RESUMO
The mitotic spindle drives chromosome movement during mitosis and attaches to chromosomes at dedicated genomic loci named centromeres. Centromeres are epigenetically specified by their histone composition, namely the presence of the histone H3 variant CENP-A, which is regulated during the cell cycle by its dynamic expression and localization. Here, we combined biochemical methods and quantitative imaging approaches to investigate a new function of CUL4-RING E3 ubiquitin ligases (CRL4) in regulating CENP-A dynamics. We found that the core components CUL4 and DDB1 are required for centromeric loading of CENP-A, but do not influence CENP-A maintenance or pre-nucleosomal CENP-A levels. Interestingly, we identified RBBP7 as a substrate-specific CRL4 adaptor required for this process, in addition to its role in binding and stabilizing soluble CENP-A. Our data thus suggest that the CRL4 complex containing RBBP7 (CRL4(RBBP7)) might regulate mitosis by promoting ubiquitin-dependent loading of newly synthesized CENP-A during the G1 phase of the cell cycle.
Assuntos
Autoantígenos/metabolismo , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteína 7 de Ligação ao Retinoblastoma/metabolismo , Proteína Centromérica A , Proteínas de Ligação a DNA/metabolismo , Humanos , Mitose , Ligação Proteica , Estabilidade Proteica , Proteína 4 de Ligação ao Retinoblastoma/metabolismoRESUMO
BACKGROUND: We previously described the inhibition of HIV-1 replication by a 54-mer hairpin-loop structured oligodeoxynucleotide (ODN) A, which binds the polypurine tract (PPT) on HIV-1 RNA. ODN A was shown to lead to reduced viral RNA in virions or early during infection. METHODS AND RESULTS: Here we demonstrated that ODN A was able to cause hydrolysis of viral RNA not only by retroviral RT-associated RNase H but also cellular RNase H1 and RNase H2 in vitro. Furthermore, ODN A reduced gene expression in a dose-dependent manner in a cell-based reporter assay where a PPT sequence was inserted in the 5' untranslated region of the reporter gene. The efficacy of ODN A was higher than that of its siRNA and antisense counterparts. By knocking down cellular RNases H, we showed that RNase H1 contributed to the gene silencing by ODN A but the possibility of a partial contribution of RNase H-independent mechanisms could not be ruled out. GENERAL SIGNIFICANCE: Our findings highlight the potential application of hairpin-loop structured ODNs for reduction of gene expression in mammalian cells and underscore the possibility of using ODN A to trigger the hydrolysis of HIV RNA in infected cells by cellular RNases H.
Assuntos
Regulação Viral da Expressão Gênica , Oligodesoxirribonucleotídeos/genética , Oligonucleotídeos Antissenso/genética , RNA Interferente Pequeno/genética , Sequência de Bases , Linhagem Celular , Linhagem Celular Transformada , Citometria de Fluxo , Inativação Gênica , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , HIV-1/genética , HIV-1/fisiologia , Humanos , Hidrólise , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/metabolismo , Poli U/genética , Poli U/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonuclease H/genética , Ribonuclease H/metabolismoRESUMO
The spindle assembly checkpoint links the onset of anaphase to completion of chromosome-microtubule attachment and is mediated by the binding of Mad and Bub proteins to kinetochores of unattached or maloriented chromosomes. Mad2 and BubR1 traffic between kinetochores and the cytosol, thereby transmitting a "wait anaphase" signal to the anaphase-promoting complex. It is generally assumed that this signal dissipates automatically upon kinetochore-microtubule binding, but it has been shown that under conditions of nocodazole-induced arrest p31(comet), a Mad2-binding protein, is required for mitotic progression. In this article we investigate the localization and function of p31(comet) during normal, unperturbed mitosis in human and marsupial cells. We find that, like Mad2, p31(comet) traffics on and off kinetochores and is also present in the cytosol. Cells depleted of p31(comet) arrest in metaphase with mature bipolar kinetochore-microtubule attachments, a satisfied checkpoint, and high cyclin B levels. Thus p31(comet) is required for timely mitotic exit. We propose that p31(comet) is an essential component of the machinery that silences the checkpoint during each cell cycle.