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The NEDD8 system is essential for cell cycle progression and morphogenetic pathway in mice.

Tateishi K, Omata M, Tanaka K, Chiba T - J. Cell Biol. (2001)

Bottom Line: Mutant embryos showed selective apoptosis of the inner cell mass but not of trophoblastic cells.However, the mutant trophoblastic cells could not enter the S phase of the endoreduplication cycle.This cell cycle arrest was accompanied with aberrant expression of cyclin E and p57(Kip2).

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Oncology, Tokyo Metropolitan Institute of Medical Science, Bunkyo-Ku, Tokyo 113-8613, Japan.

ABSTRACT
NEDD8/Rub1 is a ubiquitin (Ub)-like molecule that covalently ligates to target proteins through an enzymatic cascade analogous to ubiquitylation. This modifier is known to target all cullin (Cul) family proteins. The latter are essential components of Skp1/Cul-1/F-box protein (SCF)-like Ub ligase complexes, which play critical roles in Ub-mediated proteolysis. To determine the role of the NEDD8 system in mammals, we generated mice deficient in Uba3 gene that encodes a catalytic subunit of NEDD8-activating enzyme. Uba3(-/-) mice died in utero at the periimplantation stage. Mutant embryos showed selective apoptosis of the inner cell mass but not of trophoblastic cells. However, the mutant trophoblastic cells could not enter the S phase of the endoreduplication cycle. This cell cycle arrest was accompanied with aberrant expression of cyclin E and p57(Kip2). These results suggested that the NEDD8 system is essential for both mitotic and the endoreduplicative cell cycle progression. beta-Catenin, a mediator of the Wnt/wingless signaling pathway, which degrades continuously in the cytoplasm through SCF Ub ligase, was also accumulated in the Uba3(-/-) cytoplasm and nucleus. Thus, the NEDD8 system is essential for the regulation of protein degradation pathways involved in cell cycle progression and morphogenesis, possibly through the function of the Cul family proteins.

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Targeted disruption of the Uba3 gene. (A) The targeting vector and the targeted allele. The coding exons numbered in accordance to initiation site as exon 1 are depicted by black boxes. A probe for Southern blot analysis is shown as a striped box. The positions of PCR primers are indicated by arrows. An asterisk denotes the essential cysteine residue on exon 6. Mouse Uba3 cDNA sequence is available from GenBank/EMBL/DDBJ under accession no. AY029181. S, SpeI site; neo, neomycin-resistant gene cassette. (B) Southern blot analysis of genomic DNA extracted from mouse tail. The DNA was digested with SpeI and subjected to hybridization with 3′ external probe shown in A. Wild-type and mutant alleles are detected as 7- and 6-kb bands, respectively. (C) Genotyping of blastocysts by PCR analysis. Wild-type (a and c, 400 bp) and mutant alleles (a–b, 900 bp) are shown. The existence of homozygous embryo at E3.5 was confirmed.
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fig1: Targeted disruption of the Uba3 gene. (A) The targeting vector and the targeted allele. The coding exons numbered in accordance to initiation site as exon 1 are depicted by black boxes. A probe for Southern blot analysis is shown as a striped box. The positions of PCR primers are indicated by arrows. An asterisk denotes the essential cysteine residue on exon 6. Mouse Uba3 cDNA sequence is available from GenBank/EMBL/DDBJ under accession no. AY029181. S, SpeI site; neo, neomycin-resistant gene cassette. (B) Southern blot analysis of genomic DNA extracted from mouse tail. The DNA was digested with SpeI and subjected to hybridization with 3′ external probe shown in A. Wild-type and mutant alleles are detected as 7- and 6-kb bands, respectively. (C) Genotyping of blastocysts by PCR analysis. Wild-type (a and c, 400 bp) and mutant alleles (a–b, 900 bp) are shown. The existence of homozygous embryo at E3.5 was confirmed.

Mentions: Mouse Uba3 cDNA (Genbank AY029181) consists of 2,117 nucleotides with 94% homology to human cDNA. Deduced 441 amino acids were 99% identical to their human counterpart. Mouse Uba3 genomic DNA was obtained by screening C57BL/6J mouse genomic bacterial artificial chromosome (BAC) library using cDNA as a probe. Two independent BAC clones were obtained, and their structures were determined (Fig. 1 A). Mouse Uba3 gene was encoded by 14 exons that spanned ∼14 kb length genomic DNA. The active site cysteine residue essential for NEDD8 activation was encoded by exon 6, and the targeting vector was designed to delete exons 5–7 (Fig. 1 A). After electroporation of targeting vector into TT2 embryonic stem (ES) cells, the homologously recombined ES cells were screened by PCR and genomic Southern blot. Two independent lines of heterozygous ES cells were then transmitted into germ line.


The NEDD8 system is essential for cell cycle progression and morphogenetic pathway in mice.

Tateishi K, Omata M, Tanaka K, Chiba T - J. Cell Biol. (2001)

Targeted disruption of the Uba3 gene. (A) The targeting vector and the targeted allele. The coding exons numbered in accordance to initiation site as exon 1 are depicted by black boxes. A probe for Southern blot analysis is shown as a striped box. The positions of PCR primers are indicated by arrows. An asterisk denotes the essential cysteine residue on exon 6. Mouse Uba3 cDNA sequence is available from GenBank/EMBL/DDBJ under accession no. AY029181. S, SpeI site; neo, neomycin-resistant gene cassette. (B) Southern blot analysis of genomic DNA extracted from mouse tail. The DNA was digested with SpeI and subjected to hybridization with 3′ external probe shown in A. Wild-type and mutant alleles are detected as 7- and 6-kb bands, respectively. (C) Genotyping of blastocysts by PCR analysis. Wild-type (a and c, 400 bp) and mutant alleles (a–b, 900 bp) are shown. The existence of homozygous embryo at E3.5 was confirmed.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198877&req=5

fig1: Targeted disruption of the Uba3 gene. (A) The targeting vector and the targeted allele. The coding exons numbered in accordance to initiation site as exon 1 are depicted by black boxes. A probe for Southern blot analysis is shown as a striped box. The positions of PCR primers are indicated by arrows. An asterisk denotes the essential cysteine residue on exon 6. Mouse Uba3 cDNA sequence is available from GenBank/EMBL/DDBJ under accession no. AY029181. S, SpeI site; neo, neomycin-resistant gene cassette. (B) Southern blot analysis of genomic DNA extracted from mouse tail. The DNA was digested with SpeI and subjected to hybridization with 3′ external probe shown in A. Wild-type and mutant alleles are detected as 7- and 6-kb bands, respectively. (C) Genotyping of blastocysts by PCR analysis. Wild-type (a and c, 400 bp) and mutant alleles (a–b, 900 bp) are shown. The existence of homozygous embryo at E3.5 was confirmed.
Mentions: Mouse Uba3 cDNA (Genbank AY029181) consists of 2,117 nucleotides with 94% homology to human cDNA. Deduced 441 amino acids were 99% identical to their human counterpart. Mouse Uba3 genomic DNA was obtained by screening C57BL/6J mouse genomic bacterial artificial chromosome (BAC) library using cDNA as a probe. Two independent BAC clones were obtained, and their structures were determined (Fig. 1 A). Mouse Uba3 gene was encoded by 14 exons that spanned ∼14 kb length genomic DNA. The active site cysteine residue essential for NEDD8 activation was encoded by exon 6, and the targeting vector was designed to delete exons 5–7 (Fig. 1 A). After electroporation of targeting vector into TT2 embryonic stem (ES) cells, the homologously recombined ES cells were screened by PCR and genomic Southern blot. Two independent lines of heterozygous ES cells were then transmitted into germ line.

Bottom Line: Mutant embryos showed selective apoptosis of the inner cell mass but not of trophoblastic cells.However, the mutant trophoblastic cells could not enter the S phase of the endoreduplication cycle.This cell cycle arrest was accompanied with aberrant expression of cyclin E and p57(Kip2).

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Oncology, Tokyo Metropolitan Institute of Medical Science, Bunkyo-Ku, Tokyo 113-8613, Japan.

ABSTRACT
NEDD8/Rub1 is a ubiquitin (Ub)-like molecule that covalently ligates to target proteins through an enzymatic cascade analogous to ubiquitylation. This modifier is known to target all cullin (Cul) family proteins. The latter are essential components of Skp1/Cul-1/F-box protein (SCF)-like Ub ligase complexes, which play critical roles in Ub-mediated proteolysis. To determine the role of the NEDD8 system in mammals, we generated mice deficient in Uba3 gene that encodes a catalytic subunit of NEDD8-activating enzyme. Uba3(-/-) mice died in utero at the periimplantation stage. Mutant embryos showed selective apoptosis of the inner cell mass but not of trophoblastic cells. However, the mutant trophoblastic cells could not enter the S phase of the endoreduplication cycle. This cell cycle arrest was accompanied with aberrant expression of cyclin E and p57(Kip2). These results suggested that the NEDD8 system is essential for both mitotic and the endoreduplicative cell cycle progression. beta-Catenin, a mediator of the Wnt/wingless signaling pathway, which degrades continuously in the cytoplasm through SCF Ub ligase, was also accumulated in the Uba3(-/-) cytoplasm and nucleus. Thus, the NEDD8 system is essential for the regulation of protein degradation pathways involved in cell cycle progression and morphogenesis, possibly through the function of the Cul family proteins.

Show MeSH
Related in: MedlinePlus