Development 112, 317-326 (1991)Printed in Great Britain © The Company of Biologists Limited 1991

317

Developmental expression of fibrillarin and U3 snRNA in Xenopus laevis

MICHELE CAIZERGUES-FERRER1*, COLETTE MATHIEU1, PAOLO MARIOTTINI2,

FRANCOIS AMALRIC1 and FRANCESCO AMALDI2

1Centre de Recherche de Biochirrue el de Gin£nque Cellulaires du CNRS, 31062 Toulouse Cedex, France2 Dipartimento di Bwlogia, Univcrsita' di Roma 'Tor Vergata', 00173 Rome, Italy

* Author for correspondence

Summary

Fibrillarin is one of the protein components that togetherwith U3 snRNA constitute the U3 snRNP, a smallnuclear ribonucleoprotein particle involved in ribosomalRNA processing in eucaryotic cells. Using an antifibril-larin antiserum for protein detection and a fibrillarincDNA and a synthetic oligonucleotide complementary toU3 snRNA as hybridization probes, the expression ofthese two components has been studied during Xenopusdevelopment. Fibrillarin mRNA is accumulated early inoogenesis, like many other messengers, and translatedduring oocyte growth. Fibrillarin protein is thusprogressively accumulated throughout oogenesis to be

assembled with U3 snRNA and used for ribosomeproduction in the amplified nucleoli. After fertilization,the amount of U3 snRNA decreases while the maternallyaccumulated fibrillarin mRNA is maintained and util-ized to produce more protein. After the mid-blastulatransition, stored fibrillarin is assembled with newlysynthesized U3 snRNA and becomes localized in theprenucleolar bodies and reforming nucleoli.

Key words: fibnllann, U3 snRNA, gene expression,Xenopus laevis, development.

Introduction

Small nuclear ribonucleoprotein particles (snRNPs),which are composed of small nuclear RNAs and fewprotein components, are implicated in the processing ofvarious classes of transcripts. In the nucleolus, the bestknown and the most abundant snRNP is the U3 snRNPpresent at about ixlO5 to 106 copies per mammaliancell (for a review see Reddy and Busch, 1988). It hasbeen proposed that this snRNP participates in thematuration of pre-rRNA and its implication in theearliest cleavage event of mouse pre-rRNA wasrecently demonstrated (Kass et al. 1990). It has alsobeen shown that, in vivo, the disruption of Xenopus U3snRNA affects ribosomal processing (Savino andGerbi, 1990). The possibility that U3 snRNP may alsobe involved in subsequent rRNA maturation steps isstill debated (Bachellerie etal. 1983; Crouch etal. 1983;Prestayko et al. 1970; Tague and Gerbi, 1984; Jeppesenetal. 1988; Kupriyanova and Timofeeva, 1988; Parker etal. 1988). The secondary structure of U3 snRNA andthe protein composition of the U3 snRNP have beendetermined for human cells (Parker and Steitz, 1987).One of the protein constituents, originally described inPhysarum polycephalum (Christensen et al. 1977), isrecognized by an antiserum from scleroderma patientsand has been identified and characterized in rat andother eucaryotes (Aris and Blobel, 1988; Guiltinan et

al. 1988; Lischwe et al. 1985; Ochs et al. 1985). This34xlO3Mr protein has been named 'fibrillarin' for itslocalization in the fibrillar region of the nucleolus (Ochsetal. 1985). The yeast homolog of fibrillarin, Nopl, hasbeen shown to be essential for cell growth (Schimmanget al. 1989; Tollervey and Hurt, 1990).

We have recently isolated and characterized a cDNAclone encoding fibrillarin of Xenopus laevis (Lapeyre etal. 1990), thus providing the opportunity of studying thefunction and regulation of U3 snRNP in this organismwhich is particularly suitable for studies on manyaspects of ribosome biogenesis. In the present paper,we describe the expression of fibrillarin and U3 snRNAgenes during Xenopus oogenesis and embryogenesis,and compare it with that of other ribosomal andnucleolar components.

Materials and methods

Biological materialsXenopus laevis were purchased from CRBM, CNRSBP50-51, 34033 Montpellier Cedex, France Adult ovarieswere disaggregated with lmgmP1 collagenase (BoehnngerMannheim) at 25°C for lh Washed oocytes were staged byhand under the microscope according to Dumont (1972).Embryos, obtained as described by Brown and Littna (1964),

318 M. Caizergues-Ferrer and others

were grown at 22 °C in dechlorinated water and stagedaccording to Nieuwkoop and Faber (1973)

RNA analysisTotal RNA was isolated from oocytes or embryos accordingto Probst et al. (1979). Fibrillann mRNA was analyzed bynorthern hybridization as previously descnbed (Caizergues-Ferrer et al 1989) In vivo labeled nbosomal RNA providedsize markers. The 32P-labeled fibnllann probe was obtainedby nick translation or random priming of the 1.6 kb insert offibnllarin cDNA clone pXomFib previously isolated andcharacterized (Lapeyre et al. 1990). Prehybndization andhybridization were performed in 5xSSC, 50% formamide at42°C Filters were then washed with two changes of 2xSSC,0.1% SDS at room temperature and two changes of0 lxSSC, 0 1 % SDS at 48°C and exposed to X-ray film. ForU3 snRNA analysis, total RNA was electrophoresed ondenaturing 5 % acrylamide-urea gel and electrotransferred toAmersham's Hybond-N membrane for hybridization. In vivolabeled snRNAs provided size markers. The probe for U3snRNA was a 22mer synthetic oligonucleotide complemen-tary to nt 101-122 of Xenopus laevis U3 snRNA sequence(Jeppesen et al. 1988); this oligonucleotide was 32P-labeled atthe 3' end by terminal transferase in conditions where onlyone or two residues are added to each molecule. Prehybnd-ization and hybndization were performed in 6xSSC at 60 °C.Filters were then washed with two changes of 2xSSC, 0.1 %SDS at 48°C and two changes of O.lxSSC, 0 1% SDS at roomtemperature and exposed to X-ray film.

Polysome/mRNP distribution of mRNAPolysomes were prepared from staged embryos, fractionatedand analyzed essentially as previously descnbed (Pierandrei-Amaldi et al. 1982; Caizergues-Ferrer et al. 1989). Cytoplas-mic extracts from 10 embryos were prepared, loaded on15-50 % sucrose gradients, centnfuged in a SW41 rotor for 2 hat 37 000 revs mm"1; the collected gradient fractions wereprecipitated with 3 vol of ethanol, the RNA extracted, run onagarose gel, transferred to a filter and hybridized to differentprobes (nucleohn, fibnllann and r-protein S8). The extent ofhybridization was determined by densitometric scanning ofthe autoradiograms and the values expressed as percent ofmRNA on polysomes.

Protein analysis by immunoblottingProtein extracts from ten oocytes (or embryos) were preparedby homogenization in 100//I of 80 mM /3-glycerophosphate,15 mM NaEGTA, lmM DTT and 5/igmP1 leupeptin,5/jgmr1 aprotinin, 1/igml"1 pepstatin, IOITIM benzamidin-Cl, lmM phenylmethylsulfonyl-fluoride (PMSF) as proteaseinhibitors, and frozen in liquid nitrogen. After thawing thehomogenates were centrifuged for lOmin at 5000 g. The yolk-free supernatants were denatured by adding 1 vol of samplebuffer, containing 0.5 M DTT, 0.175 M SDS in 50 mM Tns-HClbuffer at pH6.8, and separated by SDS-polyacrylamide gelelectrophoresis according to Laemmli (1970). Electroblottingand western analysis were performed as previously descnbed(Caizergues-Ferrer et al. 1989) using human anti-fibnllannantibodies from scleroderma patients Immunoblots wereprocessed as descnbed by Reuter et al. 1989. The proteinswere transferred electrophoretically on nitrocellulose. Thefilter was saturated with 5 % new-born calf serum (NCS) inPBS and was incubated with a 1/200 dilution of fibnllannantiserum in buffer A (10mM Tris-HCl pH7.4; 150mM NaCl;100 mM MgCl2 0.5% Tween-20; 0.1% Triton X-100; 5%NCS). It was then washed 5 times with buffer B (buffer A

without Tnton and NCS) and subsequently incubated with theappropnate dilution in buffer B of goat anti-human antibodiesconjugated with alkaline phosphatase (Promega) or 125I-protein A (Amersham) AJfter a second washing cycle, thefilter was either stained with a solution of 0 12 mM5-bromo-chloro-indolylphosphate, 0.12mM nitrotetrazoliumblue, 5 mM MgCl2,100mM Tris-HCl, 100 MM NaCl, pH.9.5 orexposed to X-ray films

Immunofluorescence microscopyOocytes were fixed in 4 % formaldehyde in PBS (phosphatebuffer) pH7.2, for 2h at 4°C Dejelhed embryos were fixedovernight at 4°C in 3.5% formaldehyde in modified Barth'ssolution and soaked in 5 % to 30 % sucrose as cryoprotectant.Oocytes and embryos were embedded in OCT compound(Miles Laboratories Inc , Elkhart, IN) and cryostat sections(12/an) were collected on a cover glass coated withO^mgml"1 poly-L-lysine. For immunocytochemistry, theslides were transferred to blocking buffer (PBS with 1 % non-fat dry milk plus 0.25 % Tnton X-100) for 30 mm, then washedseveral times in the same buffer without Tnton X-100. Theblocked sections were incubated for 1 h at 37 °C with a pnmaryantibody (human anti-fibnllann antibody, diluted 1 30) or forthe control with a nonimmune serum. The bound antibodywas visualized by 1 h incubation with fluorescein lsothiocyan-ate-conjugated rabbit anti-human immunoglobulin (Sigma)diluted 1-40 in blocking buffer. After washing as before,stained sections were mounted in PBS-glycerol plus DAPINegative controls were done with nonimmune serum or byomission of the first antibody. Samples were examined with anepifluorescence microscope (Leitz) and TMax 400 film(Kodak limited) was used for photography

Glycerol gradientsSonicates of 30 staged embryos, stage 12 and 36, (5x5Os with30 s intervals at setting 1.5 using a Branson microtip) werefractionated on 10-30 % glycerol gradients containing 20 mMHepes-KOH pH7.9, 60mM KC1 and lmM MgCl2. Centnfu-gation was performed at 25000revsmin"1 for 15h at 4°C in aSW41 rotor. 20 fractions were collected from each gradient,and extracted with PCA (phenol-chloroform-isoamyl alcohol50 501). Proteins were recovered by acetone precipitation,RNA by ethanol precipitation, and analyzed by western andnorthern blot respectively

Quantification of northern blot autoradiograms andwestern blotsLKB Ultroscan XL laser densitometer was used for quantitat-ive analysis of X-ray films of northern blots and photographsof immunoblots

Results

Fibnllann and U3 snRNA expression duringoogenesisAs a first step towards studying the expression of thegenes encoding fibrillann and U3 snRNA, the RNA ofa fixed number of staged defolliculated oocytes wasanalyzed by northern blot (Fig. 1A). Using fibrillarincDNA as a probe, a strong reaction is observed with aband at 1.8 kb. Upon prolonged autoradiographicexposure a weak band of 2.7 kb is also detected; thisband corresponds to nucleolin mRNA to which thefibrillarin probe cross-hybridizes (Lapeyre et al. 1990).

Fibrillarin and U3 snRNA in Xenopus 319

i n in iv VI

2.7 kb

1.8 kb

n ni iv v vi220 nt

U3

II I IV V

S t a g e s

VI

Fig. 1. Fibrillarin mRNA and U3 snRNA accumulation during oogenesis. (A) Northern blot. Fib - Total RNA equivalentto one oocyte was separated by electrophoresis in a denaturing-formaldehyde 1 % agarose gel, electrotransferred to anitrocellulose filter and hybridized under stnngent conditions with a nick-translated Xenopus fibnllann cDNA as a probeThe slight variations of electrophoretic mobility are due to the large amount of rRNA after stage II. U3 - Total RNAequivalent to five oocytes was separated by electrophoresis in a denaturing 7 M urea-5% polyacrylamide gel, electro-transferred on a nylon filter and hybridized under stringent conditions with a 3' labeled 22-mer synthetic oligonucleotidecomplementary to U3 snRNA (nt 101-122) U3 snRNA was identified using CHO 32P RNA as markers. The 'smile' of thegel is artefactual. (B) Quantitative data. Relative expression of fibnllann mRNA, U3 snRNA, S8 rp-mRNA and rRNAdunng oogenesis. Upper panel: values, obtained by densitometnc quantitation of the experiment shown in A and ofothers, are expressed relative to the highest point. Fibrillarin mRNA ( • • ) ; U3 snRNA (O O) Lower panelalready published curves for rRNA ( ), r-protein S8 mRNA ( ); nucleohn mRNA ( ).

Hybridization with a U3 snRNA probe gives a band atabout 220 nt, which agrees with the length of 219 ntdescribed for Xenopus U3 snRNA (Jeppesen et al.1988). A quantification of the results from differentexperiments for fibrillann mRNA and for U3 snRNA isshown in Fig. IB, upper panel. In the lower panel arereported, for comparison, already published results forthe accumulation of rRNA and of r-protein andnucleolin mRNAs during oogenesis (Pierandrei-Amaldiet al. 1982; Caizergues-Ferrer et al. 1989). FibrillarinmRNA accumulates rapidly in the previtellogenicstages reaching its maximum at stage II and decreasesduring the following stages to about 50 % of the highestvalue. This pattern of accumulation is quite similar tothose of r-protein and nucleolin mRNAs (Fig. IB,lower panel). A different pattern of accumulation isobserved for U3 snRNA, which reaches its maximum atstage V and then slightly decreases at stage VI.

We have taken advantage of the availability of ahuman autoimmune serum specific for fibrillarin(Reimer et al. 1987) to quantitate the accumulation ofthis protein dunng oogenesis, and to correlate it withthe accumulation pattern of fibrillarin mRNA. For thispurpose, we have prepared protein extracts from stagedoocytes and analyzed aliquots by immunoblotting.Fig. 2 shows an example in the insert, and the average

results of different experiments quantified by densito-metnc scanmng. It appears that the amount offibrillarin increases during oogenesis from stage I tostage VI, and that the accumulation proceeds in parallelto the U3 snRNA accumulation and nbosome accumu-lation.

Fibrillann and U3 snRNA localization duringoogenesisThe localization of fibrillarin in the oocyte was followedby indirect immunofluorescence (Fig. 3A). Fibrillarinwas detected in the amplified nucleoli already in thesmall previtellogenic oocytes (stage I and II). Theintensity of fluorescence in the amplified nucleoliincreased with the size of the oocyte from stage I to VI.

The distribution of fibrillann and U3 snRNAbetween nuclei and cytoplasm was further investigatedin stage VI oocytes. The oocytes were manuallyenucleated and RNA and proteins were extracted fromthe different fractions. Proteins were analyzed bywestern blot (Fig. 3B). Fibrillarin was detected in thetotal oocyte extracts (lane T) and in the nuclear fraction(lane N), but was not detected in the cytoplasmicfraction (lane C). A similar localization was found forU3 snRNA by northern blot analysis (Fig. 3C).

320 M. Caizergues-Ferrer and others

1 -CD4—'

oo

o5 -

hP I+II HI rv v vi

/

III IV

Stages

VI

Fig. 2. Expression of fibnllann antigen during oogenesisTotal protein extracts from ten defolhculated oocytes wereprepared as descnbed in Materials and methods. Proteinscorresponding to five oocytes were run in each lane of a12 % SDS-PAGE The gel was blotted onto nitrocelluloseand probed with anti-fibnllann serum. Detection of thecomplexes was achieved by a second antiserum coupled toalkaline phosphatase The insert shows a typical westernblot, hp, hepatocyte nuclei; I to VI oocyte stages accordingto Dumont (1972). The gTaph shows quantitative dataobtained by densitometnc scanning of the western blots ofdifferent experiments.

Fibrillarin and U3 snRNA expression duringembryogenesisTo study the fate of the maternally accumulatedfibrillann mRNA and of U3 snRNA during embryogen-esis and to follow the onset of new synthesis andaccumulation of these two RNA species, total RNAwas extracted from embryos at different developmentalstages. RNA aliquots corresponding to a fixed numberof embryos were analyzed by hybridization of northernblots with fibrillarin and U3 snRNA probes. Oneexperiment is shown as an example in Fig. 4A, anddensitometric quantitations of this and other exper-iments are given in Fig. 4B, upper panel. The level ofmaternal fibrillarin mRNA (the 1.8 kb band) remainsstable through oocyte maturation and early embryogen-esis. After the 'mid-blastula transition' (stage 8), whena general transcription activation occurs (Newport andKirschner, 1982), the amount of fibrillarin mRNAincreases by 2- to 4-fold, reaches the maximum level atneurula (stage 16), then remains constant throughoutembryogenesis. After stage 12 a weak band of 2.7 kb,corresponding to nucleolin mRNA (Lapeyre et al. 1990)is also detected. However, the maternally inherited U3snRNA could not be detected during the first hours ofdevelopment and is detected only at gastrula (stage 12),after which it steadily increases until stage 36-38 toremain constant thereafter. To follow more preciselythe fate of maternal U3 snRNA, RNA corresponding to10 embryos (instead of one embryo) was analyzed by

c .-. B Fib

hp N

U3

hp T C N

Fig. 3. Nuclear localization of fibrillann and U3 snRNA in Xenopus laevis oocyte (A) Indirect immunofluorescencemicroscopy on frozen sections of Xenopus ovary (a) Using the autoimmune antiserum against fibrillann. Note the bnghtfluorescence of the amplified nucleoh as early as stage II (stages indicated in b). (b) Corresponding DAPI fluorescence.The nuclei of the follicule cells are stained (B) Western blot (Fib). Protein extracts were prepared from the differentfractions as descnbed in Materials and methods and analyzed by electrophoresis on a 12% SDS-PAGE. The gel wasblotted onto nitrocellulose and probed with antiserum against fibnllann. Detection of the complexes was achieved by 125I-protein A. hp, Xenopus hepatocyte nuclei; T, total fraction of stage VI oocytes; C, cytoplasmic fraction; N, nuclearfraction (C) Northern blot (U3). RNA was prepared from the different fractions as described in Materials and methodsand electrophoresed on a denaturing 7M urea - 5% acrylamide gel. After electrotransfer onto nylon filter, hybndizationunder stnngent conditions was earned out with a 3' labeled synthetic oligonucleotide complementary to U3 snRNA (nt101-122). hp, Xenopus hepatocyte nuclei; T, total fraction of stage VI oocytes; C, cytoplasmic fraction; N, nuclearfraction

Fibrillann and U3 snRNA in Xenopus 321

2.7 kb

1.8 kb

220 m

220 nt

VI

•

3

h p

3

9

VI

9

12

- -

0

12

-

—

3

16 26

• •

40

6 9

40

-

hp

m

12

i

1 emb.

10emb.

Fib

L'3

Doys

8 10 12 16 26 33 3« 142 Stoges

Fig. 4. Fibnllarin mRNA and U3 snRNA accumulation dunng embryogenesis (A) Northern blot. Fib - Total RNAequivalent to one embryo was separated by electrophoresis on a denaturing-formaldehyde 1 % agarose gel,electrotransferred on a nitrocellulose filter and hybridized with a nick-translated Xenopus fibnllann cDNA as a probe. U3- Total RNA equivalent to one or ten embryos was separated by electrophoresis on a denaturing 7 M urea-5 %polyacrylamide gel, electrotransferred to nylon filter and hybndized with a 3' labelled 22-mer synthetic oligonucleotidecomplementary to U3 snRNA (nt 101-122) hp, Xenopus hepatocyte nuclei, VI, stage VI oocytes; 0 to 40, stages ofembryogenesis. (B) Quantitative data. Relative expression of fibnllann mRNA, U3 snRNA, S8 rp-mRNA and rRNAdunng oogenesis. Upper panel: values, obtained by densitometnc quantitation of the several experiments, are expressedrelative to the highest point. Fibrillann mRNA ( • • ); U3 snRNA (O O). Lower panel- already published curvesfor rRNA ( ); r-protein S8 mRNA ( ), nucleolm mRNA ( )

northern blot. The amount of U3 snRNA decreases bya factor 6 after maturation, but some residual maternalRNA could still be detected (Fig. 4A). The lower panelof Fig. 4B shows, for comparison, already publishedresults for the accumulation during embryogenesis ofrRNA and of r-protein and nucleolin mRNAs (Pier-andrei-Amaldi etal. 1982,1985; Caizergues-Ferrer et al.1989). The maximum accumulation is the same fornucleolin and fibrillarin mRNA and corresponds to thereformation of the nucleoli at neurula stage.

To know whether the maternal and the newlysynthesized fibrillarin mRNA are translated in earlyembryogenesis, we have analyzed their distributionbetween polysomes and light mRNPs (Table 1). Thematernally inherited fibrillarin mRNA is already loadedonto polysomes at stage 3 Later the fibnllarin mRNA,most of which is newly synthesized, is very efficientlyutilized for translation. In fact at stage 18 more than90 % of it is loaded onto polysomes compared with only25 % of r-proteins mRNA and 60 % of nucleolinmRNA. Also, at stage 36 more than 90% of fibrillarinmRNA is translated, compared with 60% of r-proteinmRNA and 90% for nucleolin mRNA.

Spatial distribution of fibrillarin and 1)3 snRNAduring embryogenesisThe subcellular localization of fibrillarin protein was

Table 1. Poly some / mRNP distribution of fibnllarin,nucleolin and r-protein S8 mRNAs at different

development stages, expressed as percent of mRNAloaded on polysomes

mRNAs

FibnllannNucleolinr-protein S8

3

80%

Stages

18

>90%62%25%

36

>90%82%60%

For the values relative to nucleolin and r-protein S8 mRNAs cfCaizergues-Ferrer et al. 1989 The amounts of mRNA fornucleiolin and for r-protein S8 are negligible at stage 3

analyzed by indirect immunofluorescence (Fig. 5B,D,F,H). The corresponding DAPI staining is shown inFig. 5 (A,C,E,G). A control experiment is shown inFig. 5 (A-B). On frozen sections of a late blastulaembryo, nuclei (arrows) are lightly stained by the anti-fibrillarin antiserum (Fig. 5D). In a mitotic cell (arrow-head), the fibnllarin staining is distributed on thesurface of the condensed chromosomes as alreadydescribed (Ochs et al. 1985; Reimer et al. 1987). Theweak autofluorescence of yolk seen in other regions isalso observed in control experiment (Fig. 5B). Noticethat in the control no label is seen in the nuclei (arrows)(Fig. 5B). At the late neurula stage, two kinds of

322 M. Caizergues-Ferrer and others

Fig. 5. Lxicalization of fibrillann in Xenopus embryos. Indirect immunofluorescence staining, using antifibrillarin antibodies,of cryostat sections of embrybs at different stages: control experiment, without the first antibody (A,B); blastula (C,D),late neumla:ectodermal cells (E,F); swimming tadpole (G,H). B,D,F,H, FTTC images. A,C,E,G, corresponding DAPIimages. Scale bars: in A,B,C,D,E,F=50jan; G,H=100/im The autofluorescence of yolk platelets is also observed incontrol (B). s

Fibnllarin and U3 snRNA in Xenopus 323

staining are observed. In endodermal cells, the nucleiare uniformly stained like at the blastula stage (resultsnot shown), while in dorsal ectodermal cells punctatelabel is seen (Fig. 5F). These dot-like structures couldcorrespond to the prenucleolar bodies and the newlyformed nucleoli which are known to reassemble earlierin these cells (Hay and Gurdon, 1967); a diffuse nuclearlabel is still present. At the swimming-tadpole stage(Fig. 5H), all the nuclei gave a bright signal restricted tothe nucleoli. These results show an evolution of thefibrillann localization during early development thatparallels the nucleoli reformation.

At this point, we wondered whether at the differentstages fibrillann is free, in snRNPs or associated tohigher order complexes. Sonicated extracts of embryosat different stages were fractionated on glycerolgradients, proteins and RNA were extracted fromgradient fractions and analyzed either by western blot(Fig. 6 Fib) or by northern blot (Fig. 6 U3). At stage 13(gastrula), fibnllarin and U3 snRNA were recovered inthe top of the gradient at a position corresponding tothe 10S particles (fraction 4-6). At tail bud stage (stage36), fibrillarin and U3 are recovered throughout thegradient in 10S particles and in larger higher orderstructures. At this stage, the U3 RNA distribution is

. 10S 80S"P T 2 4 6 8 10 1214 161820

FIB

St. 13

St. 36

10S80S

hp 2 4 6 8 10 12 14 16 18 20

U3St. 13

St. 36

Fig. 6. Sedimentation of fibrillarin and U3 snRNA inglycerol gradients Whole sonicates of stage 13 and stage36 embryos were fractionated on 10-30 % glycerolgradients, and proteins and RNA extracted from gradientfractions as described in Materials and methods. FIB -western blot. Proteins were electrophoresed on a 12 %SDS-PAGE. The proteins were blotted onto nitrocelluloseand probed with anti-fibrillarin serum. Detection of thecomplexes was achieved by 123I-protein A.Autoradiography was carried out for 20 h (stage 36) and for72 h (stage 13). U3 - northern blot. RNA from differentgradient fractions was electrophoresed on a denaturing 7 Murea - 5 % acrylamide gel, electrotransferred onto nylonfilter (Hybond N) and hybridized with a 3' labelledsynthetic oligonucleotide complementary to U3 snRNA (nt101-122) Blots were exposed to X-ray films for 24 h (stage36) and 36h (stage 13) hp, Xenopus hepatocyte nuclei; T,whole sonicate, 2 to 20, gradient fractions.

quite similar to the distribution observed in somaticcells (Tyc and Steitz, 1989).

In addition, the experiment shown in Fig. 6 indicatesthat the amount of fibrillarin increases about 50-foldduring embryogenesis.

Discussion

The involvement of U3 snRNP in ribosome biogenesiswas investigated through the study of fibrillann and U3snRNA expression in relation with ribosome synthesisduring Xenopus development. This represents a usefulmodel system due to the existence of two developmen-tal situations in which ribosome synthesis represents amajor effort of the cell: (1) following rDNA amplifi-cation in oogenesis, when a huge amount of ribosomesis produced from the amplified nucleoli and stored foruse during early embryo development and (2) inembryogenesis, when the embryo begins to synthesizenew nbosomes. The relative timing of expressionduring Xenopus development is already well docu-mented for rRNA, 5S RNA and r-proteins (forreferences see: Davidson, 1986; Amaldi et al. 1989).

U3 snRNP and ribosome accumulation duringoogenesisWe have shown that fibrillann mRNA is activelyaccumulated in previtellogenic oocytes reaching itshighest value at stage II, after which its amountdecreases. This mRNA accumulation pattern is similarto the one of r-proteins (Pierandrei-Amaldi et al 1982)and of nucleolin (Caizergues-Ferrer at al. 1989); asimilar accumulation pattern has been described alsofor several other unrelated mRNAs and for the totalnuclear-encoded poly(A)+RNA population (Rosbashand Ford, 1974; Golden et al. 1980; Etkin, 1988). Theamount of fibrillarin protein increases progressively,reaching its maximum level at the end of oogenesis witha pattern similar to that of ribosome accumulation. Thisfibrillarin appears to be mainly localized in theamplified nucleoli. This situation is different to thatdescribed for the Sm core proteins of the snRNPs (Ul,U2, U4, U5, U6) involved in mRNA splicing (Zeller etal. 1983). These proteins are synthesized duringoogenesis and stored in the cytoplasm of fully grownoocytes; only after mid-blastula transition do theybecome associated with the newly synthesized U RNAsand enter the nuclei, after addition of the trimethyl capstructure to the 5' end of the snRNA (for a review seeLamond, 1990). The fact that fibrillarin is nuclear andnot stockpiled in the cytoplasm of the stage VI oocytelike the Sm core proteins suggest that the targeting ofU3 RNA during early embryogenesis (in absence ofprotein synthesis) is independent of fibrillarin.

During oogenesis, the amount of U3 snRNAincreases with a pattern similar to fibrillarin up to stageV, then decreases somewhat in fully grown oocytes,when ribosome biogenesis diminishes (Dixon and Ford,1982). U3 snRNA is always localized in the nuclei. Thispattern of U3 snRNA synthesis differs from that of 5S

324 M. Caizergues-Ferrer and others

RNA, implicated in ribosome function, which issynthesized in previtellogenic oocytes (stages I and II)and sequestered as a 7S particle in the cytoplasm formonths before the onset of ribosome assembly duringlater stages of oogenesis (Picard and Wegnez, 1979;Pelham and Brown, 1980).

Fibnllann mRNA stability during egg maturationThe fibrillarin mRNA present in large oocytes quanti-tatively survives maturation and fertilization, and ismaintained through the early stages of embryo develop-ment. It has been shown that in Xenopus the sequenceUUUUUAU, when present in the 3'UTR not far fromthe AAUAAA polyadenylation sequence of anmRNA, acts as a signal for maintainance of its poly (A)tail during egg maturation, and is required for thestability of maternal mRNAs and for their utilization inearly embryogenesis (Wickens, 1990; McGrew andRichter, 1990). Accordingly, fibrillarin mRNA containsmultiple repeats of UUUUUAU or related sequencesin its 3'UTR (Lapeyre et al. 1990). In contrast, mRNAsfor r-proteins and nucleohn, which lack UUUUUAU(Mariottini et al. 1988; Caizergues-Ferrer et al. 1989)are deadenylated and inactivated during oocyte matu-ration and disappear during early cleavage stages(Hyman et al. 1984; Pierandrei-Amaldi et al. 1982;Caizergues-Ferrer et al. 1989). These observationssupport the identification of the UUUUUAU sequencein the 3'UTR of the mRNA as a signal for stabilizationof maternal mRNAs.

U3 snRNP and nucleologenesis in embryogenesisAfter oocyte maturation, the germinal vesicle breaksdown and the amplified nucleoli vanish. The reforma-tion of nucleoli begins at gastrula stage and is correlatedwith a weak revival of rDNA transcription (Busby andReeder, 1982). Since, at the end of mitosis ofmammalian cells, fibrillarin is localized in the prenuc-leolar bodies that participate in nucleolar reformation(Benavente et al. 1987; Jimenez-Garcia et al. 1989), wewanted to follow the fate of maternal U3 snRNP at thatstage of embryogenesis when nucleoli reappear, theribosome production still being very low. The amountof fibrillarin mRNA, which as shown above remainsconstant during the early stages of embryogenesis,increases 3-fold during the severals hours between the'mid-blastula transition' and neurula stage then remainsconstant at the high level through the remainder ofembryogenesis. In contrast, r-protein mRNAs disap-pear during egg maturation and early cleavage stages(see above), start to be synthesized again and accumu-lated after the 'mid-blastula transition' and increaseconspicuously thereafter (Pierandrei-Amaldi et al.1982; Baum and Wormington, 1985). Thus, at variancewith r-protein mRNAs, fibrillarin mRNA is stockpiledthroughout maturation and fertilization, to be utilizedduring early embryogenesis. In fact, we have alsoshown that the mRNA for fibrillarin is efficientlyutilized for translation during early embryogenesis,more than 90 % being loaded onto polysomes after mid-blastula transition, while rp-mRNAs, being subject to a

translational control, remain unutilized in light mRNPsduring the first part of embryogenesis, up to stage 26.Fibrillarin mRNA shows an expression pattern duringembryogenesis which is quite different from that ofr-protein mRNA, but very similar to that of histonemRNAs (Woodland et al 1979). This is not surprising ifone considers that both protein products might need toincrease proportionally to the number of nuclei. Indeedwe have shown that the amount of U3 snRNA increasesat approximately the same rate as the number of nucleiper embryo, as it has already been shown for othersnRNAs (Forbes et al 1983). Furthermore we haveshown that, as early as gastrula stage, fibrillarin and U3snRNA are recovered in the nucleus as 10S particles sothat later, when the zygotic r-RNA synthesis isactivated, fibrillarin and U3 snRNA are already presentin large amounts to participate in ribosome biogenesis.Interestingly, it has recently been described in mousethat U3 snRNA increases by a factor of 50 betweenoocyte and blastocyst stages and this change has beencorrelated with changes in nucleolar structure andfunction that occur during early mouse embryogenesis(Prather et al. 1990).

Fibrillarin is first detected in the newly formednucleoli in ectodermal cells. These data suggest that theU3 snRNP could be also involved in nucleolar structureand could play a role in the step where the prenucleolarbodies fuse with the nucleolar organizer regions(NORs). Studying the expression and localization offibrillarin and U3 snRNA in the anucleolate embryosshould provide information on the function of the U3snRNP in absence of rDNA transcription.

We thank R.LUhrmann (Marburg, FRG), J. Craft (NewHaven, USA) and U. Scheer (Wurzburg, FRG) for generousgifts of human autoimmune sera, N Gas (Toulouse, France)for help on lmmuno-cytochemistry, S. Baserga (New Haven,USA) and N. Rottman (Marburg, FRG) for fruitfuldiscussions and D Cribbs (Toulouse, France) for criticalreading of the manuscript. This research was earned outunder contract SC1*-O259-C of the Science Programme of theCommission of the European Communities and was partiallysupported by grants from 'Association pour la Recherche surle Cancer, ARC and from 'Progetto Finahzzato IngegnenaGenetica, C.N R '

References

AMALDI, F., BOZZONI, I., BECCARJ, E. AND PIERANDREI-AMALDI, P(1989) Expression of nbosomal protein genes and regulation ofnbosome biosynthesis in Xenopus development TrendsBiochcm. Set 14, 175-178

ARIS, J P AND BLOBEL, G. (1988) Identification andcharacterization of a yeast nucleolar protein that is similar to arat liver nucleolar protein J Cell Btol Vfl, 17-31

BACHELLERIE, J P., MICHOT, B AND RAYNAL, F (1983)Recognition signals for mouse pre-rRNA processing A potentialrole for U3 nucleolar RNA Molec Biol Rep 9, 79-86

BAUM, E Z AND WORMINGTON, W M (1985) Coordinateexpression of nbosomal protein genes during Xenopusdevelopment Devi Biol 111,488-498

BENAVENTE, A , ROSE, K M , REIMER, G , HUGLE-DORR, B ANDSCHEER, U (1987) Inhibition of nucleolar reformation aftermicromjection of antibodies to RNA polymerase I into mitoticcells J Cell Biol 105, 1483-1491

Fibrillann and U3 snRNA in Xenopus 325

BROWN, D D AND LITTNA, E (1964) RNA synthesis during thedevelopment of Xenopus laevis, the South African clawed toadJ molec Biol 8, 669-687

BUSBY, S J AND REEDER, R H (1982) Fate of amplified nucleohm Xenopus laevis embryos Devi Biol 91, 458-467.

CAIZERGUES-FERRER, M., MARIOTTINI, P., CURIE, C , LAPEYRE, B ,GAS, N , AMALRIC, F AND AMALDI, F (1989) Nucleohn fromXenopus laevis cDNA cloning and expression duringdevelopment Genes Dev 3, 324-333

CHRISTENSEN, M E., BEYER, A L , WALKER, B ANDLESTOURGEON, W M (1977) Identification of N°,N^dimethylarginine in a nuclear protein from the lowereukaryote Physarum polycephalum homologous to the majorprotein of mammalian 40S nbonucleoprotein particule Bwchembiophys Res Commun 74, 621-629

CROUCH, R , KANAYA, J S AND EARL, P L (1983) A model forthe involvement of the small nucleolar RNA(U3) in processingeukaryotic ribosomal RNA Molec biol Rep. 9, 75-78

DAVIDSON, E H (1986) Gene Activity in Early Development 3rdedition New York' Academic Press

DIXON, L K AND FORD, P. J (1982) Regulation of proteinsynthesis and accumulation during oogenesis in Xenopus laevisDevi Biol 93, 478-497

DUMONT, J N (1972) Oogenesis in Xenopus laevis (Daudm)Stages of oocyte development in laboratory maintained animalsJ Morph 136, 153-179.

ETKIN, L D (1988) Regulation of the mid-blastula transition inAmphibians In Developmental Biology (ed L E Browder),pp 209-240, New York Plenum Publishing Corp

FORBES, D J., KORNBERG, T B AND KJRSCHNER, M W (1983)Small nuclear RNA transcription and nbonucleoproteinassembly in early Xenopus development J Cell Biol 97, 62-72

GOLDEN, L , SCHAFER, U. AND ROSBASH, M (1980) Accumulationof individual pA+RNAs dunng oogenesis of Xenopus laevisCell 22, 553-560

GUILTINAN, M. J , ScHELLrNG, M E , EHTESHAM, N Z , THOMAS,J. C. AND CHRISTENSEN, M E (1988) The nucleolar RNA-binding B-36 is highly conserved among plants Eur J Cell Biol46, 547-553

HAY, E D AND GURDON, J B (1967) Fine structure of thenucleolus in normal and mutant Xenopus embryos J Cell Sci2, 151-162

HYMAN, L E , COLOT, H V AND ROSBASH, M (1984)Accumulation and behavior of mRNA during oogenesis andearly embryogenesis of Xenopus laevis In Molecular Aspects ofEarly Development (ed G. M Malacinski and W Klein), pp142-151, New York. Plenum Publishing Co

JEPPESEN, C , STEBBINS-BOAZ, B AND GERBI, S. A (1988).Nucleotide sequence determination and secondary structure ofXenopus U3 snRNA Nucl Acids Res 16, 2127-2148

JIMENEZ-GARCIA, L F , ROTHBLUM, L I., BUSH, H AND OCHS, RL. (1989) Nucleologenesis' use of non-isotopic in suuhybridization and immunocytochemistry to compare thelocalization of rDNA and nucleolar proteins dunng mitosisBiol Cell 65, 239-246

KASS, S , TYC, K , STEITZ, J A AND SOLLNER-WEBB, B (1990).The U3 small nucleolar nbonucleoprotein functions in the firststep of prenbosomal RNA processing Cell 60, 897-908

KUPRIYANOVA, N. S AND TIMOFEEVA, M Y (1988) 32S pre-rRNAprocessing a dynamic model for interaction with U3 RNA andstructural rearrangements of spacer regions Molec biol Rep13, 91-96

LAEMMLI, U. K (1970) Cleavage of structural protein dunng theassembly of the head of bactenophage T4 Nature 256, 680-685

LAMOND, A. I (1990) The tnmethyl-guanosine cap is a nucleartargeting signal for snRNPs Trends Biochem Sci 15, 451—452

LAPEYRE, B , MARIOTTINI, P , MATHIEU, C , FERRER, P , AMALDI,F., AMALRIC, F AND CAIZERGUES-FERRER, M (1990) Molecularcloning of Xenopus fibnllann, a conserved U3 small nuclearnbonucleoprotein recognized by antisera from humans withautoimmune disease Molec cell Biol 10, 430-434.

LISCHWE, M. J , OCHS, R L , REDDY, R , COOK, R G , YEOMAN,L C , TAN, E M , REICHLIN, M AND BUSH, H (1985)Purification and partial characterization of a nucleolar

scleroderma antigen (Mr=34,000, pi 8,5) nch in N°,N°dimethylargimne. J biol Chem 260,14304-14310

MARIOTTINI, P , BAGNI, C , ANNESI, F AND AMALDI, F (1988)Isolation and nucleotide sequences of cDNAs for Xenopus laevisnbosomal protein S8' similanties in the 5' and 3' untranslatedregions of mRNAs for vanous r-proteins Gene 67, 69-74

MCGREW, L L AND RICHTER, J D (1990). Translation control bycytoplasrmc polyadenylation dunng oocyte maturationcharacterization of cis and trans elements and regulation bycychn/MPF EM BO J 9, 3743-3751

NEWPORT, J AND KIRSCHNER, M (1982) A major developmentaltransition in early Xenopus embryos I - Charactenzation andtiming of cellular changes at the midblastula stage Cell 30,675-686

NIEUWKOOP, P D AND FABER, J (1973) Normal Table ofXenopus laevis (Daudin). Amsterdam Elsevier/North HollandPublishing Co

OCHS, R L , LJSCHWE, M A., SPOHN, W H AND BUSH, H(1985) Fibnllann a new protein of the nucleolus identified byautoimmune sera Biol Cell 54, 123-134

PARKER, K A , BRUZIK, J P AND STEITZ, J A (1988) An mvitro interaction between the human U3 snRNP and 28S rRNAsequences near the sarcin site Nucl Acids Res 16,10493-10509

PARKER, K A AND STEITZ, J. A (1987) Structural analyses of thehuman U3 nbonucleoprotein particle reveal a conservedsequence available for base painng with pre-rRNA Molec cellBiol 7, 2899-2913

PELHAM, H. R B AND BROWN, D D (1980) A specifictranscnption factor that can bind either the 5S RNA gene or 5SRNA. Proc natn Acad Sci USA 7, 4170-4174

PICARD, B AND WEGNEZ, M (1979) Isolation of a 7S particlefrom Xenopus oocytes a 5S RNA-protein complex Proc natnAcad Sci USA 76, 241-245

PIERANDREI-AMALDI, P , BECCAIU, E , BOZZONI, I. AND AMALDI, F(1985) Ribosomal protein production m normal and anucleolateXenopus embryos regulation at the post-transcnptional andtranslational levels Cell 42, 317-323

PIERANDREI-AMALDI, P , CAMPIONI, N., BECCARI, E , BOZZONI, IAND AMALDI, F. (1982) Expression of nbosomal protein genesin Xenopus laevis development Cell 30, 163-171

PRATHER, R , SlMERLY, C , SCHATTEN, G , PlLCH, D R , LOBO, S.M , MARZLUFF, W F , DEAN, W L AND SCHULTZ, G A(1990) U3 snRNPs and nucleolar development dunng oocytematuration, fertilization and early embryogenesis in the mouseU3 snRNA and snRNPs. Devi Biol 138, 247-255

PRESTAYKO, A W , TONATO, M. AND BUSCH, H (1970) Lowmolecular weight RNA associated with 28S nucleolar RNA Jmolec Biol 47, 505-515

PROBST, E , KRESSMANN, A AND BIRNSTIEL, M L (1979)Expression of sea urchin histone genes in the oocyte of Xenopuslaevis. J molec Biol 135, 709-732

REDDY, R AND BUSCH, H (1988) Small nuclear RNAs RNAsequences, structure and modification In Structure and functionof major and minor small nuclear nbonucleotide particles (edM L Birnstiel), pp 1-37, Berlin Spnnger-Verlag

REIMER, G , POLLARD, K M , PENNING, C A , OCHS, R L ,LISCHWE, M A., BUSCH, H AND TAN, E M (1987)Monoclonal autoantibody from a (New Zealand black x NewZealand white) Fl mouse and some human scleroderma seratarget an Mr 34,000 nucleolar protein of the U3 RNP particle.Arthritis Rheum 30, 793-800

REUTER, R , TESSARS, G , VOHR, H W., GLEICHMANN, E ANDLUHRMANN, R (1989) Mercunc chlonde induces autoantibodiesagainst U3 small nuclear nbonucleoprotein in susceptible miceProc natn Acad Sci USA 86, 237-241

ROSBASH, M AND FORD, P. J (1974) Polyadenyhc acid-containingRNA in Xenopus laevis oocytes J molec. Biol. 85, 87-101

SAVINO, R AND GERBI, S A. (1990). In vivo disruption ofXenopus U3 snRNA affects nbosomal RNA processing EMBOJ 9, 2299-2308

SCHIMMANG, T , TOLLERVEY, D . , KERN, H , FRANK, R AND HURT,E C (1989) A yeast nucleolar protein related to mammalian

326 M. Caizergues-Ferrer and others

fibnllann is associated with small nuclear RNA and is essentialfor viability EMBO J 8, 4015^1024

TAGUE, B W AND GERBI, S A (1984) Processing of the largerRNA precursor two proposed categories of RNA-RNAinteractions in Eukaryotes J molec Evol 20, 362-367.

TOLLERVEY, D AND HURT, E C (1990) The role of smallnucleolar nbonucleoproteins in nbosome synthesis Molec bwlRep 14, 103-106

TYC, K AND STEJTZ, J A. (1989). U3, U8 and U13 comprise anew class of mammalian SnRNPs localized in the cell nucleolusEMBO J. 8, 3113-3119

WICKENS, M (1990) In the beginning is the end. regulation of

poly(A) addition and removal dunng early development TrendsBiochcm Sci 15, 320-324

WOODLAND, H. R., FLYNN, J M. AND WYLLTE, A J (1979)Utilization of stored mRNA in Xenopus embryos and itsreplacement by newly synthesized transcripts Histone HIsynthesis using interspecies hybrids Cell 18, 166-171

ZELLER, R., NYFFENEGCER, T AND DE ROBERTIS, E M (1983)Nucleocytoplasrruc distribution of snRNPs and stockpiledsnRNA-binding proteins dunng oogenesis and earlydevelopment in Xenopus laevis Cell 32, 425-434

(Accepted 7 March 1991)