RNA-protein interactions of stored 5S RNA with TFIIIA and ribosomal protein L5 during Xenopus oogenesis

DEVELOPMENTAL

BIOLOGY

144, 129-144 (1991)

RNA-Protein Interactions of Stored 5s RNA with TFIIIA and Ribosomal Protein L5 during Xenopus Oogenesis LIZABETH A. ALLISON,’ PAUL J. ROMANIUK? AND AIMEE HAYES BAKKEN Department

of Zoology, NJ-&

University

of Washington, Seattle, Washington 98195

Accepted November 20, 1990

We studied the pathway of 5s RNA during oogenesis in Xenopus laevti from its storage in the cytoplasm to accumulation in the nucleus, the sequence requirements for the 5s RNA to follow that pathway, and the 5S RNA-protein interactions that occur during the mobilization of stored 5S RNA for assembly into ribosomes. In situ hybridization to sections of oocytes indicates that 5S RNA first becomes associated with the amplified nucleoli during vitellogenesis when the nucleoli are actively synthesizing ribosomal RNA and assembling ribosomes. When labeled 5S RNA is microinjected into the cytoplasm of stage V oocytes, it migrates into the nucleus, whether microinjected naked or complexed with the protein TFIIIA as a 7S RNP storage particle. During vitellogenesis, a nonribosome bound pool of 5S RNA complexed with ribosomal protein L5 (5s RNPs) is formed, which is present throughout the remainder of oogenesis. Immunoprecipitation assays on homogenates of microinjected oocytes showed that labeled 5S RNA can become complexed either with L5 or with TFIIIA. Nucleotides 11 through 108 of the 5S RNA molecule provide the necessary sequence and conformational information required for the formation of immunologically detectable complexes with TFIIIA or L5 and for nuclear accumulation. Furthermore, labeled 5S RNA from microinjected ‘7s RNPs can subsequently become associated with L5. Such labeled 5S RNA is found in both 5S RNPs and ‘7s RNPs in the cytoplasm, but only in 5S RNPs in the nucleus of microinjected oocytes. These data suggest that during oogenesis a major pathway for incorporation of 5S RNA into nascent ribosomes involves the migration of 5S RNA from the nucleus to the cytoplasm for storage in an RNP complex with TFIIIA, exchange of that protein association for binding with ribosomal protein L5, and a return to the nucleus for incorporation into ribosomes as they are being assembled in the amplified nucleoli. o 1991 Academic

Press. Inc.

turning to the nucleus for assembly into the large 60s subunit? and What protein(s) does the 5s RNA interact Ribosomes are composed of four RNA molecules and with in its nucleocytoplasmic journeys into and out of nearly 80 ribosomal proteins. Preribosomal particles the nucleus at different stages of oogenesis? containing all of the RNAs and many of the proteins are Xenopus oocytes offer a convenient system for studyassembled in the nucleoli of somatic cells and are ex- ing nucleocytoplasmic transport of RNA molecules. ported to the cytoplasm as 40s and 60s ribosomal sub- Whether injected into the cytoplasm or the nucleus, miunits (reviewed by Hadjiolov, 1985). In Xenopus oocytes, croinjected RNAs are transported to the cellular comhowever, 5s RNA is synthesized in large amounts before partment in which they are normally found (reviewed other components of the ribosome are available and is by DeRobertis, 1983). For example, HeLa cell tRNA and stored in the cytoplasm with nonribosomal proteins as 7s RNA remain in the oocyte cytoplasm after injection, ‘7s or 42s ribonucleoprotein particles (RNPs) for many while some of the HeLa cell 5s RNA becomes associated days or weeks prior to the onset of 18S-5.8S-28s ribo- with the amplified nucleoli (DeRobertis et al., 1982). The somal RNA (rRNA) synthesis and ribosome assembly in normal pathway of 5s RNA in somatic cells, such as the amplified nucleoli of vitellogenic oocytes (Mairy and HeLa cells, may not include a journey to the cytoplasmic Denis, 1971; Picard and Wegnez, 1979; Picard et al., compartment prior to assembly into ribosomes within 1980). Xenopus oocytes thus offer the opportunity to the nucleolus (Steitz et al, 1988). Thus, it is not clear study two important sets of questions: Does the cyto- whether microinjected HeLa cell 5s RNA would perplasmically stored 5s RNA participate in ribosome bio- fectly mimic the behavior of stored, cytoplasmic 5s genesis in the typical somatic cell pathway, after re- RNA of Xenopus oocytes. Furthermore, Xenms has two families of 5s RNA genes, the oocyte type and the somatic type, which produce slightly different 5s RNAs i Current address and correspondence address: Department of Zooland are under different developmental control (reogy, University of Canterbury, Christchurch 1, New Zealand. viewed by Wolffe and Brown, 1988). Somatic-type 5s 2 Department of Biochemistry and Microbiology, University of VicRNA apparently is not accumulated or assembled into toria, P.O. Box 1700, Victoria, British Columbia, Canada. INTRODUCTION

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Q 1991 by Academic Press, Inc. of reproduction in any form reserved.

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the pool of oocyte ribosomes stockpiled for use during nucleus. Further, the large pool of nonribosome bound embryogenesis (Denis and Wegnez, 1977). It has been 5s RNA which is stored in the cytoplasm of previtellopostulated that somatic-type 5s RNA, which differs genie oocytes as 7s RNPs is gradually replaced by a free from oocyte-type 5s RNA by six nucleotide substitupool of 5s RNP complexes. Microinjected 5s RNA can tions, is less stable metabolically than oocyte-type 5s become complexed in the cytoplasm with either TFIIIA RNA (Denis and Wegnez, 1977; Romaniuk et aL, 1988). or L5; in addition, the labeled 5s RNA from microinThese sequence and stability differences could poten- jetted 7s RNPs can undergo an exchange reaction tially alter the migratory behavior and/or in viva pro- within the oocyte and bind endogenous L5. We showed tein interactions of oocyte-type 5s RNA, consequently that nucleotides (nt) 11 through 108 of the 120-nt 5s altering the site of its assembly into ribosomes. RNA molecule provide the necessary sequence and conIt is not clear from prior studies what 5s RNA-proformational information required for formation of imtein interactions occur in later stages of oogenesis. The munologically detectable complexes with TFIIIA and L5 proteins in both 42s and 7s RNPs are prominent in pre- and for nuclear accumulation. These data lead to the vitellogenic oocytes. However, the proteins in the 42s proposal that cytoplasmically stored 5s RNA becomes RNPs are no longer detectable once vitellogenesis has mobilized during vitellogenic stages through a new assobegun (Dixon and Ford, 1982), and their functions are ciation with L5 which accompanies the 5s RNA into the still being analyzed (Johnson et al., 1984; Barrett and nucleus and hence to the nucleoli for ribosome assembly. Sommerville, 1987; LeMaire and Denis, 1987; Mattaj et We recently published a report on the RNA-protein ah, 1987; Vie1 et al., 1990). In 7s RNPs, the 5s RNA is interactions of newly synthesized 5s RNA in its export complexed in a 1:l ratio with the protein TFIIIA which from the nucleus to the cytoplasm during vitellogenesis has a dual function. This protein is involved in the stor- (Guddat et aL, 1990). Together, these reports raise the interesting question as to whether ribosome biogenesis age of 5s RNA and it also acts as a positive transcription factor for expression of 5s RNA genes (Honda and in oocytes utilizes two pathways of targeting 5s RNA to Roeder, 1980). The concentration of TFIIIA has de- the nucleoli, one of direct migration to the nucleoli after creased lo- to 30-fold in mature oocytes, from its highest transcription as is proposed for somatic cells (Steitz et concentration found in stages III and IV (Pelham et ah, ah, 1988), and the presently described one of transcrip1981; Roeder et aL, 1981), and its rate of synthesis is less tion in the nucleus, transport to the cytoplasm, and a than 15% of the maximum rate observed at stage I subsequent return to the nucleus for assembly into ribo(Dixon and Ford, 1982). In contrast, the synthesis of ri- somal subunits. This study documents a novel cytoplasbosomal proteins, including L5, is initiated and becomes mic exchange of one protein for another before an RNA maximal during stage III when vitellogenesis, rRNA molecule returns to the nucleus. Such nucleocytoplasmic trafficking of an RNA and its associated proteins prosynthesis, and ribosome assembly begin (Wormington, 1989). When ribosomes are treated with EDTA, a 5s vides the cell with yet another level of genetic regulation RNA/L5 complex (5s RNP) is released (Blobel, 1971). of its biochemical activities. Previous efforts could not detect 5s RNA/TFIIIA MATERIALS AND METHODS complexes (7s RNPs) in somatic cells (Honda and Roeder, 1980), although TFIIIA has been shown to bind Plasmids and Synthesis of Probes to both somatic-type and oocyte-type 5s RNA in vitro The plasmid pXlo8G has one repeat unit of oocyte(Romaniuk et ah, 1987). Thus, the 7s RNP storage partitype 5s DNA from pXlo8 (Birkenmeier et aL, 1978) subcle may be unique to oocytes. It is of interest, therefore, cloned into pGEM4 by Dr. Pamela Hines in our laborato investigate the intracellular trafficking and RNAprotein interactions that occur in between the cytoplas- tory. pXlo8 was a gift from Dr. L. J. Korn (Protein Demic storage of oocyte-type 5s RNA when it is bound to sign Labs, La Jolla, CA). Internally labeled riboprobes TFIIIA and its eventual association with L5 in the cyto- for in situ hybridizations were synthesized by linearizing pXlo8G with SmaI (BRL, Gaithersburg, MD), folplasmically stored 80s ribosomes of the oocyte. In this study, we investigated the association of endog- lowed by transcription using 15 units of T7 RNA polyenous 5s RNA with both TFIIIA and L5, and we exam- merase (Pharmacia, Piscataway, NJ) and 50 &i rH]ined the location of such RNPs, after microinjection of CTP (27.7 Ci/mmole, NEN, Boston, MA) according to labeled oocyte-type 5s RNA or 7s RNPs into the cyto- the procedure of Wahl et al. (1987). The template DNA plasm of vitellogenic oocytes. In this manner we studied was removed with RNase-free DNAse (Worthington, the fate of cytoplasmically stored 5s RNA during Xeno- Freehold, NJ). The expected RNA transcript size was ms oogenesis. We showed that 5s RNA first appears in 714 nt. The aH complementary RNA was hydrolyzed to the amplified nucleoli in vitellogenic oocytes when the approximately 150-nt fragments (Pardue, 1985), folnucleoli become active in 18S-5.8S-28s rRNA synthesis. lowed by ammonium acetate/ethanol precipitation. For Cytoplasmic microinjection of labeled 7s RNPs results control hybridizations, the reverse strand was synthein a portion of the labeled 5s RNA migrating into the sized using the SP6 RNA polymerase promoter at the

ALLISON,ROMANIUK,ANDBAKKEN

other end of the 5s RNA gene. For RNA slot blots, a nick-translated probe was prepared using pXlo8 and azP-dCTP (800 Ci/mmole, NEN). The Hind111 5S RNA gene insert was gel purified from the plasmid and nick translated according to Meinkoth and Wahl (1987); the probe was separated from unincorporated dNTPs on Biogel P60 and quantitated by Cerenkov counting. In Situ Hybridizations

5s RNA-Protein Interactions in Oocytes

131

probe). Slides were incubated in a moist chamber at 37°C for 20 hr. Slides were treated with RNase A, to remove nonspecifically bound probe, then washed in 0.1~ SSC at 50°C for 10 min and in 0.1X SSC at room temperature for 10 min, and then dehydrated in ethanol and air-dried. Slides were dipped in Kodak NTB 2 liquid emulsion, exposed at 4°C for 5-12 days, developed in D-19, and stained with Giemsa. Preparations were photographed with Kodak Pan X, ASA 32, B&W film.

A lobe of ovary was surgically removed from an adult Isolation and Labeling of 7’5 RNPs and 5s RNA female X Zaevis (Xenopus I, Ann Arbor, MI). The ovarStored 7s RNPs were isolated from 10 to 20 immature ian tissue was rinsed thoroughly in 0.15 M NaCl, 0.05 M ovaries (lacking large vitellogenic oocytes) of young X: Tris-HCl, pH 8.0, and 1 mM EDTA to remove blood, 2aevi.s (3-5 cm in length). The procedure of Sands and then rinsed twice in phosphate-buffered saline (PBS) Bogenhagen (1987) was followed with the addition of 0.1 (68 mM NaCl, 1.3 mM KCl, 4.0 mM Na,HPO,, 0.7 mM mM phenylmethylsulfonyl fluoride (PMSF) to the hoKH,PO,, 0.35 mM CaCl,, 0.25 mM MgCl,) to remove the mogenization buffer. The 7s RNPs were further puriEDTA. The tissue was cut into small pieces and gently fied by DEAE-cellulose column chromatography digested with collagenase (Type II, Sigma, St. Louis, (Whatman DE-52) as described by Hanas et aZ. (1983). MO; 1 mg/ml in 0.1 Msodium phosphate, pH 7.4, at room temperature for 2 hr, with end over end rotation). When Column fractions were assayed for 7s RNPs by running duplicate samples in 0.1 vol glycerol-dye loading buffer most oocytes were liberated as single cells free of follicle cells, the oocytes were rinsed twice in PBS and twice in on a nondenaturing 6% polyacrylamide (PA) gel in TBE O-R2 medium (82.5 mM NaCl, 2.5 mM KCl, 10 mM buffer (89 mM Tris base, 89 mM boric acid, 2.7 mM MgCl,, 1.0 mM Na,HPO,, 3.8 mM NaOH, 5.0 mM Hepes, EDTA). One half of the gel was stained with 0.1% CoopH 7.8, at 20°C; Wallace et al, 1973). Oocytes were sepa- massie blue R250, the other half with ethidium bromide rated into the six Dumont stages (Dumont, 1972) using at 0.01 pg/ml, to detect the presence of both protein and the following criteria: stage I, clear, 50-300 pm; stage II, RNA, respectively. Pooled 7s RNP fractions were diawhite/opaque, 300-450 pm; early stage III, tan, 450-650 lyzed against 20 mM Tris-HCl, pH 7.5,20% glycerol, 40 pm; late stage III, dark brown, 450-650 pm; stage IV, mM KCl, 2 mM MgCl,, and 1 mM DTT and stored in dark brown, distinct animal hemisphere, 600-1000 pm; small aliquots at -70°C. Concentrations were deterstage V, lighter brown, 1000-1200 pm; and stage VI, un- mined by the Bio-Rad protein microassay procedure (Richmond, CA). Labeled 7s RNPs (and 5s RNA) were pigmented equatorial band, 1200-1300 pm. Oocytes were fixed for 1 hr in 5% acetic acid, 2% form- prepared as follows: 7s RNPs were S’end labeled with 20 units of T4 RNA ligase (Pharmacia or BRL) and 125 PCi aldehyde, and 250 mM NaCl and then dehydrated through an ethanol series. Staged oocytes were embed- of [5’-32P]cytidine 3’,5’-bisphosphate (3000 Ci/mmole, ded in butyl-methacrylate and methyl-methacrylate NEN) according to the methods of Andersen et al. (9:l) with 1% benzoyl peroxide and polymerized over- (1984). Reactions containing lo-13 pg RNPs or RNA in night at 65°C (Jamrich et al, 1984). Alternatively, small 30 ~1 vol were incubated at 0°C for 16-20 hr. For recovpieces of ovarian tissue (not treated with collagenase) ery of the labeled 7s RNPs, the mixture was dialyzed were fixed in toto and embedded in paraffin by standard twice, for 2 hr each, against 500 ml of microinjection histological procedures. This was required to circum- buffer (20 mM Tris-HCl, pH 7.5, 1 mM KCl, 2 mM vent infiltration difficulties for analysis of the large, MgCl,, 1 mM DTT, 10% glycerol), a modified storage yolk-laden mature oocytes. Methacrylate sections were buffer that is within the physiological salt concentracut at 1 pm; paraffin sections were cut at 7 pm. Sections tions of the oocytes. Aliquots were frozen at -70°C. Samof embedded oocytes or ovarian tissue were transferred ples were electrophoresed on a 6% PA gel, followed by to subbed slides, the embedding medium was removed, autoradiography on XAR-5 Kodak film, to verify that and sections were then treated with proteinase K and the 7s RNPs were intact and labeled. Incorporation was acetic anhydride preceding in situ hybridization as de- measured by Cerenkov counting. For recovery of 32P-lascribed by Pardue (1985). Control slides were treated beled 5s RNA, the labeled 7s RNPs were ethanol precipiwith RNase A (Sigma). Single-stranded ‘H-labeled tated, resuspended in formamide-dye loading buffer, RNA probes were prepared from the plasmid, pXlo8G, and gel purified on an 8% PA/8 M urea gel in TBE. The as described above. 5s RNA band was excised, using the autoradiogram as a Approximately 100,000cpm of riboprobe were applied template, the gel slice crushed, and incubated overnight to each slide in 40% deionized formamide, 4~ SSC, and in 2.5 M ammonium acetate, 0.1% SDS, 1 mM EDTA, 333 pg/ml tRNA (to block nonspecific binding of the followed by extraction with phenol, then chloroform,

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DEVELOPMENTALBIOLOGY

and ethanol precipitation. The recovered, labeled 5s RNA was redissolved in renaturation/microinjection buffer (20 mM Tris-HCl, pH 7.6, 2 mM MgCl,, 1 mM KCl) and stored in aliquots at -70°C. Before use in microinjection experiments, the 5s RNA was heated at 55°C for 10 min, then cooled slowly to renature the RNA.

VOLUME144,1991

identified by 6% PAGE, were pooled, brought to 100 mM KCl, and concentrated on a 0.5-ml DE-52 column by step elution with 350 mMKC1 buffer. The 5s RNPs were dialyzed against 100 mM KC1 buffer and 20% glycerol and were stored in small aliquots at -70°C. Protein concentration was determined by the Bio-Rad protein microassay.

Synthesis of Labeled 3’ Truncated SS RNAs Templates for the production of 3’-truncated 5s RNA molecules were prepared by digesting to completion pXlo-108 with StuI, FokI, or BstNI. pXlo-108 has an oocyte-type 5s RNA gene under the control of a T7 RNA polymerase promoter (Romaniuk et ak, 1987). Internally labeled RNAs were transcribed as run-off molecules from these templates using T7 RNA polymerase and 50 &i [~u-~P]GTP (800 Ci/mmole, NEN). The synthesis was incubated for 2.5 hr at 37°C and the RNAs were gel purified on an 8% PA/8 M urea gel. The RNAs were eluted from the gel, ethanol precipitated, and redissolved in renaturation/microinjection buffer as above.

Microinjection Experiments

All experiments were repeated several times with different animals since there can be variability in synthetic activity between different batches of oocytes. The frogs were anesthetized on ice, a portion of the ovary was removed, and the wound was sutured. Ovaries were treated with collagenase, as described above, and the separated oocytes were incubated in O-R2 at 18-20°C. Oocytes were immobilized in a petri dish with 0.8 mm Nitex mesh attached to the bottom of the dish, and most of the O-R2 was drawn off prior to injection. Stage V oocytes were microinjected with 50 nl of RNA or RNP solution (containing from 1 to 10 ng of RNA/RNP) into Isolatim of 5s RNPs the equatorial, vegetal cytoplasm. After injection, oocytes were kept on ice in O-R2 for 30 min to promote The ribosomal protein L5/5S RNA complex (5s RNP), wound healing, then incubated for 18-24 hr at 18-20°C. used to test anti-serum specificity, was purified from Only oocytes that were healthy in appearance (uniform EDTA-treated ribosomes isolated from the ovaries of pigment, not mottled) were analyzed. mature X laevis females. The protocol of M. Sands Nuclei were manually dissected from oocytes using (State University of New York at Stony Brook) was used watchmaker’s forceps and collected for analysis in 1% with slight modification. Ovaries were dissected from 10 TCA. Oocytes were transferred from O-R2 to ice-cold mature frogs and rinsed in 0.15 M NaCl, 0.05 M TrisTCA and kept at 4°C for 30 min. The fixed nuclei were HCl, pH 8.0, and 1 mM EDTA. They were washed thorthen dissected out in 1% TCA, denuded of any adhering oughly and then homogenized in ice-cold buffer (100 mM cytoplasmic material by pipetting in and out of an EpKCl, 1.5 mMMgCl,, 2 mMDTT, 20 mMTris-HCl, pH 7.6, pendorf Ultra Micro Tip, and transferred to a microfuge and 0.1 mMPMSF) using pestle B with a Dounce homogtube on ice. The enucleated oocytes (cytoplasms) were enizer. The homogenate was centrifuged at 20,OOOgfor collected in a separate tube. Nucleic acids from both 30 min at 0°C. The supernatant was removed and centrisamples were extracted by the guanidinium thiocyanate fuged again at 100,OOOg for 1.5 hr at 0°C. The enriched method (Chomczynski and Sacchi, 1987). The RNA was ribosome pellet was collected and resuspended at 5-6 mg analyzed on 8% PA/8 M urea gels in TBE followed by protein/ml in 20 mM Tris-HCl, pH 7.6,2 mM DTT, 100 autoradiography. mMKC1,O.l mMPMSF, and 0.5 mMMgC1,. Three milliliters of diluted ribosomes was layered over 8 ml of 30% Antisera Used in These Analyses glycerol in the above buffer conditions and centrifuged Antibodies were generously provided to us as follows: in a Beckman Ti 70.1 rotor (Fullerton, CA) at 45,000 rpm for 1.5 hr at 0°C. The pellet of washed ribosomes was anti-TFIIIA from Dr. B. Honda (Simon Fraser Univerresuspended in 12 ml of buffer and treated with 0.1 vol of sity) was made against oocyte 7s RNPs and is charac250 mM EDTA, pH 8.0, on ice to release the 5s RNPs terized in Honda and Roeder (1980); anti-La, a human autoimmune serum, was provided by Dr. P. Thomas from the large ribosomal subunit. The EDTA-treated ribosomes were layered over 23 ml of 30% glycerol in (Oklahoma Medical Research Foundation); and the buffer and centrifuged at 25,000 rpm in a Sorvall AH627 anti-5S RNP antibody is also a human autoimmune rotor (DuPont Co., Newton, CT) for 2.5 hr. The upper serum that was given to us by Dr. J. A. Steitz (Yale two-thirds of the overlay was recovered and dialyzed University) and has been well characterized (as the Fe overnight against 15 vol of 20 mM Tris-HCl, pH 7.6, 2 serum) by Steitz et al. (1988) in HeLa cells. Initial tests mM DTT, and 100 mM KCl. The dialysate was loaded on were carried out to determine whether the serum would a l-ml DEAE-cellulose (DE-52) column and the 5s cross-react with Xenopus 5s RNP complexes in the same RNPs were eluted with a lo-column volume linear gra- manner that it immunoprecipitates HeLa cell 5s RNPs. Immunoprecipitation assays to test the specificity of dient from 125 to 400 mM KCl. The 5s RNP fractions,

ALLISON, ROMANIUK, AND BAKKEN

SS RNA-Protein

Interactions

in Oocytes

133

the anti-serum were performed using 10 ~1of serum and gen) was pelleted, and the pellet was washed four times 20 pg of purified 7S RNPs (5s RNA/TFIIIA complexes), with NET-2. After the final wash, 300 ~1 of NET-2 was or 20 pg of purified 5s RNPs (5s RNA/L5 complexes) added to each pellet, followed by 2 ~1 10 mg/ml carrier released from oocyte ribosomes by EDTA treatment, as tRNA, 30 ~1 10% SDS, and 300 ~1 of a 1:l mixture of described above. The RNA and protein components of phenol and chloroform. Samples were incubated at 37°C both kinds of RNPs had been identified as single bands for 15 min with frequent vortexing. After centrifugation for 2 min, the aqueous layer was removed and the RNA on RNA and protein gels, respectively. Immunoprecipitates were analyzed on 8% PA/8 M urea gels with a 5S was recovered by ethanol precipitation. RNA was then subject to slot blot analysis, as described below. AlternaRNA marker, and the gels were stained with ethidium bromide with the following results: purified ‘i’s RNPs tively, labeled RNA molecules from microinjected oowere immunoprecipitated with anti-TFIIIA, but not cyte samples were heat denatured in formamide and rewith anti-5S RNP, or nonimmune rabbit serum, or no solved on 8% PA/8 M urea gels followed by autoradiogserum. Purified 5S RNPs were immunoprecipitated raphy. with anti-5S RNP, but not with anti-TFIIIA, or nonimmune serum, or no serum (data not shown). Additional RNA Slot Blots assays demonstrated that anti-5S RNP does not react A lobe of ovary was removed from an adult X laevis with uncomplexed 5S RNA, tRNA, or with ribosomes in under anesthesia, and the oocytes were separated by colwhich the 5S RNA/L5 complex is internally located on lagenase as described above. Whole oocyte homogenates the 60s subunit. When oocytes were microinjected with were subject to immunoprecipitation assays as delabeled 5S RNA, homogenized, and subjected to immuscribed in the previous section. After the final ethanol noprecipitation assays with 1,2,10, and 20 ~1 of anti-5S precipitation, RNA pellets were raised in 50 ~1 TE (10 RNP, all gave the same autoradiogram signal of labeled mMTris-HCl, pH 7.5,l mMEDTA). The RNA was dena5S RNA, whereas the anti-TFIIIA produced increasing tured by the addition of 30 ~1 20x SSC and 20 ~1 37% signals of labeled RNA up to 10 ~1 of serum (data not formaldehyde and incubated at 60°C for 15 min. The shown). Therefore, in the experiments shown in this RNA was applied to a 0.45-pm nitrocellulose membrane paper, 10 ~1 of serum was used to ensure that antibody was not a limiting factor in assays. While this manu- (Schleicher & Schuell, Keene, NH) using a slot-blot apscript was in preparation, two other anti-5S RNP sera paratus. Hybridizations were carried out according to from Steitz et al. (1988) have been reported to immuno- the protocol by Schleicher and Schuell (1987), using 4 precipitate 5S RNA/L5 complexes from Xenopus oocytes X lo6 cpm of nick-translated probe per blot. Blots were washed twice in 2~ SSC/O.l% SDS and twice in 0.1 (Guddat et al., 1990) and Xenopus embryos (WormingSSC/O.l% SDS at room temperature for 10 min each. A ton, 1989). final wash was done for 60 min at 65°C in 0.1X SSC and 0.1% SDS. Blots were autoradiographed on Kodak Immunoprecipitation Assays XAR-5 film at -70°C. Protein A-Sepharose (PAS; Pharmacia) was swollen in NET-2 (50 mMTris-HCl, pH 7.4,150 mMNaC1,0.05% RESULTS NP-40) at 2.5 mg/0.5 ml. PAS was aliquoted at 0.5 ml per 5s RNA First Appears in the Amplified Nucleoli When microfuge tube, followed by the addition of antisera, They Become Activated with slight modifications of the protocol by Steitz (personal communication, and Steitz et al., 1988). Antibodies To address the question of whether incorporation of were bound to the PAS for 2 hr at room temperature stored 5S RNA into ribosomes occurs in the oocyte cytowith end over end rotation. After incubation, the resin plasm or in the nucleus in the amplified nucleoli, we (PAS-Ab) was pelleted for 5 set in a microfuge and re- first determined the cellular distribution of endogenous suspended in 1 ml of NET-Z, and the wash was repeated 5S RNA throughout oogenesis. In X laevis, the genes three times. Whole oocytes, pooled nuclei (dissected in encoding 18S, 5.8S, and 28s rRNA are selectively ampli25 mM Tris-HCl, pH 8.0, 10% glycerol, 5 mM MgCl,, 2 fied during early oogenesis and extrachromosomal numM DTT), or cytoplasms were homogenized in NET-2 cleoli form around the amplified rRNA genes (Brown with 0.1 mM PMSF and 10 unit/ml RNasin (Promega, and Dawid, 1968; Gall, 1968). These nucleoli become Madison, WI) added to reduce protein and RNA degrada- arranged around the periphery of the nucleus of the tion. Whole oocyte and cytoplasm samples were pre- previtellogenic oocyte, just beneath the nuclear envecleared of yolk and pigment by microfuging for 10 min lope. The rDNA in the nucleoli does not become tranat 10,000 rpm at 4°C. The supernatant was divided into scriptionally active until the beginning of vitellogenesis 0.5 ml samples, aliquoted onto each washed PAS-Ab (Pruitt and Grainger, 1981; Mitchell and Hill, 1987). pellet, and incubated at 4°C for 1 hr with end over end Since it has long been known that 5S RNA is stored in rotation. After incubation, the resin (now PAS-Ab-antithe cytoplasm of previtellogenic oocytes (Thomas, 1974),

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FIG. 1. In situ localization of 5s RNA in previtellogenic oocytes. Methacrylate sections (1 pm) of X laevis oocytes were hybridized with *H-labeled RNA complementary to 5s RNA synthesized from a 5s RNA gene clone, pXlo8G. Slides of sections shown in this figure were all prepared at the same time, under the same hybridization conditions. Slides were deliberately overexposed (11 days) in order to see whether the amplified nucleoli exhibited enrichment of radioactivity relative to the nucleoplasm. In (a-c), the cytoplasm is uniformly heavily labeled above background, while there are relatively few silver grains in the nucleus, whereas in (d) there is clustering of silver grains over the amplified nucleoli (NO). (a) A stage I, previtellogenic oocyte. Bar = 10 pm. (b) A stage II, previtellogenic oocyte. Bar = 10 pm. (c) A stage III, early vitellogenic oocyte. Bar = 8 @cm.(d) An early stage IV, midvitellogenic oocyte. Bar = 8 pm. The arrows in (d) indicate the representative amplified nucleoli that are densely labeled with silver grains.

it was important to establish whether or not 5s RNA appears in the nucleoli when they begin actively synthesizing the other rRNAs and assembling ribosomes. In order to localize the 5s RNA, in situ hybridizations using a ‘H-labeled 5S RNA riboprobe were carried out on histological sections of all six stages of oocytes, covering previtellogenesis, vitellogenesis, and postvitellogenesis. Figure 1 compares the silver grain distribution of 5s RNA hybrids in the cytoplasm, the nucleus, and the nu-

cleoli throughout the first four stages of oogenesis. The proportion of silver grains in the nucleoli versus the nucleoplasm is tabulated for each of the four stages in Table 1.5s RNA is enriched in the cytoplasm of stage I, II, and early stage III oocytes with relatively few silver grains over the nuclei and no significant accumulation over the amplified nucleoli (Figs. la-lc and Table 1). By early stage IV, there are large clusters of silver grains over the amplified nucleoli and more 5s RNA in the

ALLISON, ROMANKJK, AND BAKKEN TABLE 1 APPEARANCE OF 5S RNA IN THE OOCYTE NWLEOLUS AS DETERMINED BY IN SITU HYBRIDIZATION

Grain number” Embedding medium

Developmental stage

No. slides

Methacrylate

I II III IV IV VI

3 2 2 2 3 Same slides as IV

Paraffin

NO

N

Ratio NO/N

2.5 0.9 1.0 27.8 22.3 3.1

2.6 1.3 1.2 9.6 7.1 2.1

0.96 0.69 0.83 2.9 3.1 1.5

a The average of grain counts in 12-20 randomly selected areas of nucleoplasm (N) and similarly sized nucleoli (NO) (40 pm’). These data represent grain counts obtained from 6 to 10 different oocytes on each slide. There was some quantitative variation in grain numbers, but there were no qualitative differences in grain distribution for each stage. All measures were made at probe concentrations and hybridization times which achieve saturation. Slides were prepared at the same time, under the same hybridization conditions. Exposure time on both methacrylate and paraffin preparations was 11 days.

nucleoplasm (compare Figs. lc and Id). The nucleoli show a threefold increase in labeling over the nucleoplasm, presumably as a result of 5s RNA being assembled into ribosomes (Table 1). Note that in earlier stages the grain density over the nucleoli is the same as that over equal-sized areas of the nucleoplasm, reflecting the inactivity of the nucleoli regarding ribosome biogenesis. Postvitellogenic, stage VI oocytes are distinguished by their large size and by the central aggregation of most of the extrachromosomal, amplified nucleoli, which are clearly seen in Fig. 2 (a, b). There is only a slight enrichment of silver grains over some of the nucleoli in the central aggregate (Fig. 2b, Table 1) as compared with the distinct accumulation of silver grains over nucleoli in a vitellogenic, stage IV oocyte (Fig. 2~). All three pictures in Fig. 2 were taken from oocytes in the same paraffin section. On average, there was a 1.5fold greater grain density over nucleoli versus nucleoplasm in stage VI oocytes, but the total amount of nuclear hybridization was significantly lower in stage VI than it was in stage IV oocytes (Table l), which actively transcribe and assemble ribosomes. Note, however, that the strong 5s RNA hybridization to stage IV oocyte nucleoli could be due either to newly synthesized 5s RNA and/or to the migration of stored 5S RNA from the cytoplasm back into the nucleus. Control slides treated with RNase A prior to hybridization resulted in negligible silver grain density (data not shown), indicating that the majority of grains seen in the data slides represent hybridization to RNA, as opposed to DNA. To determine if the hybridization was sequence specific for 5s RNA, control slides were probed

55 RNA-Protein Interactions in Oocytes

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with RNA synthesized from the coding strand of the 5s RNA gene template, which should not hybridize to the 5s RNA. As expected, there was no hybridization in the cytoplasm or at the nucleoli in oocytes, but there was hybridization to chromosomal DNA in the surrounding follicle cell nuclei (data not shown). 5.S’RNA Is Present in Nonribosome-Bound Vitellogenic Oocytes

RNPs in

To address the question of what protein(s) 5s RNA interacts with in between storage in the cytoplasm, and eventual association with L5 in ribosomes, we first determined what endogenous 5s RNA-containing RNPs are present in vitellogenic oocytes. Although all of the stored 5s RNA is known to be complexed in previtellogenie oocytes with TFIIIA as 7s RNPs, or with other proteins and tRNA as 42s RNPs, the level of TFIIIA decreases over lo-fold from stage III through stage VI (Pelham et aZ.,1981), and the concentration of 42s RNPs drops sharply at the onset of vitellogenesis (Vie1 et al., 1990). Thus, we surveyed all stages of oocytes for endogenous RNPs containing 5s RNA and any one of three proteins present during vitellogenesis and known to interact with 5s RNA, namely TFIIIA, ribosomal protein L5, and the La protein. The La protein is proposed to be involved with termination of RNA polymerase III transcripts (Rinke and Steitz, 1982; Steitz et ah, 1988; Gottlieb and Steitz, 1989). We performed immunoprecipitations in which antibodies for each of these proteins/ RNPs were bound to protein A-coated Sepharose beads, then reacted with homogenates of whole oocytes. The RNA was extracted from the immunoprecipitated beads, applied to a slot blot, and hybridized with a labeled probe for the presence of 55 RNA. The antiTFIIIA antibody recognizes both TFIIIA alone and in a 7s RNP complex. Anti-5S RNP antisera is a human autoimmune antiserum which immunoprecipitates 5s RNA/L5 complexes (5s RNPs), but does not immunoprecipitate naked 55 RNA or 5s RNA in ribosomes or 60s subunits (data not shown; Steitz et ak, 1988; Wormington, 1989). Anti-La serum is a human autoimmune serum against the 50-kDa La antigen. Immunoprecipitation assays were carried out on all six stages of oocytes, covering previtellogenesis, vitellogenesis, and postvitellogenesis (Fig. 3). Since during previtellogenesis, stages I-II, 80% of the total RNA is either 5s RNA or tRNA (Mairy and Denis, 1971), small samples (0.2 oocyte equivalents) gave a sufficient signal on the slot blot. In later stages of oogenesis, 90% of the total RNA is rRNA, therefore larger amounts of material (20 oocyte equivalents) were required in order to load a comparable amount of nonribosome bound 5s RNA, and thus be able to detect 5s RNA-protein complexes. The immunoprecipitations showed the presence of 7s RNPs throughout all stages of oogenesis. In con-

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I-I I total

III

RNA

anti -La

FIG. 3. Localization of 5s RNA in ‘7s RNPs, 5s RNPs, and/or 5s RNA/La complexes during the previtellogenic, vitellogenie, and postvitellogenic stages of oogenesis in X luevis. Homogenates of staged oocytes were incubated with protein A-Sepharose-antibody complexes in an immunoprecipitation assay. RNA was recovered, applied to nitrocellulose using a slot blot apparatus, probed with nick-translated 5s DNA, and autoradiographed. The numbers above each column refer to the oocyte stages of Dumont (1972). Rows are labeled with the antibody used. Serum from a rabbit that had not been injected with any antigen was used as a control in mock immunoprecipitation assays, to ensure that 5s RNA was not being precipitated nonspecifically by the assay conditions. Total RNA was included as a control for the hybridization reaction. Each slot for stage I-II oocytes contains 0.2 oocyte equivalents; each slot for stage III-VI oocytes contains 20 oocyte equivalents.

trast, 5S RNPs were detectable, above background, only in vitellogenic through postvitellogenic stages. We cannot rule out the possibility that we did not detect 5s RNPs in previtellogenic oocytes because of the problems inherent in looking for an RNP of low abundance amid the massive quantities of 7S RNPs and 42s RNPs. What we can conclude, however, is that most of the 5S RNA is present in 7S RNPs, and 42s RNPs, in early oocytes. In later stages there is a substantial, and previously undescribed, endogenous pool of 5S RNPs, along with residual 7S RNPs. Only a trace amount of 5S RNA was present in the immunoprecipitates of samples treated with anti-La serum. Although Guddat et al. (1990) were able to detect a nuclear 5S RNA/La complex with labeled 5S RNA transcribed from a large excess of microinjected 5S RNA gene templates, the trace amount of 5S RNA hybrids in our immunoprecipitations is more likely background hybridization. The 5S RNA/La complexes are

FIG. 2. In situ localization of 5s RNA in vitellogenic and postvitellogenie oocytes. Paraffin sections (7 pm) of X lamis oocytes were hybridized with a 5s RNA probe as described in Fig. 1. (a) A postvitellogenic oocyte, stage VI, at low magnification showing the central aggregate (CA) of amplified nucleoli (NO). Bar = 40 pm. (b) High magnification detail of the same nucleus (N) as in (a), showing few grains over the nucleoli. Bar = 8 pm. (c) High magnification detail of a vitellogenic oocyte from the same i’-pm section, showing clustering of silver grains over amplified nucleoli. The arrow indicates a nucleolus with pronounced enrichment of grains at its periphery. Bar = 8 cm. C, cytoplasm.

ALLISON,ROMANIUK, ANDBAKKEN 5S RNA-Protein

Znteractions

very transient RNPs, and the mock immunoprecipitations conducted with control rabbit serum showed a similar level of 5S RNA hybridization. In summary, the data show that 5s RNA is present as 7S RNPs early in oogenesis and later complexes with ribosomal protein L5 to form a nonribosome bound pool of 5s RNPs detectable beginning at the onset of vitellogenesis in stage III oocytes.

a123

Identification and Localization of Labeled, Microinjected 5S RNA in RNPs

b

Vast amounts of 5S RNA are made and stored as RNP particles prior to the onset of nucleolar activation and ribosome assembly in the amplified nucleoli. In addition, 5S RNA synthesis continues throughout the vitellogenic stages of oogenesis. In situ hybridization with 5S RNA probes cannot distinguish the source of the 5S RNA as it appears in the nucleoli, that is, whether it represents stored or newly synthesized 5S RNA. Therefore, we microinjected labeled 5S RNA into the oocyte cytoplasm and followed its mobilization and potential for migration into and accumulation within the nucleus. Since our in situ hybridization data showed a reduced amount of 5S RNA accumulation over the nucleoli in stage VI oocytes, all microinjections were performed in stage V oocytes which are known to still be transcriptionally active and assembling ribosomes at a high rate (Bakken and LoGerfo, unpublished). In the experiment shown in Fig. 4a, 1 ng of 32P-labeled 5S RNA (120 nt) was microinjected into the cytoplasm of each oocyte assayed, and 20% (0.2 ng) of the RNA was found in the nucleus after 18-24 hr incubation. Quantitation was performed by densitometric measurements of suitable exposures of the autoradiograms. The observed accumulation of labeled 5S RNA in the nuclear compartment suggests a nucleolar site of assembly of the stored 5S RNA into ribosomes, as opposed to a potential cytoplasmic site of assembly. The total amount of labeled 5S RNA found in the nuclei increased up to 21 hr incubation and then remained constant at 0.2 ng per nucleus in several experiments, using oocytes from several females, in which l-3 ng of labeled RNA were injected into the cytoplasm (data not shown). Presumably, this constant amount reflects the steady-state levels of endogenous 5S RNA molecules which migrate into the nucleus, are assembled into ribosomes, and then exported to the cytoplasm. We have shown in the previous section that endogenous 5S RNA is found associated either with TFIIIA or with L5 in vitellogenic oocytes. Stored 5S RNA remains in the cytoplasm until the nucleoli become active and then it appears to migrate back to the nucleus, and through some series of interactions becomes assembled into ribosomes. Microinjection of 32P-5SRNA and subsequent immunoprecipitation from the nuclear versus

4

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137

in Oocytes

5

6

34

FIG. 4. (a) Migration of 5s RNA into the oocyte nucleus. Stage V oocytes were microinjected with full-length (120 nt) =P-5S RNA into the oocyte cytoplasm, incubated for 18-24 hr, and then manually fractionated into cytoplasmic and nuclear compartments. The autoradiogram shows the 8% polyacrylamide (PA)/8 M urea gel analysis of RNA extracted from pooled cytoplasms (0) and pooled nuclei, (0). Lane 1, uninjected “P-5s RNA marker. Lane 2, 10 cytoplasms. Lane 3, 10 nuclei isolated from oocytes in lane 2. Lane 4, uninjected labeled 5s RNA marker. Lane $9 cytoplasms. Lane 6,9 nuclei from the oocytes in lane 5. (b) Localization of ‘7s RNPs and 5s RNPs in the cytoplasm or nucleus of oocytes. Stage V oocytes were microinjected with gel-purified =P-labeled 5s RNA, incubated for 21 hr, and then manually fractionated into cytoplasmic and nuclear compartments. Homogenates from each compartment were analyzed by immunoprecipitation assays as described in Fig. 3. Labeled RNAs were recovered, analyzed on 8% PA/8 M urea gels, and autoradiographed. Lane 1, cytoplasms with anti-TFIIIA. Lane 2, nuclei with anti-TFIIIA. Lane 3, cytoplasms with anti-5S RNP. Lane 4, nuclei with anti-5S RNP.

the cytoplasmic compartments allowed us to deduce the RNA-protein interactions involved in the nucleocytoplasmic trafficking of 5S RNA. Figure 4b shows that labeled 5S RNA was precipitated as a ‘7s RNP with antiTFIIIA from the cytoplasmic fraction (lane 1) but not from the nuclear fraction (lane 2). In contrast, some of the labeled 5S RNA was immunoprecipitated as a 5S RNP from both the cytoplasmic fraction (lane 3) and the nuclear fraction (lane 4). Longer exposures of the autoradiograms maintained these conclusions. Thus, 7S RNPs appear to be confined to the cytoplasm, at least when they are initially formed there or stored there, whereas 5S RNPs are found in both compartments. The 7S RNP storage particles were also identified only in the oocyte cytoplasm by Mattaj et al. (1983). These authors proposed that 7s RNPs are excluded from the nucleus because the binding of TFIIIA to 5S RNA prevents free passage of the 7%kDa RNP through nuclear pores. Passive diffusion of proteins through the pores is limited to 65 kDa or less, and the larger molecules in this size range diffuse only slowly (reviewed by Dingwall and Laskey, 1986).

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11 The data in this paper show the migration of microinjetted, labeled 5s RNA from the cytoplasm to the nui,/’ cleus and show that 5s RNA is bound to both TFIIIA 5’ eCC~ACGGCCCC @ GO and L5 in the cytoplasm, but only to L5 in the nucleus. 3 UUCGGAUGCUG I UG This indicates that both proteins are apparently present 1:o 1000 E ; in the cytoplasm in sufficient quantities for the injected A u-00 C G 5s RNA to bind them. Guddat et al. (1990) have studied C G V A U the migration and protein associations of nascent 5s u u RNA from the nucleus to the cytoplasm. In this case, A A@ ;G they find 5s RNA bound to either TFIIIA or L5 within the nuclear compartment; both types of RNP then accu,/“G c” G C mulate in the cytoplasm. They also note that nascent 5s u u C G-01 RNA appears to show a preference for binding L5 in the C G IV nucleus. A-03

i

’

5s RNA Microinjected as 23 RNPs Can Migrate into the Nucleus in Vitellogenic &&es and Can Exchange Proteins in the Cytoplasm to Form 5s RNPs

Having determined that microinjected 5s RNA can be imported into the nucleus of oocytes, we next addressed the question of whether or not the structure of the 5s RNA or its interactions with proteins are important for its nuclear migration. Since 5s RNA is stored in the cytoplasm complexed with TFIIIA as a 7s RNP complex, and is also found in ribosomes complexed with L5 as a 5s RNP complex, it seemed likely that protein interactions would be of some importance in determining the mobilization and/or migration properties of 5s RNA. In order to adhere as closely as possible to in vivo conditions, we isolated native 7s RNPs from X Zaevis ovaries, end-labeled the 5s RNA with T4 RNA ligase and =P, and microinjected these 32P-7S RNPs into the oocyte cytoplasm. When ‘7s RNPs are end-labeled with T4 RNA ligase, the 5s RNA is often nicked at precise nucleotides, thus generating several RNA fragments of discrete size and sequence. All three fragments are of a length predicted by other investigators (Anderson et aL, 1984). The identity of the fragments was confirmed (data not shown) by RNA sequencing after partial digestion with RNase T, and RNase U, as in Donis-Keller et al. (1977). Cleavages gave rise to three sets of truncated molecules: 108/110 nt in length containing the sequence from nt 11/13 through 120 (“nt ll-EO”), 80/81 nt in length containing the sequence from nt 40 through 119/120 (“nt 40-120”), and 69/74 nt in length containing the sequence from nt 11/13 through 81/83/84 (“nt 11-84”) (summarized in Fig. 5). After microinjection of s2P-7SRNPs into the cytoplasm of oocytes, we observed that these truncated 5s RNA molecules, extracted from whole oocytes and from the cytoplasms of manually dissected oocytes, were similar in size and amount to the microinjected samples, after 18 hr of incubation (Fig. 6a). However, only the two largest RNAs, nt 11-120 and nt 40-120, were found in the nuclei of the oocytes (lane 3). The shorter nt 11-84 RNA

G

U

c C

oL04 G

G

A

”

*A;G%

ACCCUG

‘I’ C

UGGGAC C

AU

AG

UCUCG

CUGA

CG GACU AA

0°C C

GU\ U A-

i0

A

aG

nt 11113- 120(108/110

nt)

nt40 - 119/120 (80/81 nt) nt11113 - 81/83/84 (69i74 nt) ntl-108 ntl -98 ntl-69

FIG. 5. Summaryof truncated 5S RNAs used in the microinjection experiments. X laeuis oocyte-type 5s RNA sequence drawn according to the consensus secondary structure model for eukaryotic 5s RNA proposed by Garrett et al. (1981). In the uncleaved 5S RNA molecule there is extensive base pairing within helices I through V, forming the single-stranded loops, A through E. The numbers in bold type indicate the 3’ and 5’ termini of the truncated 5S RNAs we have analyzed and are listed below the model.

either could not enter or accumulate in the nucleus, or alternatively, may have been preferentially degraded in the nucleus. Figures 4a and 6a taken together show that 5s RNA, whether injected as a naked 5s RNA molecule or as a 7s RNP, can enter the oocyte nucleus and potentially participate in ribosome assembly. 5s RNA is associated initially with TFIIIA and then during vitellogenesis is found associated with L5 (Fig. 3), the protein with which it is complexed in ribosomes. Since ribosomal proteins begin to be translated from existing mRNAs during vitellogenesis (Wormington, 1989), it is expected that vitellogenic oocytes have a pool of L5 protein, some of which may be free to associate with 5s RNA. Figure 4b certainly suggests this, unless the microinjected 5s RNA competes for L5 that is already bound to the endogenous 5s RNA. To determine

ALLISON, ROMANIUK, AND BAKKEN

a

123

1081110

69174

b

123

120

FIG. 6. (a) Nuclear migration of 5S RNA microinjected in the form

of a 7s RNP storage particle. Stage V oocytes were microinjected with “P-7S RNPs into the cytoplasm and analyzed as described in Fig. 4a. The 7S RNP sample showed evidence of specific nicking of the 5S RNA during labeling, since three types of truncated 5S RNAs were present in RNA extractions before or after microinjection into the oocyte: nt 11-120 (size: 1OWllO nt), nt 40-120 (size: 80/U nt), and nt 11-84 RNAs (size: 69174 nt) (see Fig. 5). Lane 1, 6 whole oocytes. Lane 2, 12 cytoplasms. Lane 3,12 nuclei from the oocytes in Lane 2. Symbols are the same as in Fig. 4, with the two circles combined to represent the nucleus and cytoplasm of whole oocytes. (b) Association of ribosomal protein L5 with 5S RNA from microinjected 7S RNPs. Stage V oocytes were microinjected with “P-7S RNPs (“P-labeled, 120 nt 5S RNA complexed with TFIIIA) and incubated for 21 hr. Homogenates were divided into 10 oocyte equivalent aliquots and analyzed by immunoprecipitation assays as described in Fig. 4. Lane 1, anti-TFIIIA. Lane 2, anti-% RNP. Lane 3, rabbit control serum.

whether this interaction, or protein exchange between TFIIIA and L5, occurs in the cytoplasm of oocytes, we analyzed the fate of labeled 5s RNA when it is microinjetted into the cytoplasm in a 7’S RNP complex. After microinjection of labeled 7s RNPs and incubation of the oocytes, homogenates were immunoprecipitated either with anti-TFIIIA to retrieve the microinjected RNPs or with anti-5S RNP to retrieve any labeled 5s RNA which has undergone an exchange reaction with L5. Note that in these labeled 7’S RNP preparations, only full-length 5s RNA was present; there were no truncated 5s RNA molecules like those shown in Fig. 6a. Some of the labeled 5s RNA was immunoprecipitated with anti-5S RNPs (lane 2), which suggests several possibilities: L5 actively displaced TFIIIA on some of the labeled 5s RNA molecules; free 5s RNA was present in the 7’SRNP

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samples before injection and subsequently complexed with L5; or the 7s RNP simply dissociated after microinjection, thus allowing L5 to bind the labeled 5s RNA. Since there is still immunoprecipitation of labeled 7s RNPs (lane l), this indicates that the exchange, or dissociation, is not immediate and total after injection. Coassociation of the two proteins with 5s RNA, as opposed to mutual exclusion, was not considered a likely possibility since TFIIIA and L5 are known to bind to similar regions of the 5s RNA (Huber and Wool, 1986). Thus, microinjection of 5s RNA, whether naked or in 7s RNPs, in all likelihood results in some of the 5s RNA binding to L5 preferentially over association with TFIIIA. This protein exchange may, thereby, mobilize that 5s RNA for migration into the nucleus, and for targeting to the nucleolus for assembly into the 60s ribosomal subunit.

Sequence Requirements for Nuclear Migration and/or Formation of Immunologically Detectable Complexes with TFIIIA and L5

We have examined the structural requirements of 5s RNA for nuclear accumulation and for protein associations by analyzing six truncated 5s RNA molecules. We studied the ability of three gel-purified partial 5s RNA molecules, which resulted from nicking during the endlabeling of 7s RNPs (see Fig. 6a), to enter the nucleus and/or to form immunoprecipitable RNP complexes within oocytes. In addition, we injected three internally labeled 5s RNA run-off transcripts of known length, which were synthesized in vitro from a 5s RNA gene clone under control of a T7 RNA polymerase promoter (summarized in Fig. 5). Figure 7a shows that the nt 11-120 and nt 40-120 RNAs accumulate in the nucleus after being injected as part of a mixture of all three gel-purified, labeled 5s RNAs. The smaller, partially deleted nt 11-84 RNA molecules may or may not enter the nuclear compartment, but they do not accumulate there. Interestingly, only the nt 11-120 RNA is immunoprecipitable in an RNP complex either with TFIIIA (Fig. 7b, lane 2) or with L5 (lane 3). If one compares Figs. 6a and ‘7a,we see that in both cases the nt 11-120 and nt 40-120 RNAs enter and accumulate in the nucleus. Since binding to L5 seems to be an important factor in nuclear import, it may be that the nt 40-120 RNA binds one or both proteins in a fashion which is no longer recognized by our antisera, but in the case of L5 is sufficient to accompany it to the nucleus (Fig. 7a, lane 2). To further analyze the sequence requirements of the 5s RNA molecule for RNP formation, particularly at the 3’ end of the molecule, we microinjected three additional truncated 5s RNAs generated by in vitro transcription. As shown in Fig. 8a, only the longest tran-

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108 of the 120 nt 5S RNA molecule provide the necessary sequence and conformational information required for 5S RNA to bind TFIIIA or L5 as a stable, immunoprecipitable complex and for nuclear accumulation of the labeled 5S RNAs. These data are summarized in Table 2.

80181 DISCUSSION 69174

108/110

80181

We studied the pathway of 5S RNA during oogenesis in X laevis from its storage in the cytoplasm to its reentry and accumulation in the nucleus, the sequence requirements for the 5S RNA to follow that pathway, and the 5S RNA-protein interactions that occur during the mobilization of stored 5S RNA for assembly into ribosomes.

a

12

3456 108

69174 98

FIG. 7. (a) Fate of partially deleted 5s RNAs in the oocyte. Stage V oocytes were microinjected into the cytoplasm with gel-purified nt 11-120 (size: 108/110 nt), nt 40-120 (size: 80/81 nt), and nt 11-84 RNAs (size: 69/‘74 nt) (see Fig. 5). These labeled RNAs were isolated from “P-end-labeled ‘7s RNPs (see Fig. 6a). Lane 1, a mixture of all three sizes of 5s RNAs was injected and analyzed from 10 cytoplasms. Lane 2, the portion of each of those RNAs found in the 10 nuclei isolated from the oocytes in lane 1. Lane 3, injection of the gel-purified nt 11-84 RNA only, 10 cytoplasms. Lane 4, RNA from 10 nuclei from the oocytes in lane 3. (b) Interaction of truncated 5s RNAs with TFIIIA and/or L5 in oocytes. Stage V oocytes were microinjected with a mixture of gel-purified, truncated 5s RNAs, as described in (a). Homogenates of whole oocytes were immunoprecipitated after 21 hr of incubation as described in Fig. 3, and the labeled 5s RNAs were analyzed on 8% PA/8 M urea gels followed by autoradiography. Lane 1, control rabbit serum. Lane 2, anti-TFIIIA. Lane 3, anti-5S RNP. Lane 4, RNA left in the supernatant after immunoprecipitation with control rabbit serum in lane 1, showing the sizes of all of the microinjected 5s RNAs.

script, nt l-108, entered and accumulated in the nucleus. Figure 8b shows that only this partially deleted 5S RNA is immunoprecipitable in an RNP complex with TFIIIA or L5 (lanes 2 and 3, respectively). Longer exposures of these autoradiograms confirmed these conclusions. The immunoprecipitation assays, while very specific for detection of the 7s and 5s RNP complexes, do not distinguish between naked RNA and RNA that is complexed with protein in a nonimmunoreactive conformation. Thus, we cannot conclude that the other two truncated RNAs do not bind TFIIIA or L5 at all. However, combining the data from both types of partially deleted 5s RNAs, it can be concluded that nucleotides 11 through

69

b 108 98

69

FIG. 8. (a) Fate of 3’-deleted 5s RNAs in the oocyte. Internally labeled, truncated =P-5S RNAs were generated by in vitro transcription from a 5s RNA gene clone, pXlo-108. Stage V oocytes were microinjetted with a mixture of the gel-purified nt l-108, nt l-98, and nt l-69 5s RNAs and analyzed as described in Fig. 4. Lane 1, 10 cytoplasms from oocytes injected with all three lengths of 5s RNA. Lane 2, 10 nuclei from the oocytes in lane 1. Lane 3, 10 cytoplasms from oocytes injected with the l-98 nt RNA alone. Lane 4, 10 nuclei from the ooeytes in lane 3. Lane 5, 10 cytoplasms from oocytes injected with the l-69 nt 5s RNA alone. Lane 6,lO nuclei from the oocytes in lane 5. (b) Protein interactions of 3’-deleted 5s RNAs in oocytes. A mixture of three internally labeled 5s RNAs as described in (a) were microinjetted into stage V oocytes and analyzed by immunoprecipitation from homogenates of whole oocytes as described in Fig. 7b. Lane 1, control rabbit serum. Lane 2, anti-TFIIIA. Lane 3, anti-5S RNP. Lane 4, RNAs left in the supernatant from the immunoprecipitation with the control rabbit serum in lane 1, showing the sizes of all the RNAs injected.

ALLISON, ROMANIUK,AND BAKKEN

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Oocytes

TABLE 2 SUMMARYOFTHE NUCLEOTIDESEQUENCESAFFECTING5s RNA BINDINGTOTFIIIA AND L5 AND AFFECTINGNUCLEARACCUMULATION Immunoprecipitation 55 RNA sequence

Nuclear accumulation after 18-24 hr

nt l-120 (full length) nt 11/13-120 (1OWllO nt) nt 40-119/120 (80/U nt) nt11/13-81/83/84(69/74 nt) nt l-108 nt l-98 nt l-69

Anti-TFIIIA + +

+ + + +

+ -

-

by Anti-5S RNP + + + -

Note. Assays were performed as described in the legend to Fig. 4.

Association

of 5S RNA with Active, Amplified

Nucleoli

In situ hybridization shows localization of 5s RNA only in the cytoplasm in previtellogenic oocytes. These results provide additional evidence that synthesis of new ribosomes containing oocyte-type 5s RNA does not occur in previtellogenic oocytes, or occurs only at very low levels. Earlier studies have shown that previtellogenie oocytes do not contain detectable levels of ribosomes with oocyte-type 5s RNA (Denis and Wegnez, 1977), although protein synthesis is taking place (Dixon and Ford, 1982). The 5s RNA found in ribosomes of immature ovaries is of the somatic type and presumably is derived from ribosomes synthesized in the follicle cells, primordial germ cells, and oogonia. In situ hybridization shows an association of 5s RNA with amplified nucleoli in vitellogenic oocytes, correlating with the onset of rRNA synthesis and ribosome assembly. In some in situ hybridizations to sections of oocytes, 5s RNA was observed to be localized at the periphery of the nucleoli, in a region that probably corresponds to the granular component where ribosomes are forming (reviewed in Hadjiolov, 1985). The accumulation of ribosomal proteins follows the pattern of rRNA synthesis, beginning late in previtellogenesis and continuing throughout the vitellogenic period, with peak synthesis occurring in mid-oogenesis when greater than 30% of the protein synthesized by the oocyte is ribosomal protein (Hallberg and Smith, 1975; Mitchell and Hill, 1987). A full complement of ribosomes, about 101’ (Brown, 1966), accumulated in postvitellogenic oocytes, but there is still synthesis and turnover of rRNA (Leonard and LaMarca, 1975). Nucleoli in the central aggregate within nuclei of mature oocytes, analyzed by Scheer et al. (19’76),still incorporate r3H]uridine, but the ultrastructure shows distinct zones of extremely dense aggregations of nucleolar fibers, indicative of widespread inactivation. In this study, the lack of pronounced hybridization to 5s RNA sequences over the central aggregate of nucleoli adds further evidence to the decline in activity of the nucleoli in postvitellogenic oocytes.

Migration

of 5S RNA from the Cytoplasm to the Nucleus

Microinjection experiments demonstrate that 5s RNA can migrate from the cytoplasm into the nucleus in stage V oocytes. These analyses, in addition to a previous study which shows that the stored 5s RNA is incorporated into ribosomes slightly less rapidly than newly synthesized 5s RNA (Mairy and Denis, 1972), are consistent with a scenario in which the cytoplasmically stored 5s RNA returns to the oocyte nucleus for incorporation into ribosomes, while newly synthesized 5s RNA could migrate through the nucleoplasm directly to the nucleoli, according to the pathway proposed for somatic cells (Steitz et al., 1988). In order to appreciate how much 5s RNA becomes concentrated in the nuclear compartment after microinjection, one has to take into account the vast differences in volume between the nucleus and the yolkfilled cytoplasm. The oocyte nucleus is much smaller than the cytoplasm, about 20 times by volume based on diameter, or at least 10 times considering that about half the cytoplasmic volume is occupied by yolk platelets; the nucleus contains approximately 12% of the volume of the yolk-free cytoplasm in mature oocytes (Bonner, 1975; DeRobertis et al., 1982). Thus, the amount of microinjected 5s RNA present in the nucleus should be multiplied by a factor of lo-20 to appreciate the difference in terms of concentration between the nuclear and cytoplasmic compartments. When 1 ng of 32P-5SRNA (full length, or the l-108 fragment) was microinjected into the cytoplasm, 20% of the 5s RNA was found in the nucleus after 24 hr. Thus, in these experiments, the 5s RNA becomes two- to fourfold concentrated in the nucleus over the cytoplasm, which is consistent with the fact that the 5s RNA is in a state of flux between the two compartments. The 5s RNA enters the nucleus, is presumably assembled into the 60s large ribosomal subunit at the nucleolus, and the 5s RNA then returns to the cytoplasm as part of this particle. In addition, since ribosome assembly continues for a protracted period of time in X laevis oocytes, only a small fraction of mi-

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croinjected 5S RNA, along with the endogenous 5S appear that L5 is present in both cellular compartments RNA, would be expected to appear in the nucleus at any of the oocyte, since newly synthesized 5S RNA from migiven time. croinjected DNA templates associates with L5 within It remains to be determined what factors limit the the nucleus and then migrates to and accumulates in the rate of migration of 5s RNA to the nucleus and/or the cytoplasm (Guddat et ah, 1990). These two sets of data amount of accumulation. Nuclear export of tRNA (Zas- suggest then that 5S RNPs can move both into and out of the nucleus, in direct contrast to the unidirectional loff, 1983) and 40s and 60s ribosomal subunits (Khanna-Gupta and Ware, 1989; Bataille et al., 1990) has movement of 7S RNPs which apparently can only move been suggested to be a carrier-mediated, saturable pro- out of the nucleus. The relatively low levels of immunocess. Further, the oocyte nucleus appears to have a lim- precipitable 5S RNPs in the nucleus are in agreement ited capacity to accumulate ribosomal proteins (Tsurugi with the persistent, low levels of nuclear 5S RNPs seen et al, 1988). Upon inhibition of rRNA synthesis, these by Guddat et al. (1990), where most of the newly syntheauthors showed that ribosomal proteins which accumu- sized 5S RNA migrates out of the nucleus and accumulated in excess over rRNA were degraded by a cysteine lates in the cytoplasm over time. In addition, once 5S protease in the nucleus. Similarly, L5 protein appears to RNPs are incorporated into the 60s ribosomal subunit, be degraded if it does not associate with 5S RNA (Wor- they are no longer recognized by the anti-5S RNP antiassays. Proposals have mington, personal communication; Bakken, Guddat, sera in immunoprecipitation and Pieler, unpublished observations). been made that the 5S RNP is a precursor to ribosome assembly within the nuclei of mammalian somatic cells (Steitz et al., 1988), in yeast (Brow and Geiduschek, Existence and Significance of the Nonribosome Bound 1987), and withinxenopus embryos (Wormington, 1989). Pool of 5S RNPs We have provided evidence that, likewise, L5 and 5S In this paper, we showed that a pool of nonribosome RNA form a stable complex prior to assembly of ribobound 5S RNPs is present in vitellogenic oocytes and somal subunits in X laevis oocytes. Further, we have that labeled 5S RNA microinjected into the cytoplasm presented strong evidence that this 5S RNP complex can of stage V oocytes became complexed with L5. Some of form in the cytoplasm of oocytes in order to mobilize stored 5S RNA for migration to the nuclei where ribothe microinjected 5S RNA also became associated with somes are being assembled. TFIIIA, which seems surprising since very little TFIIIA is synthesized in stage V oocytes (Dixon and Ford, 1982). However, it is possible that microinjected 5S RNA is 5s RNA Sequences Required for Nuclear Accumulation exchanging with endogenous 5S RNA in preexisting and for RNA-Protein Interactions Involved in stored ‘7s RNPs, which we have shown are still present Intracellular Traficking in stage V oocytes. This type of exchange reaction has The mechanisms governing entry into or exit from the been shown to occur in vitro (Andersen and Delihas, nuclear compartment of proteins, RNA, and RNPs are 1986). Further evidence for this exchange occurring within the oocyte is that in our experiments, some of the subjects of much current interest. Studies with mutant labeled 5S RNA from microinjected 7S RNPs is released tRNAs have demonstrated the requirement for particufrom the TFIIIA and complexes with L5. What is not lar nucleotide signal sequences for the export of tRNA clear is whether the L5 derives from a pool of newly from the nucleus in X Zaevis oocytes (Zasloff, 1983; Tosynthesized protein or from preexisting 5S RNPs. Be- bian et al., 1985). Further, studies with mutant U2 fore stored 5S RNA can bind L5 and become incorpo- snRNA transcripts have shown the importance of RNA rated into the 60s large ribosomal subunit, TFIIIA must sequences in the mechanism of transport into the nucleus, for RNA that is unable to bind the Sm antigen be released from the 5S RNA. This event presumably does not accumulate in the nucleus (Mattaj and DeRooccurs during vitellogenesis when L5 becomes available for the first time (Wormington, 1989). Data presented in bertis, 1985). Recently, it has been shown that the trithis paper strongly suggest that the release of 5S RNA methylguanosine cap structure of Ul snRNA has an esfrom the 7S RNP storage particle occurs within the cy- sential role in the transport of Ul snRNPs to the nucleus toplasm, but the mechanism for this exchange of 5S (Fischer and Ltihrmann, 1990; Hamm et ah, 1990). In the RNA binding proteins, whether an active or passive pro- analysis presented here, we studied the nuclear import of six truncated 5S RNAs and their interactions with cess, is not yet known. Unlike the strictly cytoplasmic distribution of labeled TFIIIA and L5. This analysis does not exclude the possi7’S RNPs after microinjection of labeled 5S RNA, la- bility that there may be other proteins which particibeled 5S RNPs were found to be present both in the pate in the transport of 5S RNA into and out of the nucleus prior to its assembly into ribosomes. cytoplasm and the nucleus, which implicates the involveFull-length, nt l-108, and nt 11-120 5S RNAs are all ment of ribosomal protein L5 in the transport of 5S RNA back to the nucleus in X Zuevis oocytes. It would immunoprecipitable with anti-TFIIIA and antidS RNP

ALLISON, ROMANIUK,AND BAKKEN

from the cytoplasmic compartment. Thus, these partial deletions support the interpretation that nucleotides 11 through 108 provide sufficient sequence information to fold the 5s RNA molecule into a secondary/tertiary structure which can recognize and bind both TFIIIA and L5, in complexes that are recognized by the antisera used in this study. The fact that a 5s RNA lacking the first 39 nt from the 5’ end of the molecule, the nt 40-120 RNA, can accumulate in the nucleus, even though it is not immunodetectable in a 5s RNP, can be explained if binding of L5 is altered due to the partially deleted sequence and thus, the complex is not recognized by our particular antibodies. Interestingly, Guddat et al. (1990) have shown that a mutant 5s RNA with a deletion of nt 11-41, transcribed from a template injected into oocytes, is immunoprecipitable in an RNP complex with La protein, but not with TFIIIA and only very weakly with L5. This mutant RNA is not exported to the cytoplasm, it remains in the nucleus and is degraded slowly with time. Thus, nucleotides 11-41 of the 5s RNA may be required for nuclear export, but are not required for nuclear import. These two particular mutant 5s RNAs illustrate the importance and the power of studying 5s RNA-protein interactions and nucleocytoplasmic transport of RNPs in order to decipher which portions of the RNA and the protein moieties are essential for 5s RNA trafficking within the oocyte. Similar studies are being carried out to define the RNA sequence requirements for targeting 5s RNA to the nucleoli for assembly into ribosomes. Also, we are currently investigating the ability of several mutant 5s RNAs to become incorporated into ribosomes and to function in translation. We are grateful to Steve Brocco for expert preparation of methacrylate sections, to Angela Nelson for technical assistance, and to Pamela Hines for subcloning pXlo8. We also thank Donna Koslowsky for experimental insights and Simon Sims, Michael Harkey, John Dennis, and anonymous reviewers for their critical reading and helpful suggestions of earlier drafts of this manuscript. This work was supported in part by a grant from the National Institutes of Health (GM-28905) to A.H.B. and by a grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) to P.J.R. P.J.R. is also the recipient of an NSERC University Research Fellowship. L.A.A. was supported by a Public Health Service National Research Services Award (5 T32 GM-07270) from the National Institute of General Medical Science. REFERENCES ANDERSEN,J., and DELIHAS, N. (1986). Characterization of RNA-protein interactions in 7’S ribonucleoprotein particles from Xenopus laevis oocytes. J. BioL Chem. 261,2912-291’7. ANDERSEN,J., DELIHAS, N., HANAS, J. S., and Wu, C.-W. (1984). 5s RNA structure and interaction with transcription factor A. 2. Ribonuclease probe of the 7s particle from Xenom~~ laevis immature oocytes and RNA exchange properties of the 7s particle. Biochemistry 23,5759-5766.

BARRETP,P., and SOMMERVILLE,J. (1987). An alternative protein factor which binds the internal promoter of Xenopus 5s ribosomal RNA genes. Nucleic Acids Res. 15,8679-8691.

5s RNA-Protein

Interactions

in Oocytes

143

BATAILL~?,N., HELSER,T., and FRIED, H. M. (1990). Cytoplasmic transport of ribosomal subunits microinjected into the Xewpus laevis oocyte nucleus: A generalized, facilitated process. J. Cell Biol. 111, 1571-1582. BIRKENMEIER,E. H., BROWN,D. D., and JORDAN,E. (1978). A nuclear extract of Xenopus laevis oocytes that accurately transcribes 5s RNA genes. Cell 15,1077-1086. BLOBEL,G. (1971). Isolation of a 5s RNA-protein complex from mammalian ribosomes. Proc. Natl. Acad. Sci USA 68,1881-1885. BONNER,W. M. (1975). Protein migration into nuclei. I. Frog oocyte nuclei in vivo accumulate microinjected histones, allow entry to small proteins, and exclude large proteins. J. Cell BioL 64,421-430. BROW,D. A., and GEIDUSCHEK,E. P. (1987). Modulation of yeast 5s RNA synthesis in vitro by ribosomal protein YL3: A possible regulatory loop. J. Biol. Chem. 2621,13,953-13,958. BROWN,D. D. (1966). The nucleolus and synthesis of ribosomal RNA during oogenesis and embryogenesis of Xenopus laevis. NatL Cancer Inst. Monogr. 23,297-309.

BROWN,D. D., and DAWID, I. B. (1968). Specific gene amplification in oocytes. Science 160,272-280. CHOMCZYNSKI,P., and SACCHI,N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162,156-159. DENIS, H., and WEGNEZ,M. (1977). Biochemical research on oogenesis: Oocytes of Xenopus laevis synthesize but do not accumulate 5s RNA of somatic type. Dev. Biol. 58,212-217. DEROBERTIS,E. M. (1983). Nucleocytoplasmic segregation of proteins and RNAs. Cell 32,1021-1025. DEROBERTIS,E. M., LIENHARD, S., and PARISOT,R. F. (1982). Intracellular transport of microinjected 5s and small nuclear RNAs. Nuture (London) 295,572-577.

DINGWALL,C., and LASKEY, R. A. (1986). Protein import into the cell nucleus. Annu. Rev. Cell Biol. 2,367-390. DIXON, L. K., and FORD,P. J. (1982). Regulation of protein synthesis and accumulation during oogenesis in Xenopus lae-vis. Da? BioL 93, 478-497.

DONIS-KELLER,H., MAXAM, A. M., and GILBERT, W. (1977). Mapping adenines, guanines, and pyrimidines in RNA. Nucleic Acids Res. 4, 2527-2538. DUMONT,J. N. (1972). Oogenesis in Xenopus Levis (Daudin). I. Stages of oocyte development in laboratory maintained animals. J. MarphoL 136,153-180. FISCHER,U., and LUHRMANN,R. (1990). An essential signaling role for the m,G cap in the transport of Ul snRNP to the nucleus. Science 249,786-790.

GALL, J. G. (1968). Differential synthesis of the genes for ribosomal RNA during amphibian oogenesis. Proc. NutL Acad Sci. USA 60, 553-560. GARRETT,R. A., DOUTHWAITE,S., and NOLLER,H. F. (1981). Structure and role of 5s RNA-protein complexes in protein biosynthesis. Trends Biochem. Sci 6,137-139.

GO~TLIEB,E., and STEITZ,J. A. (1989). Function of the mammalian La protein: Evidence for its action in transcription termination by RNA polymerase III. EMBO J. 8,851-861. GUDDAT,U., BAKKEN, A. H., and PIELER, T. (1990). Protein-mediated nuclear export of RNA: 5s rRNA containing small RNPs in Xewpus oocytes. Cell 60, 619-628. HADJIOLOV,A. A. (1985). “The Nucleolus and Ribosome Biogenesis.” Springer-Verlag, New York. HALLBERG,R. L., and SMITH,D. C. (1975). Ribosomal protein synthesis in Xenopus laevis oocytes. Dev. BioL 42,40-52. HAMY, J., DARZYNKIEWICZ, E., TAHARA, S. M., and MATTAJ, I. W. (1990). The trimethylguanosine cap structure of Ul snRNA is a component of a bipartite nuclear targeting signal. Cell 62, 569-577. HANAS, J. S., BOGENHAGEN,D. F., and WV, C.-W. (1983). Cooperative model for the binding of Xenopus transcription factor A to the 5s RNA gene. Proc. Natl. Acad. Sci. USA 80,2142-2145.

144

DEVELOPMENTALBIOLOGY

HONDA, B. M., and ROEDER,R. G. (1980). Association of a 5S gene transcription factor with 5s RNA and altered levels of the factor during cell differentiation. Cell 22, 119-126. HUBER, P. W., and WOOL,I. G. (1986). Use of the cytotoxic nuclease o-sarcin to identify the binding site on eukaryotic 5s ribosomal ribonucleic acid for the ribosomal protein L5. J. Biol. Chem. 261, 3002-3005. JAMRICH, M., MAHON, K. A., GAVIS, E. R., and GALL, J. G. (1984). Histone RNA in amphibian oocytes visualized by in situ hybridization to methacrylate-embedded tissue sections. EMBO J. 3, 19391943. JOHNSON,R. M., BARRETT,P., and SOMMERVILLE,J. (1984). Distribution and utilization of 5S-RNA-binding proteins during the development of Xenopus oocytes. Eur. J. B&hem. 144,503-508. KHANNA-GUPTA, A., and WARE, V. C. (1989). Nucleocytoplasmic transport of ribosomes in a eukaryotic system: Is there a facilitated transport process? Proc. Natl. Acad Sci USA 86,1791-1795. LEMAIRE, M., and DENIS, H. (1987). Biochemical research on oogenesis: Binding of tRNA to the nucleoprotein particles of Xenopus laevis previtellogenic oocytes. J. BioL Chem. 262,654-659. LEONARD,D. A., and LA MARCA, M. J. (1975). In viva synthesis and turnover of cytoplasmic ribosomal RNA by Stage 6 oocytes of Xenopus la&s. Dev. BioL 45, 199-202. MAIRY, M., and DENIS, H. (1971). Recherches biochimiques sur l’oogenese. 1. Synthese et accumulation du RNA pendant l’oogenese du crapaud sud-africain Xenopus laevis. Dev. BioL 24,143-165. MAIRY, M., and DENIS, H. (1972). Recherches biochimiques sur l’oogenese. 2. Assemblage des ribosomes pendant le grand accroissement des oocytes de Xenopus laevis. Eur. J Biochem. 25.535-543. MATTAJ, I. W., COPPARD,N. J., BRO~KN,R. S., CLARK, B. F. C., and DEROBERTIS,E. M. (1987). 42s p48-the most abundant protein in previtellogenic Xenopus oocytes-resembles elongation factor la structurally and functionally. EMBO J. 6,2409-2413. MATTAJ, I. W., and DEROBERTIS,E. M. (1985). Nuclear segregation of U2 snRNA requires binding of specific snRNP proteins. Cell 40, lll118. MATTAJ, I. W., LIENHARD, S., ZELLER, R., and DEROBERTIS, E. M. (1983). Nuclear exclusion of transcription factor IIIA and the 42s particle transfer RNA-binding protein in Xenopus oocytes: A possible mechanism for gene control. J. Cell BioL 97,1261-1265. MEINKOTH,J., and WAHL, G. M. (1987). Nick translation. In “Methods in Enzymology” (S. L. Berger and A. R. Kimmel, Eds.), Vol. 152, pp. 91-94. Academic Press, New York. MITCHELL, E. L. D., and HILL, R. S. (1987). The activation of amplified ribosomal RNA genes in the oocytes of Xenopus laevis: An electron microscope analysis. Hereditas 107.219-227. PARDUE, M. L. (1985). In situ hybridisation. In “Nucleic Acid Hybridisation: A Practical Approach” (B. D. Hames and S. J. Higgins, Eds.), pp. 179-202. IRL Press, Washington, DC. PELHAM, H. R. B., WORMINGTON,W. M., and BROWN,D. D. (1981). Related 5S RNA transcription factors in Xenopus oocytes and somatic cells. Proc. Natl. Acad Sci. USA 78.1760-1764. PICARD, B., and WEGNEZ, M. (1979). Isolation of a 7S particle from Xenopus laevis oocytes: A 5S RNA protein complex. Proc. Natl. Acad Sci. USA 76,241-245.

PICARD,B., LEMAIRE, M., WEGNEZ,M., and DENIS, H. (1980). Biochemical research on oogenesis: Composition of the 42-S storage particles of Xenopus laevis oocytes. Eur. J Biochem. 109,359-368. PRUITF, S. C., and GRAINGER,R. M. (1981). A mosaicism in the higher

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order structure of Xenopus oocyte nucleolar chromatin prior to and during ribosomal gene transcription. Cell 23,711-720. RINKE, J., and STEITZ,J. A. (1982). Precursor molecules of both human 5S ribosomal RNA and transfer RNAs are bound by cellular protein reactive with anti-La lupus antibodies. Cell 29.149-159. ROEDER,R. G., LEE, D. C., HONDA, B. M., and SHASTRY,B. S. (1981). Analysis of eukaryotic gene transcription in vitro. In “Developmental Biology Using Purified Genes” (D. D. Brown, Ed.), ICN-UCLA Symposium on Molecular and Cellular Biology, Vol. 23, pp. 429-446. Academic Press, New York. ROMANILJK,P. J., STEVENSON,I. L., and WONG,H.-H. A. (1987). Defining the binding site of Xenopus transcription factor IIIA on 5S RNA using truncated and chimeric 5S RNA molecules. Nucleic Acids Res. 15,2737-2754. ROMANIUK, P. J., STEVENSON,I. L., EHRESMANN,C., ROMBY,P., and EHRESMANN,B. (1988). A comparison of the solution structures and conformational properties of the somatic and oocyte 5S rRNAs of Xenopus laevis. Nucleic Acids Res. 16,2295-2312. SANDS,M. S., and BOGENHAGEN,D. F. (1987). TFIIIA binds to different domains of 5S RNA and the Xenqpus borealis 5S RNA gene. Mol. Cell Biol. 7,3985-3993.

SCHEER,U., TRENDELENBURG,M. F., and FRANKE, W. W. (1976). Regulation of genes of ribosomal RNA during amphibian oogenesis. J. Cell BioL 69,465-489.

Schleicher and Schuell, Inc. (1987). “Transfer and Immobilization of Nucleic Acids to S & S Solid Supports.” Schleicher & Schuell, Keene, NH. STEITZ, J. A., BERG, C., HENDRICK, J. P., LA BRANCHE-CHABOT,H., METSPALU,A., RINKE, J., and YARIO, T. (1988). A 5S rRNA/L5 complex is a precursor to ribosome assembly in mammalian cells. J. Cell BioL 106,545-556. THOMAS,C. (1974). RNA metabolism in previtellogenic oocytes of Xenopus Zaevis. Dev. BioL 39,191-197. TOBIAN, J. A., DRINKARD, L., and ZASLOFF,M. (1985). tRNA nuclear transport: Defining the critical regions of human tRNAp” by point mutagenesis. Cell 43, 415-422. TSURUGI,K., MOTIZUKI, M., MITSUI, K., ENDO, Y., and SHIOKAWA,K. (1988). The metabolism of ribosomal proteins microinjected into oocytes of Xenopus laevis. Exp. Cell Res. 174,177-187. VIEL, A., ARMAND, M.-J., CALLEN,J.-C., DE GRACIA, A. G., DENIS, H., and LEMAIRE, M. (1990). Elongation factor la (EF-la) is concentrated in the balbiani body and accumulates coordinately with the ribosomes during oogenesis of Xenepas laevis. Dew. BioL 141, 270278. WAHL, G. M., MEINKOTH, J. L., and KIMMEL, A. R. (1987). Northern and southern blots. In “Methods in Enzymology” (S. L. Berger and A. R. Kimmel, Eds.), Vol. 152, pp. 572-581. Academic Press, New York. WALLACE,R. A., JARED,D. W., DUMONT,J. N., and SEGA,M. W. (1973). Protein incorporation by isolated amphibian oocytes: Optimum incubation conditions. J. Exp. ZooL 184,321-334. WOLFFE,A. P., and BROW, D. D. (1988). Developmental regulation of two 5S ribosomal RNA genes. Science 241,1626-1632. WORMINGTON,W. M. (1989). Developmental expression and 5S rRNA binding activity of Xenopus ribosomal protein L5. MoL Cell. BioL 9, 5281-5288. ZASLOFF,M. (1983). tRNA transport from the nucleus in a eukaryotic cell: carrier-mediated translocation process. Proc. NatL Acad Sci. USA 80,6436-6440.