Ribonucleic acids and ribonucleoproteins from small oöcytes of Xenopus laevis

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 96624

RIBONUCLEIC ACIDS AND RIBONUCLEOPROTEINS FROM SMALL

OOCYTES OF XENOPUS LAEVIS

99

C H R I S T I A N THOMAS*

Laboratoire de Morphologie animale, Universitd libre de Bruxelles, 67, rue des Cheveaux, Rhode- Saint-Gen~se (Belgique)

(Received May 8th, 197 o)

SUMMARY

I. More than 80 % of the RNA of small o6cytes of Xenopus laevis is 4-S RNA. 2. Several constituents distinguishable by means of methylated-albumin col-

umn chromatography are found in this RNA and are present in two different ribonu- cleoproteic fractions, the sedimentation constants of which are, respectively, 5-6 S and about 20 S.

3. The ribouucleoproteic fractions are also heterogeneous, as shown by CsC1 gradient analyses and [3Hluridine incorporation; they appear from electron microscopic observations to be composed of many fibrils about Ioo ~ long, like those one can observe "in sit#' in sections of small o6cytes.

4. At this stage, o6cytes contain very few ribosomes and ribosomal RNA. 5. The possible nature and function of this low molecular weight RNA are

discussed.

INTRODUCTION

The cytoplasm from small o6cytes (less than 250/~m) of Xenopus laevis has been found to be devoid, or almost devoid, of ribosomes our evidence being based on results of electron microscope experiments 1,z. RNA is visible as fine fibrils, in fibrillogranular threads, in these o6cytes. On the contrary, o6cytes undergoing vitellogenesis contain, numerous ribosomes interspersed with the fibrillogranular threads (Figs. I and 2). These ultrastructural differences led us to extract RNA and ribonucleoproteins from the small o6cytes of X. laevis.

A short account of the present work has been published in a preliminary note s.

MATERIALS AND METHODS

Ovaries of Xenopus laevis were used soon after metamorphosis (about 2 months) since they provided homogeneous material uncontaminated by o6cytes undergoing vitellogenesis. These ovaries will he referred to as "young ovaries". As o6cyte diam- eters varied somewhat, the term "small o6cytes" has been used to define o6cytes having a diameter of 25o #m or less, as in our previous work 1-3.

* Aspirant du Fonds National de la Recherche Scientifique.

Biochim. Biophys. Acta, 224 (197 o) 9 9 - I I 3

I00 C. THOMAS

Fig. I. Section of small o6cytes of X. laevis (about lOO-2OO/~nl in diameter). Fibrillogranular threads(t) with many fibrils(f) appear in the cytoplasm (× lO8 ooo).

Fig. 2. Section of o6cytes of X. laevis undergoing vitellogenesis (about 400 #In in diameter). Many ribosomes (r) interspersed with fibrillogranular threads (t) appear in the cytoplasm ( X lO8 ooo).

(z) Homogenization and extraction o/ribonucleoproteins (a) For a s tudy of the ribonucleoproteins, pieces of X. laevis "young ovaries"

were homogenized, at 4 °, in the following medium: o.oi M phosphate buffer, o.I M KC1, o.oi M magnesium acetate, 0.25 M sucrose, I 9/0 lubrol (pH 7.4)- Pieces were also homogenized in the same medium without Mg 2+. In some cases, homogenization was performed in the presence of IO/~g/ml polyvinyl sulfate (a ribonuclease inhibitor). We were unable to detect any differences in the amounts or the properties of ribonucleoproteins, whether the inhibitor was employed or not. The homogenate was first centrifuged, at 4 °, in a 40 rotor of a "Spinco L2" ultracentrifuge at 20 ooo rev./ rain for 20 rain, to exclude most of the intracellular organelles.

(b) The supernatant obtained after the first centrifugation was layered over 15-3o °/o linear sucrose gradients in the same buffer. A second centrifugation was performed at 4 °, with a SW 50 or SW 65 rotor, in a "Spinco L2" ultracentrifuge, either at 50 ooo rev./min for 2-6 h, or at 35 ooo rev./min for lO-I5 h. The gradients were analyzed by the continuous flow method, at 260 nm, using a Cary spectrophotometer and injection of concentrated sucrose at the bot tom of the gradient.

(2) Extraction ol RN A (a) Extract ion of RNA from pieces of "young ovaries": The pieces of "young

ovaries" were homogenized, at o °, in o.oi M sodium acetate, o.I M NaC1, I mM EDTA, I ~o sodium dodecyl sulfate and IO/~g/ml polyvinyl sulfate (pH 5)-The homogenate was shaken, at o °, for 3 times IO rain with an equal volume of redistilled water- saturated phenol. RNA was precipitated from the aqueous phase, at --20 °, by addition of 2 vol. of ethanol containing NaC1 in a final concentration of 1 % .

]Siochim. Biophys. dcta, 224 (i97 o) 99-113

RNA AND RIBONUCLEOPROTEINS FROM SMALL O(~CYTES IOI

(b) Extract ion of RNA from RNA-rich fractions obtained from sucrose gradients, after homogenization in pH 7.4 medium: The fractions were either shaken with phenol, as in Method 2a, or with 1 % sodium dodecyl sulfate, for a few rain at 37 °.

(c) RNA was layered over 5-20 % or 15-3o % linear sucrose gradients made up in o.oi M sodium acetate, o.i M NaC1, I mM EDTA and IO/ ,g /ml polyvinyl sulfate (pH 5). The centrifugation was performed in a "Spinco L2" ultracentrifuge at 50 ooo rev./min for 3-4 h at 4 °. The gradients were analyzed as in lb.

(3) Incorporation o/ [3Hluridine into "young ovaries" (a) Pieces of ovaries were incubated in I ml of TC 199 medium 4,5 (Difco) con-

taining lOO-3OO #C EaHJuridine (specific activity 4-5 C/mmole) for 2-3 days at room temperature (long periods of incorporation were used to obtain highly radioactive RNA and to s tudy the stable RNA). This t reatment does not seem to damage the o6cytes, since it does not alter the properties of either the RNA or the ribonucleoproteins. Under these conditions, o6cytes of Triturus viridiscens will continue to incorporate for several days and at the end of this time many nuclei contain morpho- logically normal lampbrush chromosomes 4.

(b) Homogenized ovaries were treated and subjected to sucrose-gradient analysis as described above (see p. I). The fractions were collected after gradient centrifugation and the nucleic acids were precipitated with 5 % trichloroacetic acid. The precipitate was collected on "Millipore" filters. In some cases the fractions were filtered directly on "Millipore" filters in order to distinguish between ribonucleoproteins and "free" RNA (see ref. 6). The filters were counted in a scintillation medium (5 g 2,5-diphenyloxazole (PPO), 0.5 g (I,4-bis-(5-phenyloxazolyl-2)benzene (di- methyl POPOP) per 1 toluene) in a "Nuclear Chicago" liquid-scintillation counter.

(4) CsCl density gradient centri/ugation After homogenization of pieces of "young ovaries" in pH 7-4 medium, the

RNA-rich fractions from sucrose gradients were fixed for 24 h or more, at 4 °, with 6 % formaldehyde, by addition of 0.2 vol. formol to the fractions 7,e. In order to eliminate sucrose and fomaldehyde, these fractions were dialysed against the homogenization buffer. They were then layered onto CsC1 preformed gradients, ranging in density from 1. 3 to 1.8 g/cm 3, in the following medium: o.oi M tr iethanolamine- HC1, o.I M KC1, o.oi M magnesium acetate (pH 7-4). The gradients were centrifuged in the SW 65 rotor of a "Spinco L2" ultracentrifuge for 21 h at 48 ooo rev./min at 4 °. The gradients were read at 260 nm, under continuous flow, using a Cary or Gil- ford spectrophotometer and injection of a saturated solution of CsC1 at the bot tom of the gradient. Buoyant densities were calculated from the refractive indices.

(5) Base composition The RNA solution was incubated in 0. 3 M KOH for 18 h at 37 ° and ap-

plied to thin-layer cellulose s for two-dimensional chromatography. The solvents used at room temperature wereg: Solvent I : 250 ml isobutyric acid, 5 ml conc. NH4OH, 145 ml twice-distilled water. Solvent 2 : 8 0 ml satd. (NH4)2SO4, 18 ml I M sodium acetate, 2 ml isopropanol. The base composition of RNA was analysed by means of the absorbance at 260 nm.

Biochim. Biophys. Acta, 2z 4 (197 o) 99-ii 3

lO2 c. THOMAS

(6) RNA analysis on methylated-albumin column RNA in o.o5 M Tris-HC1 buffer (pH 7) was layered on a methylated-albumin

column 1° (I c m × 2 - 3 cm) and eluted, at room temperature, with 9O-lOO ml of a linear gradient NaC1 solution (0.2 to I M or 0.2 to 1.2 M in 0.05 M Tris-HC1 buffer, pH 7). I-ml fractions were collected. RNA radioactivity was estimated in a "Nu- clear Chicago" liquid-scintillation counter, after addition of BRAY'S 11 solution to each fraction.

(7) Electron microscopic observations RNA-rich fractions from sucrose-gradient centrifugation were treated with

6 % formaldehyde for 24 h or more, at 4 °, by addition of 0.2 vol. formol to the fractions. After dialysis against the homogenization buffer, the material was applied to carbon-coated copper grids. After being washed with water, the grids were floated upon a 1 % uranyl acetate staining solution for 2 h. Staining with lead citrate 12 was then carried out for a few seconds. Photographs were taken with an AEI EM6B electron microscope, supplied with a decontamination system, at 60 kV and a t !a direct magnification of 60 ooo.

(8) RNA extractions on isolated o6cytes o / X . laevis In order to compare RNA in o6cytes of various sizes, these were isolated by

treatment of ovaries with pronase 13. Pieces of ovaries from X. laevis were treated with pronase (200/~g/ml in Ringer buffer) for 1-2 h at room temperature. The properties of RNA from these "treated" o6cytes were not changed. 06cytes released by shaking pieces of ovaries were graded according to size and color. RNA was extracted as described in Method 2a.

(9) Preparation o / R N A used as a re[erence (a) Radioactive 28-S RNA. Pieces of ovaries from X.laevis containing o6cytes

undergoing vitellogenesis, were incubated with [3Hluridine as in Method 3a. After homogenization, following the method described in i, the ribosomes were harvested and their RNA extracted with phenol. Radioactive 28-S RNA was isolated after sucrose gradient centrifugation.

(b) Extraction o] soluble RNA and low molecular weight RNA (4 S and 5 S) bound to ribosomes [rom liver o] X. laevis. Livers of X. laevis were homogenized in the following medium at 4°: 0.05 M Tris-HC1, o.i M KC1, o.oi M magnesium acetate, 0.25 M sucrose (pH 7.4)- The homogenate was centrifuged at 4 °, in the SS 34 rotor of a "Sorvall RC2" centrifuge for 15 rain at 13 ooo rev./min. The supernatant solution was then centrifuged, at 4 °, in the 65 rotor of a "Spinco L2" ultracentrifuge for I h at 65 ooo rev/min. The supernatant of the second centrifugation was phenol- extracted following the method described in 2a, in order to obtain soluble RNA. The pellet from this second centrifugation was taken up in the same homogenization medium at 4 ° and deoxycholate was added to a final concentration of 0.5 %. A third centrifugation yielded a ribosome pellet, the RNA of which was extracted following Method 2a and filtered through a I cm × 7 ° cm Sephadex G-Ioo column, at 4 °, with a o.oi M sodium acetate buffer (pH 5)- The low molecular weight RNA was eluted from the column after the unretarded fraction.

Biochim. Biophys. Acta, 224 (197 o) 99-113

RNA AND RIBONUCLEOPROTEINS FROM SMALL OOCYTES 103

RESULTS

(z) RNA /rom "young ovaries" o~ X. laevis Most of the RNA from pieces of "young ovaries" containing small o6cytes is

low molecular weight RNA (more than 80 %) (Fig. 3)- The sedimentation coefficient of this RNA is about 4 S, because it sediments in a sucrose gradient at the same position as the soluble 4-S RNA of E. coli (Fig. 4). I8-S and 28-S ribosomal RNA peaks are obvious, but only occur in small amounts.

1.5 4S

E ~.od

: [ / i

top bottom

1.0 [

0'8 t

0.6_ // ~0.4- / /

So,

~ 0 ~ ~ o B

1250

/,, ooo? / A ",,

%,.. %-°.°.°

2'0 Fraction No.

Fig. 3. Sedimenta t ion pa t t e rn of RNA from pieces of "young ovaries" containing smal l o6cytes of X. laevis. 5-20 % sucrose gradient centr i fugat ion for 3 h a t 4 ° and 5 ° ooo rev . /min (SW 5o). "Cont inuous reading" . Sedimenta t ion from left to right.

Fig. 4. Sedimenta t ion pa t t e rn of labeled low molecular weight R N A from Peaks I and 2 of ribonucleoprotein ext rac ts f rom "young ovaries" of X. laevis toge ther wi th non-labeled 4-S soluble R N A from E. coll. 15-3o % sucrose gradient centr i fugat ion for 12 h at 4 ° and 35 ooo rev. /min (SW 50). Sedimenta t ion from left to right.

(2) Ribonueleoproteins /rom "young ovaries" o/X. laevis The sedimentation in sucrose gradients of homogenates of "young ovaries"

results in two main peaks at the top of the gradient, which are present in much larger amounts than the ribosomal peak, the sedimentation coefficient of which is about 80 S (Fig. 5). The surface ratio of the ribosomal peak to "light peaks" corresponds

Peak 1

1"5~ .,~ak i 1.0 2

Ribosomes ° ! \ _/k "~ 0

top bottom

Fig. 5. Sedimenta t ion pa t t e rn of an ex t rac t from pieces of "young ovaries" of X. laevis. 15-3o ~o sucrose gradient centr i fugat ion for 2 h a t 4 ° and 5 ° ooo rev . /min (SW 5o). "Cont inuous reading" . Sed imenta t ion from left to right.


lO4 c. THOMAS

to that of ribosomal RNA to the 4-S RNA peaks (see Fig. 3). The lighter of these two peaks (Peak I) is most abundant. The two peaks have sedimentation coeffi- cients of 5-6 S (Peak I) and about 20 S (Peak 2). Extraction with phenol or sodium dodecyl sulfate of the RNA from each of these two peaks gives in each case a sedimentation coefficient of about 4 S.

(3) Risks o / R N A degradation The presence of large quantities of low molecular weight RNA after extraction

of the ovaries suggested that the RNA may have been degraded during homogenization. In order to eludicate this point, a small amount of labeled 28-S RNA from X. laevis o6cytes undergoing vitellogenesis was added during homogenization of pieces of "young ovaries". Analysis, in a sucrose gradient, of the ribonucleoproteins from the homogenate does not show a movement of radioactivity to the top of the gradient, but rather an increase and a "spreading" of the sedimentation coefficient of the added RNA (Fig. 6). Such an increase in the sedimentation rate of added RNA by interactions with soluble proteins was described by GIRARD AND BALTIMORE 14 and BALTI- MORE AND HUANG 15.

300

E2oo-

t)

.~100 ~d 8

~ o ©

rE]- 28s

i

°." -o*'~' \ • o.° o °.o~°

. . . . . . °.o-°

16 2; Froction No.

1.5

1.0-

0.5-

o

o o

I

-60~ F

-45~ o

1'o 2'o 30 Fraction No.

Fig. 6. a. Sedimentation pattern of labeled 28-S ribosomal RNA from ovaries of X. laevis containing o6cytes undergoing vitellogenesis, b. Sedimentation pattern of labeled 28-S ribosomal RNA from ovaries of X. laevis containing o6cytes undergoing vitellogenesis together with an extract from pieces of "young ovaries" of X. laevis. 15-3o % sucrose gradient centrifugation for 2 h at 4 and 5 ° ooo rev./min (SW 5o). "Continuous reading". Sedimentation from left to right.

An electron microscopic s tudy of sections of small o6cytes of X. laevis after ribonuclease digestion and selective staining of the RNA-containing structures 2 also argues against an eventual degradation: it shows that cytoplasmic RNA is visible as fibrils having a diameter of lO-2O •. The longest fibrils are about lOO-15o A long. As section thickness is usually higher than this value, one can say that the longest fibrils we can observe are whole unsectioned molecules. This observed dimension is in agreement with that of a low molecular weight RNA.

(4) Properties o/ Peaks i and 2 /rorn the ribonucleoprotein homogenates (a) The e/]ects o/ Mg 2+ (Fig. 7) Extraction of pieces of "young ovaries" in a homogenization medium lacking

Mg ~+ does not greatly alter the absorbance outline of Peaks I and 2 in a sucrose gradient.

Biochim. Biophys. Acta, 224 (197o) 99-113


2.0

Peak I

1 . 5 - ~

1.0- E =

o l

'~ 0.5-

O-

Ribosomal subunlts ?

bottom

1.54

1.0-

E c

0.5-

o i

o

;'-,, 1 1 5 0 0

: ". i :' . ~.

. • ......... :/"°' t1°°°~ i,-.\",, i ',

500 ~

~6 ig Fraction "No.

Fig. 7. Sedimentation pat tern of an extract carried out without Mg 2+ on pieces of "young ovaries" of X . laevis. 15-3o % sucrose gradient centrifugation for 2 h at 4 ° and 5 ° ooo rev./min (SW5o). "Continuous reading". Sedimentation from left to right.

Fig. 8. Sedimentation pat tern of an extract from pieces of "young ovaries" of X. laevis labeled in vitro for 2 days with [SH]uridine. 15-3 ° % sucrose gradient centrifugation for 3.5 h at 4 ° and 5o ooo rev./min (SW 5o). Fractions 1-8 and 13-17 were collected, respectively, for methylated- albumin chromatography of Peak i RNA (Fig. 9) and Peak 2 RNA (Fig. io).

(b) [3HlUridine incorporation After [~H]uridine incorporation in pieces of "young ovaries", for several days,

radioactivity appears in Peaks I and 2 (Figs. 8 and 15). This radioactivity is acid- soluble after a t reatment with 0.3 M KOH for 18 h at 37 °. Specific activity of Peak 2 is larger than that of Peak I (Figs. 8 and 15).

(c) Study o[ RNA present in each peak ( i ) Base composition: The two Peaks (I and 2) have a high G + C content but

differ in base composition (Table I).

TABLE I

NUCLEOTIDE COMPOSITION OF IRNA FROM THE TWO PEAKS (NUCLEOTIDES °/o )

Values are means from three analyses.

Nucleotides Peak z Peak 2

A 19.5 22.6 U 19.7 17.6 G 33.7 33.7 C 27 26.1

(ii) Chromatographic properties on methylated-albumin column: The RNA of Peak I elutes as one peak from a methylated-albumin column, at a concentration of 0.5 to 0.55 M NaC1 (Fig. 9). The RNA of Peak 2 gives two peaks at 0.45-0.50 M and 0.50-0.55 M NaC1 (Fig. IO). In our experimental conditions, the RNA of Peak i and one of the two RNA's of Peak 2 (eluting with the highest NaC1 concentration) elute with a similar or nearly similar NaC1 concentration. In order to compare the chromatographic behaviour of the RNA of Peaks I and 2, with that of transfer and 5-S ribosomal RNA, chromatography of a small amount of labeled RNA from Peaks

Biochim. Biophys. Aeta, 224 (197 o) 99-113

lO6 c. THOMAS

0.15-

0,1- -0.6 E -o.5 o - 0 . 4

0.05- -0.3

0 i i i

30 40 .50 Fraction No.

/ \,,

-300 1

.c_ E

mo~

~o ~ .8_

0 w

Fig. 9. lV[ethylated-albumin column chromatography of RNA from Peak I extracted from pieces of "young ovaries" of X. laevis incubated in vitro for 2 days wi th [3H]uridine (the par t of Peak I which was analysed corresponds to Fract ions i 8 described in Fig. 8).

0'1 t ~" ,% ,%

0.084 43 : " p_°. /. '.

i 0.0 0.6 ' "° ' ' '~',

o.o4:0.4 _~ <

30 4 '0 ' 5 ' 0 ' Fraction No.

2oo 7

-150 .~

-lOO ~ o

-50 '~ 8 .9

0 a:

Fig. IO. Methylated-albumin column chromatography of RNA from Peak 2 extracted from pieces of "young ovaries" of X. laevis incubated in vitro for 2 days with [aH]uridine (the pa r t of Peak 2 which was analysed corresponds to Fract ions 13-17 described in Fig. 8). Note: The RNA which was analysed in Figs. 9 and IO is not whole RNA of Peaks I and 2, as Fract ions 9-12 described in Fig. 8 are not present.

I and 2 was s imultaneously performed together with the low molecular weight R N A from the liver of X. laevis. It was found that (Figs. II and 12): a fraction of R N A from Peak 2 (eluting at the lowest NaC1 concentration) elutes like soluble R N A (supernatant of ribosomes) and transfer R N A bound to ribosomes; the R N A of Peak

0.4-

0.3-

~0.2- E c

.~ 0.1-

o

So / " "\. / \ 05 / -0.4 ~ \ . / ",,o -o.3 - t T . . . . . . . . . . . . . . . . ;<.- "

/ , /

- I I ~ i i i " i i

25 30 35 40 45 50 Fraction No.

8oo T £

-6CX3 .~

~oo

-200 >

o_

0

Fig. i I. Methylated-albumin column chromatography of soluble RNA from the liver of X . laevis together wi th labeled 4-S RNA from Peaks i and 2 of an extract from "young ovar ies" of X. laevis incubated in vitro with [SH]uridine.


RNA AND RIBONUCLEOPROTEINS FROM SMALL OOCYTES lO7

0.2

.~ 0.12-

o.o~- o.~ ~ . o oA i/ ,,,. \

" o.o4:-o3 y " " ' ~ " \, \.

2s 3o 3~ 4'0 ~5 3b FPaction No.

000%.

800 :

8

r oo o

I 0 rr

Fig. 12. M e t h y l a t e d - a l b u m i n co l umn c h r o m a t o g r a p h y of low molecu la r weight r ibosomal R N A f rom the l iver of X. laevis t oge the r wi th labeled 4-S R N A f rom Peaks I and 2 of an ex t rac t f rom the " y o u n g ovar ies" of X. laevis i ncuba t ed in vitro wi th pH]ur id ine .

I and the remaining fraction of that of Peak 2 (eluting at the highest NaC1 concentration) elute between transfer RNA and 5-S ribosomal RNA.

(d) RNA/proteins ratio of Peaks z and ~. Since the fractions (Peaks I and 2) to be studied occur at the top of the sucrose

gradients, it was impossible to make a direct analysis of the RNA/protein ratio in this region; the results would be distorted by the presence of abundant soluble proteins in the same region. However, the RNA/protein ratio of the several constituents could then be estimated by their buoyant density in CsC1 gradients and the use of nitrocellulose membrane filters enabled a distinction to be made between ribonucleoproteins and "free" RNA owing to the adsorption of the former by membrane filters 6.

(i) CsC1 gradients: Peak I shows several components (Fig. I3), the largest of which has a high buoyant density (greater than 1.65) and is probably composed of "free" RNA or (and) of ribonucleoproteins with a high RNA/protein ratio. However, ribonucleoproteins having a buoyant density of 1.3-1. 5 g/cm 3 are also present in smaller amounts. A large proportion of ribonucleoproteins with a low RNA/protein

o.6 I

0.4 i

/ \

1.3 1.4 1.5 1.(5 Density(g/cm 3)

°ltA ~ 0.05

\ 1.3 1.4 1.5 1.6

Density (g/crn3)

Fig. 13. Sed imen ta t i on pa t t e rn , in a CsC1 pre - formed gradient , of t he fomaldehyde- f ixed Peak I f r om an ex t r ac t of " y o u n g ovar ies" of X. laevis (see Note, Fig. 14). Cent r i fuga t ion for 21 h a t 4 ° and 48 ooo r ev . /min (SW 65). "C on t i nuous read ing" .

Fig. 14. Sed imen ta t i on pa t t e rn , in a CsCI pre - formed gradient , of fo rmaldehyde- f ixed Peak 2 f rom an ex t r ac t of " y o u n g ovar ies" of X. laevis (see Note). E x p e r i m e n t a l condi t ions as in Fig. 13. Note: High d e n s i t y c o m p o n e n t s did no t necessar i ly reach the i r equ i l ib r ium b u o y a n t densi ty , as t h e y were cen t r i fuged in a CsC1 pre - formed g rad ien t for per iods of t ime below those requi red for 4-S R N A to reach t he b o t t o m of t he gradient . R u n s were no t prolonged fu r the r in order to avoid ex tens ive degrada t ion of r ibonuc leopro te ins in t he presence of CsC1.

Bioehim. Biophys. Acta, 224 (197o) 99-113

lO8 C. THOMAS

ratio is present in Peak 2 (Fig. 14). However, a high buoyant density component accounting for more than 50 % of the RNA of this peak is also present. I t is difficult to see if this "light" component is a real structure or a dissociation product of Peak 2. Indeed, Peak 2 seems unstable after dialysis or formaldehyde treatment and one must also take into account the possibility of degradation of ribonucleoprotein components by CsC1, in spite of fixation with formaldehyde.

(ii) Use of "Millipore" nitrocellulose filters (Fig. I5): A part of Peak I label cannot be retained on "Milipore" membrane filters and may probably be akin to "free" RNA without proteins. In contrast, the whole Peak 2 label is bound to membrane filters.

(iii) Possibility of RNA interactions with cellular proteins during homogenization: Addition of a cellular extract increases the sedimentation rate of RNA through the interactions of proteins with RNA ~4,~5. As we had to take this effect into account, we added radioactive 4-S RNA from Peaks I and 2, during homogenization of pieces from "young ovaries". Fig. 16 shows a change in the sedimentation rate of a great amount of added RNA, the label outline of which partially fuses with the absorbance outline of Peaks I and 2. A part of the radioactive RNA can be retained on nitrocellulose membrane filters and so must be bound to proteins.

2 . 5 I - - -

2.o2 i ",, / .;'/I,.o /., i ].s, i ' "~ "Z° ";"

1.o~ ]. ! \ '~, . ° ,~/ '° . , - ' ' \ " o L ..... "? ~ "~">" / X • ] : --.-~'"

o s~ / ' i

0 i 10 ~ 30 Frac t ion No. -

5000 2.0

~4oooi i

"~ 1.0 - c E

-2000 o ~

4 0 0 0 > 0 . 5 -

0 ~ 0

] 5 0 0

40011

, ~ ._c

\ / / ! /

; / '- %.o ,- ~'~ "~

lb 2'0 3b Fraction No.

Fig. 15. Sucrose gradient centrifugation of acid-insoluble RNA and " b o u n d " RNA (RNA retained by nitrocellulose membrane filters) from an extract of "young ovaries" of X. laevis incubated for 2 days with [3H]uridine. 15-3o % sucrose gradient centrifugation for 15 h at 4 ° and 35 ooo rev./min. Centrifugation from left to right.

Fig. 16. Sucrose gradient centrifugation of labeled ovarian 4-S RNA (extracted from Peaks I and 2) together wi th an extract of "young ovaries" of X. laevis. 15-3o °/o sucrose gradient centrifugation for 15 h at 4 ° and 35 ooo rev./min. "Cont inuous reading". Centrifugation from left to right. O - - - O , acid-insoluble RNA. A - - - ~., bound RNA.

Such modifications of 4-S RNA behaviour during homogenization we showed, have an affect upon the interpretation of the reality of ribonucleoproteins and con- firm the possible formation of artificial ribonucleoproteins. However, artificial aggre- gations of RNA and proteins do not explain the overall properties of the Peaks I and 2, the RNA's of which are different as was shows in previous sections.

To conclude, ribonucleoproteins present in the two peaks could, at the same time, not only come from artificial interactions between RNA and proteins during homogenization but also from cellular structures present "in situ". A part of Peak I could be "free" RNA.



(e) Electron microscopic studies Electron microscopic studies of components of Peaks I and 2 (Figs. 17, 18),

after positive staining, showed that they are chiefly composed of fine fibrils, although one can also see many granules. These fibrils, the diameter of which is lO-3O A, are about lOO-15o A long and could be 4-S RNA "free" or associated with proteins. This value is in agreement with that of fibrils observed "in situ" in sections of small o6cytes 1,2. One must take into account that formaldehyde fixation leads to a large decrease in the sedimentation rate of Peak 2. Then, the appearance of that peak, after formaldehyde fixation and electron microscopic examination, could not depict the real aspect of the 2o-S component isolated from a sucrose gradient.

Fig. 17. E lec t ron microscopic aspec t of P eak I c o m p o n e n t s f rom an ex t r ac t of " y o u n g ovar ies" of X. laevis, f, fibrils. ( x 270 ooo).

Fig. 18. E lec t ron microscopic aspec t of Peak 2 c o m p o n e n t s f rom an ex t r ac t of "young ovar ies" of X. laevis, f, fibrils. (× 270 ooo).

(5) Low molecular weight RN A evolution in X. laevis oi~cytes 200-400 [zm in diameter We studied these o6cytes in order to see what is the fate of the low molecular

weight RNA of small o6eytes from X. laevis at the onset of vitellogenesis, a stage at which numerous ribosomes are visible with the electron microscope in the o6cyte cytoplasm 1, 2.

(a) Amount o / R N A present in the ob'cytes In order to estimate approximately the amounts of low molecular weight RNA

present in o6cytes, we extracted RNA from an identical number of o6cytes isolated by t reatment with pronase. Three classes of o6cytes were selected: (i) o6cytes about 200-250 #m in diameter: t ransparent (small o6cytes); (ii) o6cytes about 300-350/~m in diameter: lightly whitish (o6cytes at the onset of vitellogenesis); (iii) o6cytes about 35o-4oo/ ,m in diameter: whitish (o6cytes at the onset of vitellogenesis).

The observations (Fig. 19) show that the amount of 4-S RNA present in the small o6cytes does not decrease. A slight increase actually oc:urs at the onset of

Biochim. Biophys. Acta, 224 (I97 o) 99-113

I I 0 C. THOMAS

0.'~-

0.3-

0.2-

0.1-

0

0.4

0.3 E c 0.2 8

o,1

O-

0.6.

0.5-

0.4"

0.3-

0.2-

0.1-

0

o

4s

18S 28S

b

28S

c

28S

18S

top bottom

Fig. 19. a, b, c, Sucrose gradient centrifugation of RNA from a same number of isolated o6cytes of X . laevis. 15-3o ~o Sucrose gradient centrifugation for 3 h at 4 ° and 5 ° ooo rev. /min. "Conti- nuous reading". Sedimentat ion from left to right, a. 20o small ohcytes about 2oo--25ol, m in d iameter ( t ransparent) . b. 20o o6cytes at the onset of vitellogenesis about 3oo-35 ° ffm in diameter. c. 20o ohcytes at the onset of vitellogenesis about 35o-4oo ffm in diameter.

vitellogenesis (3oo-4oo ffm), yet, there is a considerable increase in I8-S and 28-S RNA at this time.

(b) The chromatographic properties, on methylated-albumin column, of low molecular weight RNA present in o6cytes at the onset of vitellogenesis (300-400/,in) are similar to those of 4-S RNA in small o6cytes (Fig. 20).

008 ~ :-.

o , , , , o - o s ..// 'o. " . . . . .

25 30 35 40 45 50 Fraction No.

Fig. 20. Methylated-albumin column chromatography of low molecular weight 1R:NA (lion-labeled) f rom o6cytes of X. laevis at the onset of vitellogenesis together with labeled 4-S RNA of Peaks i and 2 extracted from "young ovaries" of X . laevis.

Biochim. Biophys . Acta, 224 (i97 o) 99-113

RNA AND RIBONUCLEOPROTEINS FROM SMALL OOCYTES I I I

The amount and chromatographic properties of 4-S RNA present in the small o6cytes of X. laevis are maintained in spite of the large increase of I8-S and 28-S ribosomal RNA in oScytes at the onset of vitellogenesis. This supplies further evidence against an eventual degradation of RNA.

DISCUSSION

We have shown that in pieces from X. laevis "young ovaries" there is a large amount of low molecular weight RNA, a part of which seems to be present as ribo- nucleoproteic fractions. The possibility of RNA degradation during homogenization seems unlikely, as shown by addition of high molecular weight RNA, electron mi- croscopy and evolution of RNA in oScytes of different sizes.

Our observations could have a general value: most of the RNA in the small oScytes of other amphibian might also be low molecular weight RNA. Preliminary experiments with extracts of small oScytes from Rana [usca and Axolotl have shown us that the major part of the RNA is present in low sedimentation range particles (like the peaks which were found in Xenopus). Besides, electron microscopic observations showed us that the cytoplasm of small oScytes from R. ]usca has the same fibrillar components as in X. laevis.

Several authors 16,17 have shown that there is a preferential synthesis and a considerable accumulation of ribosomal RNA during amphibian oSgenesis, which seems to be related to the selective replication of the "ribosomal DNA" of the nucleolar organizer and to the appearance of many nucleoli in the germinal vesicle of small oScytes, during the first weeks after metamorphosis (in oScytes less than 5 ° #m in diameter) ~,19,13,2°. Despite this early replication of "ribosomal DNA" the small oScytes of X. laevis, less than 250 ttm in diameter, are distinguished by the accumulation of 4-S RNA and contain very little ribosomal RNA. This accumulation could be related to a repression, at least partial, of "nucleolar ribosomal DNA" in these small oScytes. Inhibition would be removed at the onset of vitellogenesis.

It is not easy to identify the 4-S RNA of small oScytes of X. laevis with one of the several low molecular weight cytoplasmic or nuclear RNA's described in most cells and present in small amounts as compared with I8-S and 28-S ribosomal RNA 2~-z2. Some properties of oScyte 4-S RNA (high quantity, presence in the "soluble cellular fraction") would induce us, at first sight, to compare it with low molecular weight RNA present in great amounts (sometimes more than 9 ° °/o of cellular RNA) in cells which have a low metabolic rate ~3-3s. In these cells, low molecular weight RNA might result from a ribosomal degradation. However, there are many discre- pancies between these cells and X. laevis small oScytes which are metabolically growing cells. I t is therefore very unlikely that 4-S RNA of small o6cytes results from ribosomal degradation. It is important to note that the protein synthesis occurring in the cytoplasm of small oScytes of X. laevis is very low, when compared with that occurring in the cytoplasm of oScytes at the onset of vitellogenesis ~,ag.

The result of the elution on a methylated albumin column of 4-S RNA from small oScytes of X. laevis or from oScytes at the onset of vitellogenesis, suggests that a part of this 4-S RNA could be transfer RNA.

Biochim. Biophys. Acta, 224 (197o) 99-113

112 c. THOMAS

The greater part of low molecular weight RNA from small o6cytes and from o6cytes at the onset of vitellogenesis has an NaC1 elution concentration between that of transfer RNA and that of 5-S ribosomal RNA. These elution conditions could correspond with those of the hypothetical transfer RNA precursors 24-27. But these are rapidly converted into transfer RNA, whereas the 4-S RNA in small o6cytes of X. laevis seems to be metabolically stable.

I t is difficult to assert that 5-S ribosomal RNA is present in small o6cytes. However, at the onset of vitellogenesis, there is probably a progressive accumulation of 5-S ribosomal RNA in the o6cytes, as the rate at which 5-S RNA accumulates is apparent ly regulated by the rate of synthesis of I8-S and 28-S ribosomal RNA in o6genesis40,19, 41.

The nature and the functions of the low molecular weight RNA from small o6cytes of X. laevis and the relations between this RNA, transfer RNA and 5-S ribosomal RNA of X. laevis o6cytes at the end of vitellogenesis are very difficult to define. I t is important to add that the properties of 4-S RNA from small o6cytes of X. laevis make it a very unlikely precursor of I8-S and 28-S ribosomal RNA, in con- t rast with the previous hypothesis based on electron microscopic observations 1.2.

Preliminary observations from other authors corroborated the presence of a high percentage of low molecular weight RNA in small o6cytes of X. laevis:

FoRI~ 42 described in the smallest o6cytes of X. laevis a ribonucleoprotein particle having a sedimentation constant of 42-S and comprising over 60 °/o of the total ribonucleoproteins in the ovarian tissue. The RNA of this particle is predominantly 5-S RNA but there is also a unique species 4.7 S. The difference between the sedimentation constant of the ribonucleoproteins described by FORD 42 and those which we found, could arise from a difference in the homogenization medium.

According to MAIRY-VoN ]TRENCKELL 43, low molecular weight RNA from small o6cytes of X. laevis could be formed by a mixture of transfer RNA and 5-S ribosomal RNA, according to the elution position of RNA on Sephadex. Transfer RNA and 5-S ribosomal RNA would, respectively constitute 35 % and 4 ° °/o of whole RNA in o6cytes 15o/~m in diameter.

These observations are, in the main, in agreement with ours. However, FORD 42, like MAIRY-VON I~RENCKELL 43, depicts a great amount of 5-S RNA in small oScytes of X. laevis. In contrast with this, methylated-albumin column chromatography showed us that a high proportion of low molecular weight RNA in small o6cytes of X. laevis which probably corresponds to the RNA described as 5-S by the previous authors, elutes at an NaC1 concentration between that of transfer RNA and of 5-S ribosomal RNA from liver of X. laevis. The nature of this RNA eluting between 4 and 5 S is enigmatic: could it be a 5-S ribosomal RNA with a molecular conformation different from that of 5-S ribosomal RNA from the liver of X. laevis? New experiments would be needed to resolve this problem.

ACKNOWLEDGEMENTS

We wish to thank Dr. P. MALPOIX for help in the translation. This work was done with financial support of contracts Euratom-U.L.B. and Belgian State U.L.B.


RNA AND RIBONUCLEOPROTEINS FROM SMALL OOCYTES i13

R E F E R E N C E S

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Ribonucleic acids and ribonucleoproteins from small oöcytes of Xenopus laevis

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