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First published online 20 March 2007
doi: 10.1242/jcs.000166

Journal of Cell Science 120, 1412-1422 (2007)
Published by The Company of Biologists 2007
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Regulatory mechanisms governing the oocyte-specific synthesis of the karyoskeletal protein NO145

Susanne Voltmer-Irsch1, Sandra Kneissel1,2, Pierre G. Adenot3 and Marion S. Schmidt-Zachmann1,*

1 Division of Cell Biology, German Cancer Research Center, 69120 Heidelberg, Germany
2 Abbott GmbH & Co. KG, Diagnostics Division, 65205 Wiesbaden, Germany
3 Unité de Biologie du Développement et Reproduction, Institut National de la Recherche Agronomique, 78350 Jouy-en-Josas, France


Figure 1
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  		Fig. 1. Karyoskeletal protein NO145 in Xenopus oocytes and eggs. (A) Laser scanning confocal microscopy showing double label immunolocalization on spread preparations of GV contents of X. laevis stage VI oocytes. The specific localization of NO145 protein in the nucleolar cortex (green) and of nucleolar protein NO38/B23 (red) in the granular component are shown in the merged picture. Bar, 15 µm. (B,C) Synthesis of NO145 protein and its mRNA in X. laevis oocytes and eggs. (B) Western blot of total proteins of one oocyte (VI) and egg (E), probed with antibody NO145-H or antibody X8 directed against protein NO38/B23. (C) Northern blot of total RNA from stage VI oocytes (VI) and eggs (E) was hybridized with a random prime-labeled NO145-specific probe (left panel); the ethidium bromide-stained gel serves as a loading control (right panel). RNA size markers of 4, 3, 2, 1.5 and 1 kb are indicated.

 

Figure 2
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  		Fig. 2. Cell-type-specific expression of the gene encoding NO145 and alterations of the mRNA poly(A) tail during maturation. (A) RT-PCR analyses of NO145 and NO38 expression from various tissues of X. laevis (lanes 1-4: ovary, testis, muscle, and heart, respectively). (B) Scheme of the RACE-PAT assay adapted from Sallés et al. (Sallés et al., 1999Go). Total cellular RNA was reverse-transcribed using an oligo d(T) primer fused to a G/C-rich anchor sequence. Subsequent PCR amplification with a NO145-specific primer and the oligo d(T) anchor yielded a mixture of PCR-products representing the length of the poly(A) tail. (C) Top: ethidium bromide-stained PCR products obtained by the RACE-PAT assay on total RNA of X. laevis staged oocytes (I-VI) and eggs (E). Bottom: immunoblot of SDS-PAGE-separated total proteins from one oocyte of stages I–VI and from an unfertilized egg, per lane, and probed with NO145 and Xp-54 antibodies. (D) Alignment of oocyte and egg NO145 gene transcripts amplified by the RACE-PAT assay. Vertical lines represent identical residues between the transcripts. The stop codon is underlined and the nuclear polyadenylation signal is boxed. Poly(A) stretches are shown in bold letters.

 

Figure 3
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  		Fig. 3. Distribution of NO145 mRNAs in mRNPs and ribosomes of X. laevis oocytes and eggs. Homogenates from X. laevis stage VI oocytes (A) and eggs (B) were separated on 20-60% Nycodenz gradients to separate 42S particles (fractions 1-7), mRNPs (fractions 9-15) and ribosomes (fractions 17-23). RNA was isolated from the obtained fractions and subjected to RACE-PAT analysis using the appropriate primer sets to analyze the distribution of NO145 and histone H4 mRNAs.

 

Figure 4
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  		Fig. 4. Degradation of NO145 protein during oocyte maturation by the proteasome system. (A) Gradual disappearance of endogenous NO145 protein during oocyte maturation. Defolliculated stage VI oocytes were treated with progesterone (P; after 2 hours). Mature oocytes were collected at the indicated times (M0-120; in minutes). Lanes VI and E indicate immature stage VI oocytes and eggs, respectively. An amount equivalent to one oocyte per lane was loaded and immunoblotted with antibodies against NO145 and NO38. Analogous oocyte stages were analyzed by the RACE-PAT method. (B) Phosphorylation of NO145 in maturing oocytes. Lysates from stage VI oocytes, matured oocytes (M0) and eggs (E), were treated with calf intestine alkaline phosphatase (CIP) as indicated and analyzed by immunoblotting with antibodies against NO145 and NO38. The positions of fast- and slow-migrating protein variants are indicated by close and open arrowheads, respectively. (C) Schematic representation of the experimental protocol to analyze the involvement of the proteasome pathway in the degradation of protein NO145. (D) Methylated ubiquitin affects the degradation process of NO145 during maturation. Methylated ubiquitin or injection buffer were injected into oocytes at GVBD50. Samples were taken 0, 30, 60, 120 and 180 minutes after appearance of the white spot (M0-M180) and protein extracts were analyzed by immunoblotting using NO145 antibodies. (E) Degradation of the endogenous NO145 protein is blocked by proteasome inhibitors. Immature stage VI oocytes (VI) were induced to mature by addition of progesterone. At GVBD50 oocytes with (+) or without (–) a white spot were taken as controls. At this time, non-white spot oocytes were injected with either MG132, lactacystin or injection buffer alone (lanes indicated by +) or left uninjected (lanes indicated by –). All oocytes were incubated until GVBD100 or overnight (E) and crude extracts from five oocytes each were prepared, and the equivalent of one oocyte or egg per lane was separated by SDS-PAGE. The resulting immunoblot was probed with antibodies against NO145 and finally re-probed with Xp54 antibodies as loading control.

 

Figure 5
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  		Fig. 5. NO145 protein and its mRNA during X. laevis embryogenesis. (A) Immunoblot of SDS-PAGE-separated total proteins isolated from stage VI oocytes and from embryos of stages 1-25, probed with NO145-specific antibodies (top panel) and antibodies against the nucleolar protein NO66 (bottom). (B) Total RNA from stage VI oocytes and embryos of stages 1-10 were separated by agarose gel electrophoresis. The corresponding northern blot was hybridized with a random prime-labeled NO145-specific probe. (C) RACE-PAT assays performed on total RNA from X. laevis oocytes (VI), embryos (stages 1-25), and eggs (E) with NO145-specific primers. The resulting PCR products were separated on a 2% agarose gel and stained with ethidium bromide. Re-amplified PCR products from embryonic stages 12, 15 and 25, respectively, are shown in the box below. Bars over the lane number indicate midblastula transition (MBT; fraction 8 in A,B,C).

 

Figure 6
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  		Fig. 6. NO145 mRNA 3' UTRs affect the translation of reporter mRNAs as well as the stability of the encoded proteins in Xenopus oocytes and upon maturation. (A) A firefly luciferase reporter construct (Luc) was either fused to the short (Luc-s) or the long (Luc-l) version of the NO145-3' UTR. The mRNAs were synthesized in vitro with a poly(A)50 tail, capped and subsequently injected at a sixfold excess over Renilla luciferase mRNA in stage VI oocytes. After 4 hours of incubation, the oocytes were homogenized in pools of four oocytes each and the firefly and Renilla luciferase activities were measured. (B) Histogram showing the normalized luciferase activity of two independent experiments each. The translational efficiencies of the three constructs are compared. (C) Capped mRNAs were transcribed in vitro from constructs consisting of a His-tagged version of the NO38 coding sequence followed by either the authentic 3' UTR region of NO38 (His-NO38wt), the short NO145-3' UTR (His-NO38-s) or the long NO145 3' UTR (His-NO38-l). (D) Stage VI oocytes were injected with capped mRNAs synthesized from the different His-NO38 constructs. After incubation overnight, oocyte extracts were analyzed by immunoblotting using the His antibody to allow the specific detection of the reporter proteins encoded by the different mRNA variants. (E) Some of the injected oocytes were induced to maturation by progesterone treatment. Upon GVBD, egg extracts were separated by SDS-PAGE and the corresponding immunoblot was incubated with the His antibody.

 

Figure 7
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  		Fig. 7. The human orthologue of the NO145 protein. (A) Sequence comparison between human NO145 (hsNO145) and its Xenopus homolog (X.l. NO145). Identical amino acids (aa) are marked by vertical bars and are indicated in bold letters. Peptide sequences used for generating antibodies are indicated by boxes, the putative nuclear localization signal is shaded grey and putative sequence motifs involved in protein degradation are indicated in red. (B) Autoradiogram of SDS-PAGE-separated rabbit reticulocyte lysate after in vitro transcription and translation in the presence of the pBT-hsNO145. Bars indicate the position of reference proteins: 205, 116, 97.4 and 66 kDa (from top to bottom). (C,C') Tissue protein lysates of human origin (C; ovary, testis, intestine; shown in lanes 1-3, respectively, after Coomassie Blue staining) were analyzed by western blotting (C') using NO145-specific antibodies. A parallel blot was probed for vimentin using the mAb Vim3B4 (Herrmann et al., 1989Go). Reference proteins are the same as in B. (D-D'') Immunofluorescent localization of NO145 protein on paraffin sections through human ovary using hsNO145-specific antibodies. (D) Phase-contrast micrograph; (D') DAPI staining; (D'') corresponding immunofluorescence micrograph using hsNO145-5 antibody. Bar, 50 µm.

 

Figure 8
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  		Fig. 8. NO145 protein in bovine oocytes. (A) Immunoblot on proteins extracted from isolated bovine oocytes, probed with hsNO145-1A antibody. (B-C'') Immunolocalization in the nucleus of bovine oocytes using hsNO145-specific antibodies (green) and DNA counterstaining (red). (B) Overview of an oocyte surrounded by the cumulus cell layers. (B') Magnification of this nucleus shows that NO145 protein is enriched in dot-like structures (arrowheads) associated with dense chromatin but not with the partly `vacuolated' nucleolus (arrow). (C-C'') Magnification of another GV showing NO145 protein in granular structures (arrowheads) that are not closely associated to nucleoli (arrows). Bars, 20 µm (B) and 10 µm (B' and C).

 

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© The Company of Biologists Ltd 2007