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Characterization and targeted disruption of murine Nup50, a p27(Kip1)-interacting component of the nuclear pore complex.

ABSTRACT: p27(Kip1) is a member of the Cip-Kip family of cyclin-dependent kinase (Cdk) inhibitors that binds to cyclin-Cdk complexes and inhibits their catalytic activity in response to antiproliferative stimuli. p27(Kip1) is regulated by several posttranscriptional mechanisms, including subcellular localization. We have identified a component of the nuclear pore complex (NPC), termed Nup50, through its two-hybrid interactions with p27(Kip1). Nup50 is a nucleoplasmically oriented component of the nuclear pore complex with a role in protein export (T. Guan, R. H. Kehlenbach, E. C. Schirmer, A. Kehlenbach, F. Fan, B. E. Clurman, N. Arnheim, and L. Gerace, Mol. Cell. Biol. 20:5619-5630, 2000). We found that murine Nup50 is a widely expressed nucleoporin and that Nup50 expression is highest in the developing neural tube and adult testes. We have also examined interactions between Nup50 and the NPC and found specific two-hybrid interactions between Nup50 and several well-defined components of the NPC, as well as coimmunoprecipitation of Nup50 with the nucleoporin Nup153 from transfected mammalian cells. In order to study Nup50 function in vivo, we cloned the mouse Nup50 genomic locus and created a targeted Nup50 deletion in the mouse germ line. Nup50 disruption resulted in a complex phenotype characterized by late embryonic lethality, neural tube defects, and intrauterine growth retardation. Although Nup50-null mouse embryo fibroblasts exhibited no defects in either cell cycle control or p27(Kip1) regulation, Nup50 deletion was associated with abnormalities in p27(Kip1) expression and cell proliferation in the developing neuroepithelium. We conclude that Nup50 is a nucleoporin with essential functions during mouse development.

SUBMITTER: Smitherman M

PROVIDER: S-EPMC86029 | biostudies-literature | 2000 Aug

REPOSITORIES: biostudies-literature

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Characterization and targeted disruption of murine Nup50, a p27(Kip1)-interacting component of the nuclear pore complex.

Smitherman M M Lee K K Swanger J J Kapur R R Clurman B E BE

Molecular and cellular biology 20000801 15

p27(Kip1) is a member of the Cip-Kip family of cyclin-dependent kinase (Cdk) inhibitors that binds to cyclin-Cdk complexes and inhibits their catalytic activity in response to antiproliferative stimuli. p27(Kip1) is regulated by several posttranscriptional mechanisms, including subcellular localization. We have identified a component of the nuclear pore complex (NPC), termed Nup50, through its two-hybrid interactions with p27(Kip1). Nup50 is a nucleoplasmically oriented component of the nuclear ...[more]

PMID: 10891500

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Project description:Mice lacking the p27(Kip1) Cdk inhibitor (Cdkn1b) exhibit increased susceptibility to lymphomas from the Maloney murine leukemia virus (M-MuLV), and exhibit a high frequency of viral integrations at Xpcl1 (Kis2), a locus on the X-chromosome. Xpcl1 encodes miR-106a~363, a cluster of microRNAs that are expressed in response to adjacent retroviral integrations. We report the first large-scale profile of microRNA expression in MuLV-induced lymphomas, in combination with microarray gene expression analysis. The source material was T-cell lymphomas induced by M-MuLV in p27(Kip1) knockout mice and normal thymus. Surprisingly, the overall levels of miRNA expression were equivalent in lymphomas and normal thymus. Nonetheless, the expression of specific microRNAs was altered in tumors. The miR-106a~363 miRNA were over-expressed in lymphomas, particularly those with viral integrations at the Xpcl1 locus. In contrast, p27(Kip1) deletion itself was associated with a different pattern of microRNA expression. Gene expression was dramatically altered in lymphomas, yet paralleled data from T-cell lymphomas induced by other mechanisms. Genes with altered expression in association with the p27(Kip1) null genotype were of similar functional classes to those associated with Xpcl1 integration, but with the opposite pattern of expression. Thus, the effect of p27(Kip1) deletion may be to oppose an anti-oncogenic effect of Xpcl1 rather than enhancing its oncogenic functions. A subset of miR-106a~363 target genes was consistently reduced in lymphomas with Xpcl1 integrations, particularly genes with cell cycle and immune functions. We identify four predicted target genes of miR-106a~363 miRNA, including N-Myc (Mycn), and the TGF-beta receptor (Tgfbr2) using 3'UTR reporter assays. Still, bioinformatic miRNA target predictions were poor predictors of altered gene expression in lymphomas with Xpcl1 integration. Confirmation of miR-106a~363 gene targeting relevant to the tumor phenotype requires in vivo validation, because only a subset of predicted targets are consistently reduced in tumors that overexpress miR-106a~363.

Dataset Information

Characterization and targeted disruption of murine Nup50, a p27(Kip1)-interacting component of the nuclear pore complex.

Publications

Characterization and targeted disruption of murine Nup50, a p27(Kip1)-interacting component of the nuclear pore complex.

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