polypeptides synthesized in a wheat germ lysate by preformed mRNA stored in the cysts. MATERIALS
AND
METHODS
13HlLysine (50 Ci/mmole) was obtained from New England Nuclear, and [35Slmethionine (196 Ci/mmole) was from Amersham; [3H]poly(U) (61.9 $X/~mole P) was from Miles; ATP, GTP, CTP, creatine phosphate, creatine phosphokinase, and dithiothreitol (DTT) were purchased from Calbiochemicals; oligo(dT)-cellulose T, was from Collaborative Research; sodium dodecyl sulfate (SDS), acrylamide, N,N’-methylenebisacrylamide, and ammonium persulfate were from Serva; ampholines (pH 5-7 and pH 3.5-10) were from LKB; and pancreatic RNAse was from Worthington. Fractionation of the Postmitochondrial Supernatant from Undeveloped and Developed Embryos Artemia salina cysts (7.5 g) were incubated in 2 liter of artificial seawater (Hultin and Morris, 1968) at 28°C for different lengths of time (1, 5, and 8 hr). The postmitochondrial supernatant was prepared as previously described (Felicetti et al., 1975) and was treated with 1% deoxycholate and 1% Brij 58 for 30 min at 0°C. The detergent-treated supernatant, 1.5 ml, was layered on top of 16.5-ml 15-30% linear sucrose gradients in TNM buffer (30 mM Tris-HCl, pH 7.4, 100 mM NaCl, and 10 mM MgCl,) and was centrifuged at 23,000 rpm for 14 hr in a SW 27.1 Spinco rotor at 4°C. The A,,, was automatically recorded by pumping the gradients from
VOLUME
59, 1977
the bottom through a 0.2-cm flow cell of a Gilford spectrophotometer. Fractions of 1.5 ml were collected from three identical sucrose gradients. RNA Extraction ization
and [3HlPoly(U)
Hybrid-
RNA was extracted by the phenol-sodium dodecylsulfate procedure (pH 9) as previously described (Felicetti et al., 1975). The [3Hlpoly(U)-hybridization test was performed according to Rosbash and Ford (1974). Polyribosome Preparation from Undeveloped and Developed Embryos and CellFree Assay in the Presence of Heterologous Supernatant The postmitochondrial supernatant derived from unincubated cysts or embryos incubated at 28°C for 10, 20, 30, and 60 min (15 g) was treated with 1% deoxycholate and 1% Brij 58 as before. Ten milliliters of detergent-treated supernatant were layered on a discontinuous sucrose gradient consisting of 1 ml each of 2 M and 1 M sucrose in TNM buffer and was centrifuged at 100,OOOg for 14 hr in a 50 Ti Spinco rotor at 4°C. The polyribosome pellets were resuspended in TKM buffer (50 mM Tris-HCl, pH 7.4, 25 mM KCl, and 5 mM MgCl,) and were immediately used for the cell-free assay. The incubation mixture contained in a final volume of 250 ~1: 50 mM Tris-HCl, pH 7.4, 100 mM KCl, 3 mM MgCl*, 10 mM dithiothreitol, 2 mM ATP, 0.5 mM GTP, 2 mM creatine phosphate, 2 pg of creatine phosphokinase, a mixture of essential amino acids (each at 40 mM, except lysine), 40 +i of 13Hllysine, 10.5 A,,, unit of polysomes, and 100 pg of the S 150 fraction from young rat brain (Amaldi et al., 1973). Incubation was at 37°C for 60 min. Aliquots, 5 ~1, were hydrolyzed with 0.2 ml of 0.1 N KOH (20 min at 37”C), precipitated with 1 ml of 10% trichloroacetic acid (TCA), collected on Whatman GF/C filters, and counted in 5 ml of butyl-
AMALDI,
FELICETTI,
AND
CAMPIONI
Flow of Informational
RNA
51
with 1 vol of TKM buffer, centrifuged at 100,OOOgfor 2 hr, treated with RNAase as before, and lyophilized. Acrylamide Gel Electrophoresis of the (I, Slab gel electrophoresis. The samples Polypeptides Synthesized by Embryo were dissolved in 50 ~1 of solution containPolysomes during Cell-Free Incubation ing 6.7% SDS, 16.7% P-mercaptoethanol, Each incubation mixture was diluted up 16.7% glycerol, and trace amounts of pyronin G. The samples were heated at 100°C to 1 ml with TKM buffer (20 mM Trisfor 1 min and were loaded on a 1.5-mmHCl, pH 7.4, 80 n-&f KCl, 3 m&I MgCl,) and was centrifuged at 100,OOOgfor 2 hr in thick slab gel containing a 7-20% acrylamorder to remove the ribosomes. The super- ide gradient. Electrophoresis was pernatant was treated with 50 pg/ml of pan- formed at 150 V, and the run was stopped creatic ribonuclease for 20 min at 37”C, when pyronin G reached the end of the gel. and labeled proteins were precipitated Gel and running buffers were prepared acwith 5% trichloroacetic acid. Denaturation cording to Laemmli (1970). (ii) Two-dimensional gel electrophoreof labeled samples, gel preparation, gel fractionation by a Savant auto gel divider, sis. The lyophilized samples were suband counting were as described by Jacobs- jected to isoelectrofocusing, followed by Lorena et al. (1972). SDS-slab gel electrophoresis as described by O’Farrtil (1975). First-dimension 2.5Oligo(dT) Cellulose Chromatography of mm gels were loaded on 1.5-mm-thick slab RNA gels containing a 7-20% acrylamide gradient and were sealed with agarose. RNA extracted from the postmitochonThe unequilibrated first-dimension gels drial fraction of unincubated cysts and were prerun for 20 min at 20 mA with high from polysomes of embryos incubated 1 SDS buffer, and then regular running and 4 hr was separated into poly(A)+ and poly(A) - fractions by oligo(dT)-cellulose buffer was applied as described by chromatography as previously described O’Farrel (1975). Power was turned off (Felicetti et al. 1975). when the bromophenol blue front reached the end of the gel. The slabs were stained Translation of Poly(A)-Containing RNA with Coomassie blue, destained, dried unFractions from Undeveloped and Develder vacuum, and exposed to Kodak Kodioped Embryos in a Wheat Germ Lysate rex X-ray films. A wheat germ lysate was prepared as RESULTS described by Roberts and Paterson (1973). The incubation mixture, in a final volume Study of the Distribution of Poly(A)-Conof 50 ~1, contained: ‘0.8A,,, units of lysate, taining RNA in Undeveloped and De25 mM Hepes-HCl, pH 7.4, 3 mM Mgveloped Embryos by the [3H]Poly(U)acetate, 64 mM KCl, 1.4 mM DTT, 1 mM Hybridization Technique ATP, 0.1 mM GTP, 0.6 n-&I CTP, 10 mM creatine phosphate, 8 Fg of creatine phosThe postmitochondrial supernatant dephokinase, each of 19 nonradioactive rived from unincubated cysts or cysts incuamino acids (each at 40 fl, except methibated at 28°C for different lengths of time onine) and 4.3 &i of [35S]methionine. In(1, 5, and 8 hr) was treated with detergents and was fractionated on sucrose gradients cubation was at 27°C for 60 min. as described in Materials and Methods. Electrophoretic Analysis of Translational The amount of RNA extracted from the Products in a Wheat Germ Lysate polysomal pellet, monosomes, and subriThe incubation mixtures were diluted bosomal particles and its ability to hybridPBD toluene (6 g/liter) scintillation counter.
in a Beckman
52
DEVELOPMENTAL
VOLUME
BIOLOGY
ize with [3H]poly(U) was determined as shown in Table 1. At time 0, most of the RNA is extracted from the monomers (72%), with only a small amount from the polysomes (5.5%). When cysts are incubated, the amount of monomers progressively decreases, while a parallel increase in the amount of polysomes is observed. There is no significant variation in the amount of RNA extracted from different developmental stages, suggesting that synthesis of ribosomal or transfer RNA does not occur to a measurable extent. As would be expected, the variation in the amount of poly(A)-containing RNA free in the cytoplasm or bound to polysomes reflects the process of activation of protein synthesis in the embryos: There is a decrease in the mRNA present in the subribosomal particles and a parallel increase in that bound to the polysome fraction. The amount of poly(A)-containing RNA bound to monosomes does not change significantly: The fluctuations observed at different times of incubation could be due either to polysome degradation during
fractionation of the extracts or to defective separation on sucrose gradients of the monosomes from the lighter ribosomal aggregates. If one calculates the counts per minute of [:lHlpoly(U) hybridized per A,,,, unit of total RNA extracted, there is no significant change in the specific activity of the RNA prepared from unincubated cysts or from embryos incubated for different lengths of time. This result suggests that, at least in the early stages of development, the average amount of poly(A)-containing RNA per embryo is constant, and a peculiar feature of this system seems to lie in the transfer of mRNA from the cytoplasmic particles to the polysomes. The results of Table 1 concerning the variation in the absorbance and in the [“H]poly(U)-hybridization capacity of different RNA fractions in the course of development can be expressed as a percentage of the total and can be plotted against hours of incubation (Fig. 1). The plot gives a clear picture of the modifications induced in these parameters by incubation of cysts in seawater.
TABLE TOTAL
AMOUNT
FRACTIONS
Gradient
AND
[3H]Po~~(U)
HYBRIDIZATION
OF POSTMITOCHONDRIAL
SUPERNATANT
fraction
RNA
59, 1977
1 CAPACITY FROM
OF RNA
UNDEVELOPED
EXTRACTED AND
extracted
FROM
DEVELOPED
(3H)Poly(U)
(A,,,, unit) 0 hr Polysomes
(pellet)
Monosomes
(I-4)
Subribosomal (5-12) Total
particles
6.4 (5.54) 83.15 (72.07) 25.83 (22.4) 115.38 (100)
1 hr 12.9 (12.17) 66.6 (62.83) 26.5 (25.0) 106.0 (100)
5 hr 28.2 (25.38) 62.47 (56.23) 20.43 (18.39) 111.1 (100)
8 hr
0 hr
35.2 (35.26) 48.3 (48.39) 16.32 (16.35) 99.82 (100)
4,290 (7.54) 5,380 (9.46) 47,210 (83) 56,880 (100)
GRADIENT EMBRYOS”
hybridized (cpm)
1 hr
5 hr
8 hr
5,750 (11.44) 8,500 (16.92) 36,000 (71.64) 50,250 (100)
12,680 (24.4) 10,800 (20.8) 28,460 (54.8) 51,940 (100)
17,340 (35.16) 6,620 (13.42) 25,360 (51.42) 49,320 (100)
0 The detergent-treated postmitochondrial supernatant was fractionated on sucrose gradients as described in Materials and Methods. The polyribosomes sediment to the bottom of the tubes. The monosome peak (fractions l-4) is clearly separated from subribosomal particles (fractions 5-12) as shown in Fig. 2. The RNA was extracted from the polysomal pellets and gradient fractions by the SDS-phenol procedure and was resuspended in 100 ~1 of sterile water, and aliquots (20 ~1 for polysome pellets, 40 ~1 for gradient fractions) were hybridized with 15,000 cpm of [3H]poly(U) (specific activity: 1.5 x lo5 cpm/pg) as described in Materials and Methods. The values in the table are the average values calculated from two different experiments. The ranges from 468 to 494 cpm at different amount of [3HlpolyW) hybridized per A 260 unit of total RNA extracted developmental stages.
AMALDI,
FELICETTI,
AND
CAMPIONI
Flow
of Informational
FIG. 1. Percentage of variation of amount of RNA and [3H]poly(U) hybridization capacity in the polyribosomes, monosomes, and subribosomal particles of unincubated and incubated cysts. The experimental data reported in Table 1 are expressed as a percentage of the total and are plotted against hours of incubation. MO, monosomes; PO, polyribosomes; SP, subribosomal particles.
The decrease in the amount of poly(A)containing RNA present in the subribosoma1 particles is very rapid for the first 5 hr (see Fig. 11, but then slows down and reaches a plateau at longer incubation times (10 and 18 hr, not shown). The easier interpretation of this phenomenon is that development does not resume synchronously in all incubated cysts: In fact, under our experimental conditions, only 50-60% of the cysts reach the nauplius stage after 20 hr of incubation. The fate of cytoplasmic mRNA upon resumption of development can be studied in more detail by submitting the postmitochondrial supernatant of undeveloped and developed embryos (1 and 5 hr) to sucrose density gradient centrifugation and by measuring the distribution of poly(A)-containing particles across the gradient. As shown in Fig. 2, most of the poly(A)-containing RNA in the unincubated cysts is found in the 40s region of the gradient as previously reported (Felicetti et al., 1975). Incubation of embryos results in a dramatic decrease in the mRNA sedimenting in the 40s region of the gradient, while RNA particles of lower S values are rela-
53
RNA
I hr
I
I
5 hr
5
IO Fractions
FIG. 2. Distribution of poly(A)-containing RNA in gradient fractions of a postmitochondrial supernatant derived from different developmental stages of Artemia salina embryos. The detergent-treated postmitochondrial supernatant was submitted to sucrose density gradient centrifugation as described in Materials and Methods. RNA was extracted from pooled gradient fractions and was hybridized with 13Hlpoly(U) as described in Table 1. In this particular experiment, the amount of 13H1polyKJ) hybridized to RNA derived from polysome pellets was 644 (0 time), 767 (1 hr), and 1690 (5 hr), cpm, respec. tively.
54
DEVELOPMENTAL
BIOLOGY
tively preserved. This may suggest that mRNA in the 40s region is bound to the small ribosomal subunits in the form of a blocked initiation complex and, upon resumption of development, is the first species to be activated and join the polyribosomes. However, the incubation-dependent decrease in free cytoplasmic mRNA does not necessarily mean that it has moved to the polysomes. One could argue that, soon after incubation, preformed mRNA is degraded, and, since the total amount of poly(A)-containing RNA remains constant in the early stages of development, new mRNA species coding for different proteins are transcribed and directly enter the polyribosome cycle. In order to check this possibiuty, we have tried to compare the pattern of proteins synthesized in a wheat germ lysate by cyst-preformed mRNA (Felicetti et al., 1975) with the translational products of polyribosomes derived from embryos at very early stages of development (10, 20, 30, and 60 min).
A2601D
t:m
ISO
VOLUME
59, 1977
Cell-Free Incubation of Polyribosomes from Unincubated and Incubated Embryos in the Presence of Heterologous Supernatant Polyribosome-enriched pellets were prepared by centrifugation through a two-step discontinuous sucrose gradient as described in Materials and Methods. The pattern of polysomes at different times of incubation is shown in Fig. 3. In unincubated cysts there is a prevalence of heavier-size aggregates, with very little material in the region of dimers, trimers, and other light particles. After 10 min of incubation, a shift of the heavy aggregates toward the middle region of the gradient is observed. At 20 min, the formation of light polysomes on the left of the monomer peak is evident. At 30 and 60 min, the polysomes amass in the lighter region of the gradient, the class of ribosomes most represented having a peak in the tetramer or examer region, respectively. A certain amount of heavy aggregates is still present at longer incubation times. In conclusion,,
t;20
I.60
t;10
7
.6
.6
A
:i .2 A
I
Al 5
IO
Flactlons
:I,
15
5
10
15
A 5
10
I5
FIG. 3. Polyribosome patterns from Artemia salina embryos at different stages of development. Ten A,,,, units of polyribosome-enriched pellets obtained from the detergent-treated postmitochondrial supernatant, as described in Materials and Methods, were layered on top of 11.5ml 15-50% linear sucrose gradients in TNM buffer and were centrifuged at 37,000 rpm for 85 min in a SW 41 rotor at 4°C. The A,, was automatically recorded in a Gilford spectrophotometer.
AMALDI,
FELICETTI,
AND
CAMPIONI
it seems that resumption of development is accompanied by the appearance of polysomes in the lighter region of the gradient which increase in size as incubation proceeds. Polyribosome pellets from early developmental stages were incubated in a cell-free system in the presence of heterologous supernatant in order to examine the pattern of early translational products. Heterologous S-150 from rat brain was used in place of the homologous one to avoid interference by inhibitory or activating factors present in different amounts in the supernatant of embryos in the course of differentiation. The rate of incorporation of 13Hllysine into hot TCA-insoluble material was very low for polyribosomes derived from unincubated or lo-min-incubated embryos (data not shown). Higher incorporation rates were observed for polysomes from 20-, 30-, and 60-min-incubated embryos. Translational products were analyzed by acrylamide gel electrophoresis after removal of the ribosomes. The electrophoretie pattern of polypeptides synthesized in vitro at early developmental stages is very similar (Fig. 41, with the majority of low molecular weight proteins ranging from 25,700 to 10,000 daltons. This result suggests that there is no preferential translation of any mRNA species within the first hour of development. It is interesting to note that polyribosomes derived from unincubated cysts are totally inactive in the cell-free system, and no radioactive material is found across the gel. It is quite surprising that the heavy aggregates detected in the cysts by sucrose gradient centrifugation (see Fig. 3) do not function in protein synthesis. It is likely that the heavy particles extracted are either mRNP particles of unusually high sedimentation values or ribosomal complexes which, for some unknown reason (presence of bound inhibitory components, lack of mRNA activity), cannot participate in the biosynthetic process. That these heavy
Flow of
RNA
Informational
20
40
60
55
80
Gel fractions FIG. 4. Acrylamide gel electrophoresis in SDSurea of polypeptides synthesized in a cell-free system by polysomes of embryos at different developmental stages. Processing of labeled samples, preparation of gels, electrophoresis, gel fractionation, and counting are as described in Materials and Methods. Standard solutions of ovalbumin (MW 43,000), chymotrypsinogen (MW 25,700), and cytochrome c (MW 11,700) were used as reference markers.
particles represent an extreme physiological adaptation to the drastic changes suffered by cell metabolism during encystment and dehydration cannot be ignored. If one compares the electrophoretic pattern of proteins synthesized at very early
56
DEVELOPMENTAL
BIOLOGY
stages of development (Fig. 4) with that obtained in a wheat germ lysate by using cyst-cytoplasmic mRNA (Felicetti et al., 1975), the analogies are very remarkable. However, the number of proteins synthesized under these conditions is so high that it does not allow a clear resolution of closely migrating peaks. In order to draw a definite conclusion, more sophisticated techniques of product analysis are required. Therefore, we decided to translate poly(A)-containing RNA extracted from both unincubated and incubated embryos in a wheat germ lysate and to analyze the cell-free products by two-dimensional slab gel electrophoresis. Two-Dimensional Slab Gel Electrophoresis of Translational Products Obtained in a Wheat Germ Lysate by Using Poly(A) +RNA from Undeveloped and Developed Embryos We have found that the oligo(dT)-cellulose poly(A)+ RNA derived from the postmitochondrial supernatant of unincubated cysts is less efficiently translated in a wheat germ lysate than the corresponding fractions from polysomes of incubated cysts. This is most likely due to the presence of low molecular weight inhibitory components contaminating mRNA preparations. For this reason, in the experiments to be described, poly(A)-containing RNA from unincubated cysts was submitted to sucrose density gradient centrifugation, and purified RNA fractions of different S values were tested individually in a wheat germ lysate (Table 2). The tubes corresponding to the most active RNA fractions (2-11) were pooled, the ribosomes were removed, and the supernatant was treated with RNAse and lyophilized as described in Materials and Methods. On the other hand, oligo(dT)-cellulose poly(A)containing RN-4 extracted from the polysomes of l- and 4-hi--incubated cysts was directly tested in the wheat germ lysate, and the postribosomal supernatant was processed as before. Since poly(A)-contain-
VOLUME
59, 1977
ing RNA from both unincubated and incubated embryos directs the synthesis of polypeptides of low molecular weight (ranging from 8000-40,000), a 7-20% acrylamide gel gradient was used in the preparation of the slab gels. By comparing the pattern obtained with preformed mRNA (0 time) to that obtained with polysomal mRNA from l- and 4-hrincubated embryos (Fig. 51, one observes a striking similarity, except for one band missing at 0 time (see arrow) and a weaker intensity of the radioactive bands corresponding to polypeptides with a molecular weights greater than 25,700 in the O-time plate. A better resolution of the translational products was achieved by submitting the 35S-labeled samples to the first step of isoelectric focusing (isoelectric points in the range of 4.5-71, followed by the second step of SDS-slab gel electrophoresis through 720% acrylamide gradients (Fig. 6). About 70-80 radioactive spots can be identified in the plates corresponding to different developmental stages. The positions of the spots in the gels are fairly constant, and most of the spots do coincide. However, at least three to five new spots (see arrows) appear in the samples corresponding to polysomal mRNA from incubated embryos. These novel spots migrate in the upper region of the slab gel and have molecular weights greater than 43,000. Although the intensity of the spots with molecular weights greater than 43,000 is weaker in the O-time plate so that some of them could escape detection by the most accurate photographic reproduction, it can be assumed that at least 5% of the spots identified in the l- and 4-hr plates are missing at 0 time, probably due to the appearance on the polysomes of a new species of mRNA absent (or inactive) in the mRNA population of unincubated cysts. DISCUSSION
Sierra et al. (1976) have studied the distribution of mRNA activities among dif-
AMALDI,
FELICETTI,
AND
Flow of Informational
CAMPIONI TABLE
TRANSLATION
OF PoLY(A)-CONTAINING RNA
P;‘?;,:’
Postmitochondrial unincubated
cysts
supernatant (12 g)
of
DERIVED FROM GERM LYSATE~
Poly(A) tested/50
covered (Am unit) 4.5
57
2
RNA FRACTIONS EMBRYOS IN A WHEAT
source
RNA
+ RNA ~1 of lysate (A,,, unit)
0.035-0.04”
UNINCUBATED
AND INCUBATED
V5S1Methionine incorporated/50 ~1 of lysate
In;~be~; (ml)
Fraction
“fo?6)x
1 2 3 4 5 6 I 8 9 10 11 12
0.56 2.10 2.65 2.56 1.96 2.51 2.38 2.95 2.58 2.08 1.17 0.72
0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
Polyribosomes from embryos (40 g)
1-hr-incubated
0.5
0.025
6.01
0.4
Polyribosomes from embryos (40 g)
4-hr-incubated
1.45
0.036
6.02
0.4
0 Poly(Akontaining RNA, 4.5 A,,, unit, from unincubated cysts was applied on top of 17-ml 15-30% linear sucrose gradients in SDS buffer and was centrifuged at 26,000 rpm for 20 hr in the SW 27.1 rotor at 20°C (Felicetti et al., 1975). Gradient fractions, 1.5 ml, were precipitated with ethanol, and the RNA was dried under vacuum and dissolved in 100 ~1 of sterile water. Aliquots of each RNA fraction were tested in a wheat germ lysate as described in Materials and Methods. After incubation, the most active fractions (tubes 2-11) were pooled and processed for electrophoretic analysis as described in Materials and Methods. Poly(A) + RNA from the polysomes of incubated embryos was tested directly in a wheat germ lysate, and the reaction mixtures were processed as above. In this experiment, the endogenous incorporation of wheat germ lvsate in the absence of anv RNA addition was 0.3 x 10e5 cpm/50 ~1. b Of each gradient fraction.
ferent cytoplasmic fractions of Artemia salina embryos in the course of development (unincubated and 16-hr-incubated cysts). They found that the total mRNA activity [poly(A)+ and poly(A)RNA) of the embryos increased threefold, but the most pronounced change with development was observed in the poly(A) + RNA activity which increased over sixfold. The discrepancy with our results can be explained if one considers in detail the different experimental approaches used: (a) We have focused our attention on the very early stages of development (up to 8 hr), and one can assume that such profound changes in the activity of poly(A) + mRNA have not occurred yet; (b) we neglected the
amount of poly(A) + RNA found in the mitochondrial fraction P,, since we have previously found that it does not account for more than 10-E% of the total preformed mRNA; Cc) Sierra et al. (1976) measured activity of poly(A)+mRNA fractions in a wheat germ lysate, while we chose a more quantitative approach by measuring the amount of [3Hlpoly(U) hybridized. We have constantly found that poly(A)+ mRNA from unincubated cysts is less efficiently translated in a wheat germ lysate than the corresponding fractions derived from polysomes of incubated embryos, and this is due to the presence of low molecular weight inhibitory components in the oligo(dT)-cellulose RNA frac-
58
DEVELOPMENTAL
BIOLOGY
VOLUME
59, 1977
13Uk -
25X
FIG. 5. Autoradiography of %&labeled proteins synthesized in a wheat germ lysate and analyzed by SDS-slab gel electrophoresis. Poly(A)-containing RNA fractions from undeveloped and developed embryos were translated in wheat germ lysate as described in Table 2. The incubation mixtures corresponding to poly(A)-containing RNA from unincubated cysts (0 time) and from incubated embryos (1 and 4 hr) were processed as described in Materials and Methods. The amount of TCA-insoluble radioactivity applied to each slot was 102 x 10e3 cpm for the O-time sample (100 ~1 of incubation mixture) and 230 x 1O-3 cpm for the I- and 4-hr samples (50 ~1 of incubation mixture). The autoradiograms were exposed for 15 and 7 days respectively. fl-Galactosidase, (MW 130,000), ovalbumin (MW 43,000), and chymotrypsinogen (MW 25,700) were used as molecular weight standards.
tions. It is likely that mRNA activity in some of the cytoplasmic fractions of unincubated embryos has been underestimated: Practically, they found no detectable activity in the S-105 and ribosomal fraction. In agreement with our results is the work by Grosfeld and Littauer (1975)
who found that the postmitochondrial supernatants of dormant and incubated developing cysts contain approximately the same quantities of poly(A)-rich mRNA. The facts discussed extensively above are pertinent to the following question. As soon as development resumes, is it a burst
FIG. 6. Autoradiography of YS-labeled proteins synthesized in a wheat germ lysate and analyzed by twodimensional gel electrophoresis. The amount of TCA-insoluble radioactivity applied to first-dimension gels (see Materials and Methods) was 204 x 10m3 cpm for the O-time sample (200 ~1 of incubation mixture) and 460 x 10m3 cpm for the l- and 4-hr samples (100 ~1 of incubation mixture). The autoradiograms were exposed for 10 days (l- and 4-hr samples) and 20 days (O-time sample).
59
60
DEVELOPMENTAL
BIOLOGY
of synthesis of new mRNA species which occurs or is the mRNA stored in the cysts what is mainly utilized by the embryos? We are more inclined to support the second hypothesis since our results have clearly shown that, first, there is a flow of informational RNA from the subribosomal poly(A)-containing particles to the polysomes of developing embryos; second, there is a close similarity between the translational patterns at very early stages of development and the polypeptides synthesized in a wheat germ lysate by preformed mRNA stored in the cysts. Our results do not rule out the possibility that breakdown and resynthesis of the same family of mRNA sequences occur after cyst incubation. This event seems very unlikely to us, since there would be no reason for encysted embryos to preserve a defined set of mRNA sequences in their cytoplasm and destroy them as soon as development is resumed. Conventional slab gel electrophoresis revealed that at least one band with a molecular weight less than 27,500 daltons was missing in the O-time slab (Fig. 5). The fact that this finding was not confirmed by careful examination of the spots resolved by two-dimensional gel electrophoresis suggests that the labeled polypeptide(s1 does not enter the firstdimension gel and is probably stuck at the origin (as if the isoelectric point was higher than 7). Despite this limitation, two-dimensional slab gel electrophoresis proved to be an essential tool in unraveling the complex pattern of proteins synthesized in the embryos and allowed us to detect any subtle change among the hundreds of spots revealed by autoradiography. The extra band and the new spots observed in the slabs corresponding to l- and 4-hr-incubated embryos may derive either from early transcription of a new mRNA species or simply by post-transcriptional modifications of pre-existing mRNA molecules. It has been shown that mRNAs from both
VOLUME
59,
1977
developed and undeveloped embryos contain 5-terminal 7-methyl-guanosine, in an inverted linkage through three phosphate groups to the rest of polynucleotide chains, and that extracts from both developmental stages possess &terminal methylase activity (Muthukrishnan et al., 1975). It has been concluded that the low level of protein synthesis in dormant embryos cannot be explained by a defective mRNA methylation. However the process of capping and methylation at the 5 terminus of mRNA could still be invoked as a regulatory mechanism to activate translation of discrete species of cyst-preformed mRNA in early. development. The mechanism of activation of protein synthesis in the encysted embryos is still obscure. If one carefully examines the polyribosome patterns in the embryos incubated for short periods of time (see Fig. 31, it appears that active polysomes are progressively increasing in size, as if, at the beginning, the rate of initiation is impaired and preformed mRNAs are not fully charged with ribosomes. This observation is in contrast with the hypothesis that preformed mRNA is intrinsically unable to interact with ribosomes and suggests that a limitant amount of initiation factors is present in unincubated cysts. Similar conclusions have been drawn by Filipowicz et al. (1975, 1976) who found that the activity of IF-MP, an initiation factor that promotes binding of met tRNA, to 40s subunits, increases over 20-fold following resumption of development. REFERENCES P., RUSCA, G., and CALISSANO, P. (1973). In vitro synthesis of a brain-specific protein (S-100) by free and membrane bound polysomes. Biochin. Biophys. Actu 299, 634-641. FELICETTI, L., PIERANDREI AMALDI, P., MORETTI, S., CAMPIONI, N., and URBANI, C. (1975). Intracellular distribution, sedimentation values and template activity of polyadenilic acid-containing RNA stored in Artemia salina cysts. Cell Differ. 4, 339354. AMALDI,
AMALDI,
FELICETTI,
AND
CAMPIONI
SIERRA, J. M. and OCHOA, S. (1975). chain initiation in eukaryotes: InitiaMP in Arremia salina embryos. Proc. Sci. USA, 72, 3947-3951. W., SIERRA, J. M., NOMBELA, C., OCHOA, S., MERRICK, W. C., and FRENCH ANDERSON, W. (1976). Polypeptide chain initiation in eukaryotes: Initiation factor requirements for translation of natural messengers. Proc. Nat. Acad. Sci. USA 73, 44-48. FINAMORE, F. J., and CLEGG, J. S. (1969). Biochemical aspects of morphogenesis in the Brine shrimp, Artemia salina. In “The Cell Cycle” (G. M. Padilla, G. L. Whitson, and I. L. Cameron, eds.), pp. 249-278. Academic Press, New York. GROSFELD, H., and LITTAUER, U. Z. (19751. Cryptic form of mRNA in dormant Artemia salina cysts. Biochem. Biophys. Res. Commun. 67, 176-181. HULTIN, T., and MORRIS, J. E. (1968). The ribosomes of encysted embryos of Artemia salina during cryptobiosis and resumption of development. Deuelop. Biol. 17, 143-164. JACOBS-LORENA, M., BAGLIONI, C., and BORUN, T. W. (1972). Translation of messenger RNA for histones from HeLa cells by a cell-free extract from mouse ascite tumor. Proc. Nat. Acad. Sci. USA 69, 2095-2099. FILIPOWICZ,
Polypeptide tion factor Nat. Acad. FILIPOWICZ,
W.,
Flow
of Informational
RNA
61
LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of Bacteriophage T 4. Nature (London) 227, 680-685. MUTHUKRISHNAN, S., FILIPOWICZ, W., SIERRA, J. M., BOTH, G. W., SHATKIN, A. J., and OCHOA, S. (1975). mRNA methylation and protein synthesis in extracts from embryos of brine shrimp, Artemia salina. J. Biol. Chem. 250, 9836-9341. NILSSON, M. O., And HULTIN, T. (1974). Characteristics and intracellular distribution of messenger like RNA in encysted embryos of Artemia salina. Deuelop. Biol. 38, 138-149. O’FARREL, P. N. (1975). High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007-4021. ROBERTS, B. E., and PATERSON, B. M. (1973). Efficient translation of tobacco mosaic virus RNA and rabbit globin 9s RNA in a cell-free system from commercial wheat germ. Proc. Nat. Acad. Sci. USA 70, 2330-2334. ROSBASH, M. and FORD, P. J. (1974). Polyadenylic acid-containing RNA in Xenopus laeuis oocytes. J. Mol. Biol. 85, 87-101. SIERRA, J. M., FILIPOWICZ, W., and OCHOA, S. (1976). Messenger RNA in undeveloped and developing Artemia salina embryos. Biochem. Biophys. Res. Commun. 69, 181-189.
BIOLOGY
59, 49-61
(1977)
Flow of Informational RNA from Cytoplasmic Poly(A)-Containing Particles to Polyribosomes in Artemia salina Cysts at Early Stages of Development PAOLA
PIERANDREI
National
Research Received
AMALDI, Council November
LUCIANO (C.N.R.J,
2.1976:
FELICETTI,
Laboratory nrcrpted
of Cell
in rwwpd
AND
NADIA
Biology,
00196
form
March
CAMPIONI Rome,
Italy
28, 1977
When Artemia salina cysts are incubated at 28°C in artificial seawater, the pool of cytoplasmic mRNA as measured by the [“H]poly(U) hybridization technique is depleted, and a parallel increase in the polysome bound mRNA is observed. The shift of informational RNA from subribosomal particles to polyribosomes occurs without a significant increase in the amount of poly(A)-containing RNA per embryo, at least during the first hours of development. In order to ascertain whether resumption of development is due mainly to utilization of preformed mRNA, the polypeptides synthesized in a wheat germ lysate under the direction of cyst cytoplasmic mRNA were compared with those obtained using polysomal mRNA from developed embryos (1 and 4 hr). The complex pattern of proteins synthesized in vitro was resolved into 70-80 radioactive spots by two-dimensional gel electrophoresis and was shown to be almost identical in unincubated and incubated cysts, except for a few novel spots. Our results do not exclude that early transcription of a few mRNA species could play a role in the activation of metabolic processes which occur when development is resumed. INTRODUCTION
et al., 1975; Sierra et al., 1976). In a previous paper (Felicetti et al., 19751, we have shown that poly(A)-containing particles with sedimentation values ranging from 4 to 80s are present in the cyst postmitochondrial supernatant. In addition, poly(A)-containing RNA can be fractionated by sucrose density gradient centrifugation, and RNA fractions of different S values can be translated in a wheat germ lysate, resulting in the formation of a characteristic pattern of proteins (BOOO-40,000 M. W.) The question arises as to whether the activation of protein synthesis in the early stages of development is mainly due to utilization of cytoplasmic preformed mRNA or whether an early transcriptional event is also required to cope with the growing needs of developing embryos. No direct evidence of RNA synthesis in early development can be achieved due to the fact that the inner cuticle layer of the cysts is extremely impermeable to amino
Artemia salina cysts contain primarily dessiccated embryos at the gastrula stage, and, under this form, they can survive for long periods of time in unfavorable environments. In the extracts of dry cysts or cysts hydrated in seawater at O”C, the level of protein synthesis is very low, the polyribosomes are almost absent, and the ribosomal population is almost entirely composed of monosomes. If the cysts are hydrated in seawater and incubated at 3O”C, relatively few polysomes are detected within 5 min (Finamore and Clegg, 1969). The temperature-dependent activation of protein synthesis in the early stages of development can be exploited as a model system to study regulatory mechanisms at the translational level, since preformed mRNA not associated with ribosomes has been detected in subcellular fractions of the cyst homogenate (Nilsson and Hultin, 1974; Grosfeld and Littauer, 1975; Felicetti 49 Copyright All
rights
0 1977 by Academic of reproduction in
Press, any form
Inc. reserved.
ISSN
0012-1606
50
DEVELOPMENTAL
BIOLOGY
acids, nucleic acid precursors, and antibiotics (Finamore and Clegg, 1969). We have tried to circumvent the failure of in vivo labeling with two experimental approaches: (a) measurement of the amount of poly(A)-containing RNA extracted from the postmitochondrial supernatant of undeveloped and developed cysts; (b) comparison of translational products at early stages of development with polypeptides synthesized in a wheat germ lysate by preformed mRNA stored in the cysts. MATERIALS
AND
METHODS
13HlLysine (50 Ci/mmole) was obtained from New England Nuclear, and [35Slmethionine (196 Ci/mmole) was from Amersham; [3H]poly(U) (61.9 $X/~mole P) was from Miles; ATP, GTP, CTP, creatine phosphate, creatine phosphokinase, and dithiothreitol (DTT) were purchased from Calbiochemicals; oligo(dT)-cellulose T, was from Collaborative Research; sodium dodecyl sulfate (SDS), acrylamide, N,N’-methylenebisacrylamide, and ammonium persulfate were from Serva; ampholines (pH 5-7 and pH 3.5-10) were from LKB; and pancreatic RNAse was from Worthington. Fractionation of the Postmitochondrial Supernatant from Undeveloped and Developed Embryos Artemia salina cysts (7.5 g) were incubated in 2 liter of artificial seawater (Hultin and Morris, 1968) at 28°C for different lengths of time (1, 5, and 8 hr). The postmitochondrial supernatant was prepared as previously described (Felicetti et al., 1975) and was treated with 1% deoxycholate and 1% Brij 58 for 30 min at 0°C. The detergent-treated supernatant, 1.5 ml, was layered on top of 16.5-ml 15-30% linear sucrose gradients in TNM buffer (30 mM Tris-HCl, pH 7.4, 100 mM NaCl, and 10 mM MgCl,) and was centrifuged at 23,000 rpm for 14 hr in a SW 27.1 Spinco rotor at 4°C. The A,,, was automatically recorded by pumping the gradients from
VOLUME
59, 1977
the bottom through a 0.2-cm flow cell of a Gilford spectrophotometer. Fractions of 1.5 ml were collected from three identical sucrose gradients. RNA Extraction ization
and [3HlPoly(U)
Hybrid-
RNA was extracted by the phenol-sodium dodecylsulfate procedure (pH 9) as previously described (Felicetti et al., 1975). The [3Hlpoly(U)-hybridization test was performed according to Rosbash and Ford (1974). Polyribosome Preparation from Undeveloped and Developed Embryos and CellFree Assay in the Presence of Heterologous Supernatant The postmitochondrial supernatant derived from unincubated cysts or embryos incubated at 28°C for 10, 20, 30, and 60 min (15 g) was treated with 1% deoxycholate and 1% Brij 58 as before. Ten milliliters of detergent-treated supernatant were layered on a discontinuous sucrose gradient consisting of 1 ml each of 2 M and 1 M sucrose in TNM buffer and was centrifuged at 100,OOOg for 14 hr in a 50 Ti Spinco rotor at 4°C. The polyribosome pellets were resuspended in TKM buffer (50 mM Tris-HCl, pH 7.4, 25 mM KCl, and 5 mM MgCl,) and were immediately used for the cell-free assay. The incubation mixture contained in a final volume of 250 ~1: 50 mM Tris-HCl, pH 7.4, 100 mM KCl, 3 mM MgCl*, 10 mM dithiothreitol, 2 mM ATP, 0.5 mM GTP, 2 mM creatine phosphate, 2 pg of creatine phosphokinase, a mixture of essential amino acids (each at 40 mM, except lysine), 40 +i of 13Hllysine, 10.5 A,,, unit of polysomes, and 100 pg of the S 150 fraction from young rat brain (Amaldi et al., 1973). Incubation was at 37°C for 60 min. Aliquots, 5 ~1, were hydrolyzed with 0.2 ml of 0.1 N KOH (20 min at 37”C), precipitated with 1 ml of 10% trichloroacetic acid (TCA), collected on Whatman GF/C filters, and counted in 5 ml of butyl-
AMALDI,
FELICETTI,
AND
CAMPIONI
Flow of Informational
RNA
51
with 1 vol of TKM buffer, centrifuged at 100,OOOgfor 2 hr, treated with RNAase as before, and lyophilized. Acrylamide Gel Electrophoresis of the (I, Slab gel electrophoresis. The samples Polypeptides Synthesized by Embryo were dissolved in 50 ~1 of solution containPolysomes during Cell-Free Incubation ing 6.7% SDS, 16.7% P-mercaptoethanol, Each incubation mixture was diluted up 16.7% glycerol, and trace amounts of pyronin G. The samples were heated at 100°C to 1 ml with TKM buffer (20 mM Trisfor 1 min and were loaded on a 1.5-mmHCl, pH 7.4, 80 n-&f KCl, 3 m&I MgCl,) and was centrifuged at 100,OOOgfor 2 hr in thick slab gel containing a 7-20% acrylamorder to remove the ribosomes. The super- ide gradient. Electrophoresis was pernatant was treated with 50 pg/ml of pan- formed at 150 V, and the run was stopped creatic ribonuclease for 20 min at 37”C, when pyronin G reached the end of the gel. and labeled proteins were precipitated Gel and running buffers were prepared acwith 5% trichloroacetic acid. Denaturation cording to Laemmli (1970). (ii) Two-dimensional gel electrophoreof labeled samples, gel preparation, gel fractionation by a Savant auto gel divider, sis. The lyophilized samples were suband counting were as described by Jacobs- jected to isoelectrofocusing, followed by Lorena et al. (1972). SDS-slab gel electrophoresis as described by O’Farrtil (1975). First-dimension 2.5Oligo(dT) Cellulose Chromatography of mm gels were loaded on 1.5-mm-thick slab RNA gels containing a 7-20% acrylamide gradient and were sealed with agarose. RNA extracted from the postmitochonThe unequilibrated first-dimension gels drial fraction of unincubated cysts and were prerun for 20 min at 20 mA with high from polysomes of embryos incubated 1 SDS buffer, and then regular running and 4 hr was separated into poly(A)+ and poly(A) - fractions by oligo(dT)-cellulose buffer was applied as described by chromatography as previously described O’Farrel (1975). Power was turned off (Felicetti et al. 1975). when the bromophenol blue front reached the end of the gel. The slabs were stained Translation of Poly(A)-Containing RNA with Coomassie blue, destained, dried unFractions from Undeveloped and Develder vacuum, and exposed to Kodak Kodioped Embryos in a Wheat Germ Lysate rex X-ray films. A wheat germ lysate was prepared as RESULTS described by Roberts and Paterson (1973). The incubation mixture, in a final volume Study of the Distribution of Poly(A)-Conof 50 ~1, contained: ‘0.8A,,, units of lysate, taining RNA in Undeveloped and De25 mM Hepes-HCl, pH 7.4, 3 mM Mgveloped Embryos by the [3H]Poly(U)acetate, 64 mM KCl, 1.4 mM DTT, 1 mM Hybridization Technique ATP, 0.1 mM GTP, 0.6 n-&I CTP, 10 mM creatine phosphate, 8 Fg of creatine phosThe postmitochondrial supernatant dephokinase, each of 19 nonradioactive rived from unincubated cysts or cysts incuamino acids (each at 40 fl, except methibated at 28°C for different lengths of time onine) and 4.3 &i of [35S]methionine. In(1, 5, and 8 hr) was treated with detergents and was fractionated on sucrose gradients cubation was at 27°C for 60 min. as described in Materials and Methods. Electrophoretic Analysis of Translational The amount of RNA extracted from the Products in a Wheat Germ Lysate polysomal pellet, monosomes, and subriThe incubation mixtures were diluted bosomal particles and its ability to hybridPBD toluene (6 g/liter) scintillation counter.
in a Beckman
52
DEVELOPMENTAL
VOLUME
BIOLOGY
ize with [3H]poly(U) was determined as shown in Table 1. At time 0, most of the RNA is extracted from the monomers (72%), with only a small amount from the polysomes (5.5%). When cysts are incubated, the amount of monomers progressively decreases, while a parallel increase in the amount of polysomes is observed. There is no significant variation in the amount of RNA extracted from different developmental stages, suggesting that synthesis of ribosomal or transfer RNA does not occur to a measurable extent. As would be expected, the variation in the amount of poly(A)-containing RNA free in the cytoplasm or bound to polysomes reflects the process of activation of protein synthesis in the embryos: There is a decrease in the mRNA present in the subribosomal particles and a parallel increase in that bound to the polysome fraction. The amount of poly(A)-containing RNA bound to monosomes does not change significantly: The fluctuations observed at different times of incubation could be due either to polysome degradation during
fractionation of the extracts or to defective separation on sucrose gradients of the monosomes from the lighter ribosomal aggregates. If one calculates the counts per minute of [:lHlpoly(U) hybridized per A,,,, unit of total RNA extracted, there is no significant change in the specific activity of the RNA prepared from unincubated cysts or from embryos incubated for different lengths of time. This result suggests that, at least in the early stages of development, the average amount of poly(A)-containing RNA per embryo is constant, and a peculiar feature of this system seems to lie in the transfer of mRNA from the cytoplasmic particles to the polysomes. The results of Table 1 concerning the variation in the absorbance and in the [“H]poly(U)-hybridization capacity of different RNA fractions in the course of development can be expressed as a percentage of the total and can be plotted against hours of incubation (Fig. 1). The plot gives a clear picture of the modifications induced in these parameters by incubation of cysts in seawater.
TABLE TOTAL
AMOUNT
FRACTIONS
Gradient
AND
[3H]Po~~(U)
HYBRIDIZATION
OF POSTMITOCHONDRIAL
SUPERNATANT
fraction
RNA
59, 1977
1 CAPACITY FROM
OF RNA
UNDEVELOPED
EXTRACTED AND
extracted
FROM
DEVELOPED
(3H)Poly(U)
(A,,,, unit) 0 hr Polysomes
(pellet)
Monosomes
(I-4)
Subribosomal (5-12) Total
particles
6.4 (5.54) 83.15 (72.07) 25.83 (22.4) 115.38 (100)
1 hr 12.9 (12.17) 66.6 (62.83) 26.5 (25.0) 106.0 (100)
5 hr 28.2 (25.38) 62.47 (56.23) 20.43 (18.39) 111.1 (100)
8 hr
0 hr
35.2 (35.26) 48.3 (48.39) 16.32 (16.35) 99.82 (100)
4,290 (7.54) 5,380 (9.46) 47,210 (83) 56,880 (100)
GRADIENT EMBRYOS”
hybridized (cpm)
1 hr
5 hr
8 hr
5,750 (11.44) 8,500 (16.92) 36,000 (71.64) 50,250 (100)
12,680 (24.4) 10,800 (20.8) 28,460 (54.8) 51,940 (100)
17,340 (35.16) 6,620 (13.42) 25,360 (51.42) 49,320 (100)
0 The detergent-treated postmitochondrial supernatant was fractionated on sucrose gradients as described in Materials and Methods. The polyribosomes sediment to the bottom of the tubes. The monosome peak (fractions l-4) is clearly separated from subribosomal particles (fractions 5-12) as shown in Fig. 2. The RNA was extracted from the polysomal pellets and gradient fractions by the SDS-phenol procedure and was resuspended in 100 ~1 of sterile water, and aliquots (20 ~1 for polysome pellets, 40 ~1 for gradient fractions) were hybridized with 15,000 cpm of [3H]poly(U) (specific activity: 1.5 x lo5 cpm/pg) as described in Materials and Methods. The values in the table are the average values calculated from two different experiments. The ranges from 468 to 494 cpm at different amount of [3HlpolyW) hybridized per A 260 unit of total RNA extracted developmental stages.
AMALDI,
FELICETTI,
AND
CAMPIONI
Flow
of Informational
FIG. 1. Percentage of variation of amount of RNA and [3H]poly(U) hybridization capacity in the polyribosomes, monosomes, and subribosomal particles of unincubated and incubated cysts. The experimental data reported in Table 1 are expressed as a percentage of the total and are plotted against hours of incubation. MO, monosomes; PO, polyribosomes; SP, subribosomal particles.
The decrease in the amount of poly(A)containing RNA present in the subribosoma1 particles is very rapid for the first 5 hr (see Fig. 11, but then slows down and reaches a plateau at longer incubation times (10 and 18 hr, not shown). The easier interpretation of this phenomenon is that development does not resume synchronously in all incubated cysts: In fact, under our experimental conditions, only 50-60% of the cysts reach the nauplius stage after 20 hr of incubation. The fate of cytoplasmic mRNA upon resumption of development can be studied in more detail by submitting the postmitochondrial supernatant of undeveloped and developed embryos (1 and 5 hr) to sucrose density gradient centrifugation and by measuring the distribution of poly(A)-containing particles across the gradient. As shown in Fig. 2, most of the poly(A)-containing RNA in the unincubated cysts is found in the 40s region of the gradient as previously reported (Felicetti et al., 1975). Incubation of embryos results in a dramatic decrease in the mRNA sedimenting in the 40s region of the gradient, while RNA particles of lower S values are rela-
53
RNA
I hr
I
I
5 hr
5
IO Fractions
FIG. 2. Distribution of poly(A)-containing RNA in gradient fractions of a postmitochondrial supernatant derived from different developmental stages of Artemia salina embryos. The detergent-treated postmitochondrial supernatant was submitted to sucrose density gradient centrifugation as described in Materials and Methods. RNA was extracted from pooled gradient fractions and was hybridized with 13Hlpoly(U) as described in Table 1. In this particular experiment, the amount of 13H1polyKJ) hybridized to RNA derived from polysome pellets was 644 (0 time), 767 (1 hr), and 1690 (5 hr), cpm, respec. tively.
54
DEVELOPMENTAL
BIOLOGY
tively preserved. This may suggest that mRNA in the 40s region is bound to the small ribosomal subunits in the form of a blocked initiation complex and, upon resumption of development, is the first species to be activated and join the polyribosomes. However, the incubation-dependent decrease in free cytoplasmic mRNA does not necessarily mean that it has moved to the polysomes. One could argue that, soon after incubation, preformed mRNA is degraded, and, since the total amount of poly(A)-containing RNA remains constant in the early stages of development, new mRNA species coding for different proteins are transcribed and directly enter the polyribosome cycle. In order to check this possibiuty, we have tried to compare the pattern of proteins synthesized in a wheat germ lysate by cyst-preformed mRNA (Felicetti et al., 1975) with the translational products of polyribosomes derived from embryos at very early stages of development (10, 20, 30, and 60 min).
A2601D
t:m
ISO
VOLUME
59, 1977
Cell-Free Incubation of Polyribosomes from Unincubated and Incubated Embryos in the Presence of Heterologous Supernatant Polyribosome-enriched pellets were prepared by centrifugation through a two-step discontinuous sucrose gradient as described in Materials and Methods. The pattern of polysomes at different times of incubation is shown in Fig. 3. In unincubated cysts there is a prevalence of heavier-size aggregates, with very little material in the region of dimers, trimers, and other light particles. After 10 min of incubation, a shift of the heavy aggregates toward the middle region of the gradient is observed. At 20 min, the formation of light polysomes on the left of the monomer peak is evident. At 30 and 60 min, the polysomes amass in the lighter region of the gradient, the class of ribosomes most represented having a peak in the tetramer or examer region, respectively. A certain amount of heavy aggregates is still present at longer incubation times. In conclusion,,
t;20
I.60
t;10
7
.6
.6
A
:i .2 A
I
Al 5
IO
Flactlons
:I,
15
5
10
15
A 5
10
I5
FIG. 3. Polyribosome patterns from Artemia salina embryos at different stages of development. Ten A,,,, units of polyribosome-enriched pellets obtained from the detergent-treated postmitochondrial supernatant, as described in Materials and Methods, were layered on top of 11.5ml 15-50% linear sucrose gradients in TNM buffer and were centrifuged at 37,000 rpm for 85 min in a SW 41 rotor at 4°C. The A,, was automatically recorded in a Gilford spectrophotometer.
AMALDI,
FELICETTI,
AND
CAMPIONI
it seems that resumption of development is accompanied by the appearance of polysomes in the lighter region of the gradient which increase in size as incubation proceeds. Polyribosome pellets from early developmental stages were incubated in a cell-free system in the presence of heterologous supernatant in order to examine the pattern of early translational products. Heterologous S-150 from rat brain was used in place of the homologous one to avoid interference by inhibitory or activating factors present in different amounts in the supernatant of embryos in the course of differentiation. The rate of incorporation of 13Hllysine into hot TCA-insoluble material was very low for polyribosomes derived from unincubated or lo-min-incubated embryos (data not shown). Higher incorporation rates were observed for polysomes from 20-, 30-, and 60-min-incubated embryos. Translational products were analyzed by acrylamide gel electrophoresis after removal of the ribosomes. The electrophoretie pattern of polypeptides synthesized in vitro at early developmental stages is very similar (Fig. 41, with the majority of low molecular weight proteins ranging from 25,700 to 10,000 daltons. This result suggests that there is no preferential translation of any mRNA species within the first hour of development. It is interesting to note that polyribosomes derived from unincubated cysts are totally inactive in the cell-free system, and no radioactive material is found across the gel. It is quite surprising that the heavy aggregates detected in the cysts by sucrose gradient centrifugation (see Fig. 3) do not function in protein synthesis. It is likely that the heavy particles extracted are either mRNP particles of unusually high sedimentation values or ribosomal complexes which, for some unknown reason (presence of bound inhibitory components, lack of mRNA activity), cannot participate in the biosynthetic process. That these heavy
Flow of
RNA
Informational
20
40
60
55
80
Gel fractions FIG. 4. Acrylamide gel electrophoresis in SDSurea of polypeptides synthesized in a cell-free system by polysomes of embryos at different developmental stages. Processing of labeled samples, preparation of gels, electrophoresis, gel fractionation, and counting are as described in Materials and Methods. Standard solutions of ovalbumin (MW 43,000), chymotrypsinogen (MW 25,700), and cytochrome c (MW 11,700) were used as reference markers.
particles represent an extreme physiological adaptation to the drastic changes suffered by cell metabolism during encystment and dehydration cannot be ignored. If one compares the electrophoretic pattern of proteins synthesized at very early
56
DEVELOPMENTAL
BIOLOGY
stages of development (Fig. 4) with that obtained in a wheat germ lysate by using cyst-cytoplasmic mRNA (Felicetti et al., 1975), the analogies are very remarkable. However, the number of proteins synthesized under these conditions is so high that it does not allow a clear resolution of closely migrating peaks. In order to draw a definite conclusion, more sophisticated techniques of product analysis are required. Therefore, we decided to translate poly(A)-containing RNA extracted from both unincubated and incubated embryos in a wheat germ lysate and to analyze the cell-free products by two-dimensional slab gel electrophoresis. Two-Dimensional Slab Gel Electrophoresis of Translational Products Obtained in a Wheat Germ Lysate by Using Poly(A) +RNA from Undeveloped and Developed Embryos We have found that the oligo(dT)-cellulose poly(A)+ RNA derived from the postmitochondrial supernatant of unincubated cysts is less efficiently translated in a wheat germ lysate than the corresponding fractions from polysomes of incubated cysts. This is most likely due to the presence of low molecular weight inhibitory components contaminating mRNA preparations. For this reason, in the experiments to be described, poly(A)-containing RNA from unincubated cysts was submitted to sucrose density gradient centrifugation, and purified RNA fractions of different S values were tested individually in a wheat germ lysate (Table 2). The tubes corresponding to the most active RNA fractions (2-11) were pooled, the ribosomes were removed, and the supernatant was treated with RNAse and lyophilized as described in Materials and Methods. On the other hand, oligo(dT)-cellulose poly(A)containing RN-4 extracted from the polysomes of l- and 4-hi--incubated cysts was directly tested in the wheat germ lysate, and the postribosomal supernatant was processed as before. Since poly(A)-contain-
VOLUME
59, 1977
ing RNA from both unincubated and incubated embryos directs the synthesis of polypeptides of low molecular weight (ranging from 8000-40,000), a 7-20% acrylamide gel gradient was used in the preparation of the slab gels. By comparing the pattern obtained with preformed mRNA (0 time) to that obtained with polysomal mRNA from l- and 4-hrincubated embryos (Fig. 51, one observes a striking similarity, except for one band missing at 0 time (see arrow) and a weaker intensity of the radioactive bands corresponding to polypeptides with a molecular weights greater than 25,700 in the O-time plate. A better resolution of the translational products was achieved by submitting the 35S-labeled samples to the first step of isoelectric focusing (isoelectric points in the range of 4.5-71, followed by the second step of SDS-slab gel electrophoresis through 720% acrylamide gradients (Fig. 6). About 70-80 radioactive spots can be identified in the plates corresponding to different developmental stages. The positions of the spots in the gels are fairly constant, and most of the spots do coincide. However, at least three to five new spots (see arrows) appear in the samples corresponding to polysomal mRNA from incubated embryos. These novel spots migrate in the upper region of the slab gel and have molecular weights greater than 43,000. Although the intensity of the spots with molecular weights greater than 43,000 is weaker in the O-time plate so that some of them could escape detection by the most accurate photographic reproduction, it can be assumed that at least 5% of the spots identified in the l- and 4-hr plates are missing at 0 time, probably due to the appearance on the polysomes of a new species of mRNA absent (or inactive) in the mRNA population of unincubated cysts. DISCUSSION
Sierra et al. (1976) have studied the distribution of mRNA activities among dif-
AMALDI,
FELICETTI,
AND
Flow of Informational
CAMPIONI TABLE
TRANSLATION
OF PoLY(A)-CONTAINING RNA
P;‘?;,:’
Postmitochondrial unincubated
cysts
supernatant (12 g)
of
DERIVED FROM GERM LYSATE~
Poly(A) tested/50
covered (Am unit) 4.5
57
2
RNA FRACTIONS EMBRYOS IN A WHEAT
source
RNA
+ RNA ~1 of lysate (A,,, unit)
0.035-0.04”
UNINCUBATED
AND INCUBATED
V5S1Methionine incorporated/50 ~1 of lysate
In;~be~; (ml)
Fraction
“fo?6)x
1 2 3 4 5 6 I 8 9 10 11 12
0.56 2.10 2.65 2.56 1.96 2.51 2.38 2.95 2.58 2.08 1.17 0.72
0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
Polyribosomes from embryos (40 g)
1-hr-incubated
0.5
0.025
6.01
0.4
Polyribosomes from embryos (40 g)
4-hr-incubated
1.45
0.036
6.02
0.4
0 Poly(Akontaining RNA, 4.5 A,,, unit, from unincubated cysts was applied on top of 17-ml 15-30% linear sucrose gradients in SDS buffer and was centrifuged at 26,000 rpm for 20 hr in the SW 27.1 rotor at 20°C (Felicetti et al., 1975). Gradient fractions, 1.5 ml, were precipitated with ethanol, and the RNA was dried under vacuum and dissolved in 100 ~1 of sterile water. Aliquots of each RNA fraction were tested in a wheat germ lysate as described in Materials and Methods. After incubation, the most active fractions (tubes 2-11) were pooled and processed for electrophoretic analysis as described in Materials and Methods. Poly(A) + RNA from the polysomes of incubated embryos was tested directly in a wheat germ lysate, and the reaction mixtures were processed as above. In this experiment, the endogenous incorporation of wheat germ lvsate in the absence of anv RNA addition was 0.3 x 10e5 cpm/50 ~1. b Of each gradient fraction.
ferent cytoplasmic fractions of Artemia salina embryos in the course of development (unincubated and 16-hr-incubated cysts). They found that the total mRNA activity [poly(A)+ and poly(A)RNA) of the embryos increased threefold, but the most pronounced change with development was observed in the poly(A) + RNA activity which increased over sixfold. The discrepancy with our results can be explained if one considers in detail the different experimental approaches used: (a) We have focused our attention on the very early stages of development (up to 8 hr), and one can assume that such profound changes in the activity of poly(A) + mRNA have not occurred yet; (b) we neglected the
amount of poly(A) + RNA found in the mitochondrial fraction P,, since we have previously found that it does not account for more than 10-E% of the total preformed mRNA; Cc) Sierra et al. (1976) measured activity of poly(A)+mRNA fractions in a wheat germ lysate, while we chose a more quantitative approach by measuring the amount of [3Hlpoly(U) hybridized. We have constantly found that poly(A)+ mRNA from unincubated cysts is less efficiently translated in a wheat germ lysate than the corresponding fractions derived from polysomes of incubated embryos, and this is due to the presence of low molecular weight inhibitory components in the oligo(dT)-cellulose RNA frac-
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DEVELOPMENTAL
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FIG. 5. Autoradiography of %&labeled proteins synthesized in a wheat germ lysate and analyzed by SDS-slab gel electrophoresis. Poly(A)-containing RNA fractions from undeveloped and developed embryos were translated in wheat germ lysate as described in Table 2. The incubation mixtures corresponding to poly(A)-containing RNA from unincubated cysts (0 time) and from incubated embryos (1 and 4 hr) were processed as described in Materials and Methods. The amount of TCA-insoluble radioactivity applied to each slot was 102 x 10e3 cpm for the O-time sample (100 ~1 of incubation mixture) and 230 x 1O-3 cpm for the I- and 4-hr samples (50 ~1 of incubation mixture). The autoradiograms were exposed for 15 and 7 days respectively. fl-Galactosidase, (MW 130,000), ovalbumin (MW 43,000), and chymotrypsinogen (MW 25,700) were used as molecular weight standards.
tions. It is likely that mRNA activity in some of the cytoplasmic fractions of unincubated embryos has been underestimated: Practically, they found no detectable activity in the S-105 and ribosomal fraction. In agreement with our results is the work by Grosfeld and Littauer (1975)
who found that the postmitochondrial supernatants of dormant and incubated developing cysts contain approximately the same quantities of poly(A)-rich mRNA. The facts discussed extensively above are pertinent to the following question. As soon as development resumes, is it a burst
FIG. 6. Autoradiography of YS-labeled proteins synthesized in a wheat germ lysate and analyzed by twodimensional gel electrophoresis. The amount of TCA-insoluble radioactivity applied to first-dimension gels (see Materials and Methods) was 204 x 10m3 cpm for the O-time sample (200 ~1 of incubation mixture) and 460 x 10m3 cpm for the l- and 4-hr samples (100 ~1 of incubation mixture). The autoradiograms were exposed for 10 days (l- and 4-hr samples) and 20 days (O-time sample).
59
60
DEVELOPMENTAL
BIOLOGY
of synthesis of new mRNA species which occurs or is the mRNA stored in the cysts what is mainly utilized by the embryos? We are more inclined to support the second hypothesis since our results have clearly shown that, first, there is a flow of informational RNA from the subribosomal poly(A)-containing particles to the polysomes of developing embryos; second, there is a close similarity between the translational patterns at very early stages of development and the polypeptides synthesized in a wheat germ lysate by preformed mRNA stored in the cysts. Our results do not rule out the possibility that breakdown and resynthesis of the same family of mRNA sequences occur after cyst incubation. This event seems very unlikely to us, since there would be no reason for encysted embryos to preserve a defined set of mRNA sequences in their cytoplasm and destroy them as soon as development is resumed. Conventional slab gel electrophoresis revealed that at least one band with a molecular weight less than 27,500 daltons was missing in the O-time slab (Fig. 5). The fact that this finding was not confirmed by careful examination of the spots resolved by two-dimensional gel electrophoresis suggests that the labeled polypeptide(s1 does not enter the firstdimension gel and is probably stuck at the origin (as if the isoelectric point was higher than 7). Despite this limitation, two-dimensional slab gel electrophoresis proved to be an essential tool in unraveling the complex pattern of proteins synthesized in the embryos and allowed us to detect any subtle change among the hundreds of spots revealed by autoradiography. The extra band and the new spots observed in the slabs corresponding to l- and 4-hr-incubated embryos may derive either from early transcription of a new mRNA species or simply by post-transcriptional modifications of pre-existing mRNA molecules. It has been shown that mRNAs from both
VOLUME
59,
1977
developed and undeveloped embryos contain 5-terminal 7-methyl-guanosine, in an inverted linkage through three phosphate groups to the rest of polynucleotide chains, and that extracts from both developmental stages possess &terminal methylase activity (Muthukrishnan et al., 1975). It has been concluded that the low level of protein synthesis in dormant embryos cannot be explained by a defective mRNA methylation. However the process of capping and methylation at the 5 terminus of mRNA could still be invoked as a regulatory mechanism to activate translation of discrete species of cyst-preformed mRNA in early. development. The mechanism of activation of protein synthesis in the encysted embryos is still obscure. If one carefully examines the polyribosome patterns in the embryos incubated for short periods of time (see Fig. 31, it appears that active polysomes are progressively increasing in size, as if, at the beginning, the rate of initiation is impaired and preformed mRNAs are not fully charged with ribosomes. This observation is in contrast with the hypothesis that preformed mRNA is intrinsically unable to interact with ribosomes and suggests that a limitant amount of initiation factors is present in unincubated cysts. Similar conclusions have been drawn by Filipowicz et al. (1975, 1976) who found that the activity of IF-MP, an initiation factor that promotes binding of met tRNA, to 40s subunits, increases over 20-fold following resumption of development. REFERENCES P., RUSCA, G., and CALISSANO, P. (1973). In vitro synthesis of a brain-specific protein (S-100) by free and membrane bound polysomes. Biochin. Biophys. Actu 299, 634-641. FELICETTI, L., PIERANDREI AMALDI, P., MORETTI, S., CAMPIONI, N., and URBANI, C. (1975). Intracellular distribution, sedimentation values and template activity of polyadenilic acid-containing RNA stored in Artemia salina cysts. Cell Differ. 4, 339354. AMALDI,
AMALDI,
FELICETTI,
AND
CAMPIONI
SIERRA, J. M. and OCHOA, S. (1975). chain initiation in eukaryotes: InitiaMP in Arremia salina embryos. Proc. Sci. USA, 72, 3947-3951. W., SIERRA, J. M., NOMBELA, C., OCHOA, S., MERRICK, W. C., and FRENCH ANDERSON, W. (1976). Polypeptide chain initiation in eukaryotes: Initiation factor requirements for translation of natural messengers. Proc. Nat. Acad. Sci. USA 73, 44-48. FINAMORE, F. J., and CLEGG, J. S. (1969). Biochemical aspects of morphogenesis in the Brine shrimp, Artemia salina. In “The Cell Cycle” (G. M. Padilla, G. L. Whitson, and I. L. Cameron, eds.), pp. 249-278. Academic Press, New York. GROSFELD, H., and LITTAUER, U. Z. (19751. Cryptic form of mRNA in dormant Artemia salina cysts. Biochem. Biophys. Res. Commun. 67, 176-181. HULTIN, T., and MORRIS, J. E. (1968). The ribosomes of encysted embryos of Artemia salina during cryptobiosis and resumption of development. Deuelop. Biol. 17, 143-164. JACOBS-LORENA, M., BAGLIONI, C., and BORUN, T. W. (1972). Translation of messenger RNA for histones from HeLa cells by a cell-free extract from mouse ascite tumor. Proc. Nat. Acad. Sci. USA 69, 2095-2099. FILIPOWICZ,
Polypeptide tion factor Nat. Acad. FILIPOWICZ,
W.,
Flow
of Informational
RNA
61
LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of Bacteriophage T 4. Nature (London) 227, 680-685. MUTHUKRISHNAN, S., FILIPOWICZ, W., SIERRA, J. M., BOTH, G. W., SHATKIN, A. J., and OCHOA, S. (1975). mRNA methylation and protein synthesis in extracts from embryos of brine shrimp, Artemia salina. J. Biol. Chem. 250, 9836-9341. NILSSON, M. O., And HULTIN, T. (1974). Characteristics and intracellular distribution of messenger like RNA in encysted embryos of Artemia salina. Deuelop. Biol. 38, 138-149. O’FARREL, P. N. (1975). High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007-4021. ROBERTS, B. E., and PATERSON, B. M. (1973). Efficient translation of tobacco mosaic virus RNA and rabbit globin 9s RNA in a cell-free system from commercial wheat germ. Proc. Nat. Acad. Sci. USA 70, 2330-2334. ROSBASH, M. and FORD, P. J. (1974). Polyadenylic acid-containing RNA in Xenopus laeuis oocytes. J. Mol. Biol. 85, 87-101. SIERRA, J. M., FILIPOWICZ, W., and OCHOA, S. (1976). Messenger RNA in undeveloped and developing Artemia salina embryos. Biochem. Biophys. Res. Commun. 69, 181-189.