Caizergues-Ferrer M et al. (1989), Nucleolin from Xenopus laevis: cDNA cloning and...

XB-ART-26892

Genes Dev 1989 Mar 01;33:324-33. doi: 10.1101/gad.3.3.324.

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Nucleolin from Xenopus laevis: cDNA cloning and expression during development.

Caizergues-Ferrer M , Mariottini P , Curie C , Lapeyre B , Gas N , Amalric F , Amaldi F .

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Nucleolin is a key nucleolar protein in higher eukaryotic cells and is involved directly in ribosome biogenesis. Using an antiserum raised against hamster nucleolin, the homologous protein was detected in nucleoli of Xenopus laevis hepatocytes as well as in the amplified nucleoli of oocytes. A cDNA encoding Xenopus nucleolin has been isolated and sequenced. The deduced protein sequence reveals similar domains in Xenopus and in mammals, but they have undergone separate evolutions. In particular, each of the four RNA-binding domains has evolved differently--the carboxy-proximal domain is twice as conserved (87%) as the amino-proximal domain (42%). These data shed some light on the possible roles of each domain. The expression of nucleolin has been followed throughout oogenesis and embryogenesis. The appearance of nucleolin during early development precedes the transcription of rDNA and the synthesis of ribosomal proteins. The maximal accumulation of nucleolin at gastrulation coincides with nucleolar reformation. Furthermore, when ribosomal synthesis is activated during oogenesis and embryogenesis, peptides immunorelated to nucleolin appear and accumulate. The results suggest that nucleolin plays a role not only in ribosome assembly but also in nucleologenesis.

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Species referenced: Xenopus laevis
Genes referenced: eif3a rps8 tbx2

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	Figure 1. Nucleolin localization. Indirect immunofluorescence microscopy on isolated liver nuclei from Xenopus using the purified antiserum raised against CHO nucleolin (A) and corresponding phase contrast (B). Nucleoli are stained positively. Erythrocytes are not stained (e). Frozen section of Xenopus ovary (C) showing part of an oocyte nucleus (N) with numerous nucleoli after incubation with anti-nucleolin antibody. The amplified nucleoli (nu) at this stage (st. IV) are strongly fluorescent. (D) Phase contrast: the nucleus is circled. Bar, 50 ~m.
	Figure 2. Detection of nucleolin in X. laevis. Protein analysis. (Lanes 1, 2, 4, and 5) Protein extracts separated on a 10% SDSPAGE and electroblotted were probed using an antiserum raised against CHO nucleolin. Detection of the complexes was achieved either by 12SI-labeled protein A {lanes I and 2), or by a second antiserum coupled to alkaline phosphatase (lanes 4 and 5). (Lane 3) Protein phosphorylation was obtained with an endogenous kinase contained in a low-ionic-strength extract of Xenopus hepatocyte nuclei and addition of [~/-32P]ATP. Proteins were separated as before, then the gel was dried and autoradiographed for 4 hr at - 70~ with an intensifying screen. (Lane 1) As a control, 0.2 ~g of nucleolin purified from CHO cells was run in parallel. (Lane 2) Low-ionic-strength extract from nuclei of Xenopus hepatocytes. (Lane 4) Protein extract corresponding to 10 oocytes from stage I to III, roughly separated from the yolk [see Materials and methods). [Lane 5) Same as in lane 4, but with oocytes from stage IV to VI. Numbers on the left refer to the molecular weight of the peptides, in kD.
	Figure 3. Northern blot analysis. RNA was isolated, separated under denaturing conditions in a formaldehyde-1% agarose gel, blotted onto nitrocellulose filter, and then hybridized with different probes. CHO nucleolin cDNA was used to probe total RNA purified either from CHO cells as a control (lane 1) or from Xenopus oocytes (lane 2). Xenopus nucleolin cDNA was used to probe Xenopus oocyte RNA [lane 2').
	Figure 4. Xenopus nucleolin: pXomN1 DNA sequence and deduced amino acid sequence. Numbers above the DNA sequence refer to the nucleotides while numbers underlined on the right refer to the amino acid residues. Acidic tracts are in boldface type. The three serine residues that are candidates for the phosphorylation sites are boxed. The potential nuclear translocation signal, similar to the prototype signal of SV40 large T antigen (Kalderon et al. 1984) is underlined by stars. This signal is located just downstream from the last acidic stretch as in the Xenopus protein N038 tSchmidt-Zachmann et al. 1987). The four RNA-binding consensus sequences, centered on the residues at postions 149, 238, 327, and 417, are in boldface type and underlined. The glycine-rich domain at the carboxyl terminus is in boldface type. The polyadenylation signal is boxed.
	Figure 5. Alignment of the four RNA-binding domains of Xenopus nucleolin with those of CHO nucleolin (R I to R IV). Numbers on the left refer to the position of the first residue of a line. The consensus sequence indentified previously is in boldface type. Sequences were aligned allowing some gaps (.) to get the best matches (*).
	Figure 6. Northern blot showing nucleolin mRNA accumulation during oogenesis and embryogenesis. Total RNA corresponding to 0.5 oocyte or embryo was separated by electrophoresis in a denaturing formaldehyde-1% agarose gel, blotted onto nitrocellulose filter, and hybridized under stringent conditions with nick-translated Xenopus nucleolin cDNA as a probe. (a) Stages I to VI of oogenesis according to Dumont 1972. Autoradiography was carried out for 20 hr at - 70~ with an intensifying screen (XM film from 3M). (b) Stages 3 to 40 of embryogenesis according to Nieuwkoop and Faber (1956). Autoradiography was carried out for 10 hr.
	Figure 7. Relative expression of rRNA, nucleolin, and r-protein mRNAs during embryogenesis. [A) Total RNA corresponding to two embryos of each stage was analyzed by dot-blot and hybridization with different DNA probes: nucleolin (Nucl.), two r-proteins (rp-S8 and rp-L1 }, and rDNA (rRNAJ. (BJ Quantitative data were obtained for nucleolin and r-protein $8 by densitometric scanning of the autoradiograms, the values from four independent experiments have been plotted. Average values for each stage are expressed in percent of the value of the stage with the highest score, rpS8 mRNA {O-O); nucleolin mRNA [@--O).
	Figure 8. Polysome/mRNP distribution of nucleolin and r-protein mRNA. Cytoplasmic fractions of 15 embryos of stage 18 and 36 were run through sucrose gradients. RNA was extacted from gradient fractions and analyzed by dot-blot hybridization with probes for nucleolin, or for r-protein ($8} mRNA. The percent of mRNA in polysomal or mRNP fraction was determined by densitometric scanning of the autoradiogram.
	Figure 9. Fate of nucleolin during embryogenesis. Total protein extracts of 10 embryos were prepared in the presence of protease inhibitors as described in Materials and methods. (A) Proteins corresponding to one embryo per lane were electrophoresed on a 10% SDS-PAGE. The amount of protein per lane increases from 10 v-g for stage 8 to 100 ~g at stage 46. The gel was blotted onto nitrocellulose, probed with purified IgG directed against nucleolin of CHO, then stained with 12SI-labeled protein A. The filter was exposed for 20 hr at - 70~ with an intensifying screen. Lanes: (C) Control lane, 0.2 wg of purified CHO nucleolin; (8-46) embryo stage according to Nieuwkoop and Faber (1956). (B) Endogenous phosphorylation of nucleolin in embryonic extracts. Same protein extracts as in A were incubated in the presence of [~-32p]ATP. Proteins were separated on a 10% SDS-PAGE. The gel was dried and autoradiographed for 4 hr at room temperature. (Lanes 8-32) Embryo stages. Numbers in the middle refer to the peptides in kD.