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1.
Cell ; 164(1-2): 69-80, 2016 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-26724866

RESUMO

Long noncoding RNAs (lncRNAs) have emerged as regulators of diverse biological processes. Here, we describe the initial functional analysis of a poorly characterized human lncRNA (LINC00657) that is induced after DNA damage, which we termed "noncoding RNA activated by DNA damage", or NORAD. NORAD is highly conserved and abundant, with expression levels of approximately 500-1,000 copies per cell. Remarkably, inactivation of NORAD triggers dramatic aneuploidy in previously karyotypically stable cell lines. NORAD maintains genomic stability by sequestering PUMILIO proteins, which repress the stability and translation of mRNAs to which they bind. In the absence of NORAD, PUMILIO proteins drive chromosomal instability by hyperactively repressing mitotic, DNA repair, and DNA replication factors. These findings introduce a mechanism that regulates the activity of a deeply conserved and highly dosage-sensitive family of RNA binding proteins and reveal unanticipated roles for a lncRNA and PUMILIO proteins in the maintenance of genomic stability.


Assuntos
Instabilidade Genômica , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Sequência de Bases , Instabilidade Cromossômica , Células HCT116 , Humanos , Camundongos , Ploidias , RNA Longo não Codificante/química , RNA Longo não Codificante/genética
2.
Nat Rev Mol Cell Biol ; 16(2): 82-94, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25604195

RESUMO

The appropriate timing of events that lead to chromosome segregation during mitosis and cytokinesis is essential to prevent aneuploidy, and defects in these processes can contribute to tumorigenesis. Key mitotic regulators are controlled through ubiquitylation and proteasome-mediated degradation. The APC/C (anaphase-promoting complex; also known as the cyclosome) is an E3 ubiquitin ligase that has a crucial function in the regulation of the mitotic cell cycle, particularly at the onset of anaphase and during mitotic exit. Co-activator proteins, inhibitor proteins, protein kinases and phosphatases interact with the APC/C to temporally and spatially control its activity and thus ensure accurate timing of mitotic events.


Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Mitose/fisiologia , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos/fisiologia , Citocinese/fisiologia , Humanos
3.
Elife ; 122023 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-37096669

RESUMO

Enhancers orchestrate gene expression programs that drive multicellular development and lineage commitment. Thus, genetic variants at enhancers are thought to contribute to developmental diseases by altering cell fate commitment. However, while many variant-containing enhancers have been identified, studies to endogenously test the impact of these enhancers on lineage commitment have been lacking. We perform a single-cell CRISPRi screen to assess the endogenous roles of 25 enhancers and putative cardiac target genes implicated in genetic studies of congenital heart defects (CHDs). We identify 16 enhancers whose repression leads to deficient differentiation of human cardiomyocytes (CMs). A focused CRISPRi validation screen shows that repression of TBX5 enhancers delays the transcriptional switch from mid- to late-stage CM states. Endogenous genetic deletions of two TBX5 enhancers phenocopy epigenetic perturbations. Together, these results identify critical enhancers of cardiac development and suggest that misregulation of these enhancers could contribute to cardiac defects in human patients.


Assuntos
Cardiopatias Congênitas , Miócitos Cardíacos , Humanos , Miócitos Cardíacos/metabolismo , Sequências Reguladoras de Ácido Nucleico , Diferenciação Celular/genética , Cardiopatias Congênitas/genética
4.
STAR Protoc ; 3(4): 101682, 2022 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-36115024

RESUMO

Human embryonic stem cells (hESCs) continuously self-renew in culture and can be induced to differentiate into multiple cell types, including neural progenitor cells (NPCs). Here, we present a protocol to perform a CRISPR-Cas9 screen in hESCs to identify regulators that promote SOX1 expression during NPC formation. This screening protocol can be adapted with other endpoint reporters for the identification of genes involved in the commitment of hESCs to other cell lineages. For complete details on the use and execution of this protocol, please refer to Sivakumar et al. (2022).


Assuntos
Células-Tronco Embrionárias Humanas , Células-Tronco Neurais , Humanos , Sistemas CRISPR-Cas , Diferenciação Celular
5.
Methods Mol Biol ; 2415: 61-86, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34972946

RESUMO

Live-cell fluorescence microscopy is an effective tool for characterizing aberrant mitotic phenotypes resulting from exposure to chemical inhibitors and after RNA interference-mediated or CRISPR knockout-mediated depletion of protein targets. Live imaging of cultured cells during mitotic progression presents challenges in maintaining optimal health of cells while achieving the temporal and spatial resolution to accomplish the goals of the study. Herein are strategies to monitor and analyze mammalian cell mitosis utilizing either a wide field or a light sheet, inverted, fluorescence microscope.


Assuntos
Segregação de Cromossomos , Mitose , Células Cultivadas , Microscopia de Fluorescência/métodos , Imagem Óptica
6.
Cell Rep ; 38(7): 110395, 2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-35172133

RESUMO

Aneuploidy, defective differentiation, and inactivation of the tumor suppressor TP53 all occur frequently during tumorigenesis. Here, we probe the potential links among these cancer traits by inactivating TP53 in human embryonic stem cells (hESCs). TP53-/- hESCs exhibit increased proliferation rates, mitotic errors, and low-grade structural aneuploidy; produce poorly differentiated immature teratomas in mice; and fail to differentiate into neural progenitor cells (NPCs) in vitro. Genome-wide CRISPR screen reveals requirements of ciliogenesis and sonic hedgehog (Shh) pathways for hESC differentiation into NPCs. TP53 deletion causes abnormal ciliogenesis in neural rosettes. In addition to restraining cell proliferation through CDKN1A, TP53 activates the transcription of BBS9, which encodes a ciliogenesis regulator required for proper Shh signaling and NPC formation. This developmentally regulated transcriptional program of TP53 promotes ciliogenesis, restrains Shh signaling, and commits hESCs to neural lineages.


Assuntos
Linhagem da Célula , Cílios/metabolismo , Proteínas Hedgehog/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Organogênese , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Motivos de Aminoácidos , Animais , Sistemas CRISPR-Cas/genética , Diferenciação Celular , Linhagem Celular , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Genoma Humano , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Teratoma/patologia , Proteína Supressora de Tumor p53/química
7.
Elife ; 82019 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-30735131

RESUMO

Although numerous long noncoding RNAs (lncRNAs) have been identified, our understanding of their roles in mammalian physiology remains limited. Here, we investigated the physiologic function of the conserved lncRNA Norad in vivo. Deletion of Norad in mice results in genomic instability and mitochondrial dysfunction, leading to a dramatic multi-system degenerative phenotype resembling premature aging. Loss of tissue homeostasis in Norad-deficient animals is attributable to augmented activity of PUMILIO proteins, which act as post-transcriptional repressors of target mRNAs to which they bind. Norad is the preferred RNA target of PUMILIO2 (PUM2) in mouse tissues and, upon loss of Norad, PUM2 hyperactively represses key genes required for mitosis and mitochondrial function. Accordingly, enforced Pum2 expression fully phenocopies Norad deletion, resulting in rapid-onset aging-associated phenotypes. These findings provide new insights and open new lines of investigation into the roles of noncoding RNAs and RNA binding proteins in normal physiology and aging.


Assuntos
Senilidade Prematura/genética , Envelhecimento/genética , RNA Longo não Codificante/genética , Proteínas de Ligação a RNA/genética , Envelhecimento/fisiologia , Senilidade Prematura/patologia , Animais , Regulação da Expressão Gênica/genética , Homeostase/genética , Humanos , Camundongos , Mitocôndrias/genética , Mitose/genética , Fenótipo , Fatores de Transcrição/genética
8.
Biol Open ; 6(11): 1672-1679, 2017 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-28982702

RESUMO

Kinetochores move chromosomes on dynamic spindle microtubules and regulate signaling of the spindle checkpoint. The spindle- and kinetochore-associated (Ska) complex, a hexamer composed of two copies of Ska1, Ska2 and Ska3, has been implicated in both roles. Phosphorylation of kinetochore components by the well-studied mitotic kinases Cdk1, Aurora B, Plk1, Mps1, and Bub1 regulate chromosome movement and checkpoint signaling. Roles for the opposing phosphatases are more poorly defined. Recently, we showed that the C terminus of Ska1 recruits protein phosphatase 1 (PP1) to kinetochores. Here we show that PP1 and protein phosphatase 2A (PP2A) both promote accumulation of Ska at kinetochores. Depletion of PP1 or PP2A by siRNA reduces Ska binding at kinetochores, impairs alignment of chromosomes to the spindle midplane, and causes metaphase delay or arrest, phenotypes that are also seen after depletion of Ska. Artificial tethering of PP1 to the outer kinetochore protein Nuf2 promotes Ska recruitment to kinetochores, and it reduces but does not fully rescue chromosome alignment and metaphase arrest defects seen after Ska depletion. We propose that Ska has multiple functions in promoting mitotic progression and that kinetochore-associated phosphatases function in a positive feedback cycle to reinforce Ska complex accumulation at kinetochores.

9.
Curr Biol ; 27(10): 1477-1484.e4, 2017 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-28479321

RESUMO

The spindle and kinetochore-associated (Ska) protein complex is required for accurate chromosome segregation during mitosis [1-6] and consists of two copies each of Ska1, Ska2, and Ska3 proteins [4, 7]. The Ska complex contains multiple microtubule-binding elements and promotes kinetochore-microtubule attachment [8-11]. The Ska1 C-terminal domain (CTD) recruits protein phosphatase 1 (PP1) to kinetochores to promote timely anaphase onset [12]. The Ska complex regulates, and is regulated by, Aurora B [13]. Aurora B phosphorylates both Ska1 and Ska3 to inhibit the kinetochore localization of the Ska complex [14]. Despite its multitude of functions at kinetochores, how the Ska complex itself is recruited to kinetochores is unclear. It is unknown whether any mitotic kinases positively regulate the localization of the Ska complex to kinetochores. Here, we show that Cdk1 phosphorylates Ska3 to promote its direct binding to the Ndc80 complex (Ndc80C), a core outer kinetochore component. We also show that this phosphorylation occurs specifically during mitosis and is required for the kinetochore localization of the Ska complex. Ska3 mutants deficient in Cdk1 phosphorylation are defective in kinetochore localization but retain microtubule localization. These mutants support chromosome alignment but delay anaphase onset. We propose that Ska3 phosphorylated by Cdk1 in mitosis binds to Ndc80C and recruits the Ska complex to kinetochores where Ska1 can bind both PP1 and microtubules to promote anaphase onset.


Assuntos
Proteína Quinase CDC2/metabolismo , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose , Proteínas Nucleares/metabolismo , Aurora Quinase B/metabolismo , Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos , Proteínas do Citoesqueleto , Células HeLa , Humanos , Microtúbulos/metabolismo , Fosforilação
10.
Elife ; 52016 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-26981768

RESUMO

The spindle- and kinetochore-associated (Ska) complex is essential for normal anaphase onset in mitosis. The C-terminal domain (CTD) of Ska1 binds microtubules and was proposed to facilitate kinetochore movement on depolymerizing spindle microtubules. Here, we show that Ska complex recruits protein phosphatase 1 (PP1) to kinetochores. This recruitment requires the Ska1 CTD, which binds PP1 in vitro and in human HeLa cells. Ska1 lacking its CTD fused to a PP1-binding peptide or fused directly to PP1 rescues mitotic defects caused by Ska1 depletion. Ska1 fusion to catalytically dead PP1 mutant does not rescue and shows dominant negative effects. Thus, the Ska complex, specifically the Ska1 CTD, recruits PP1 to kinetochores to oppose spindle checkpoint signaling kinases and promote anaphase onset. Microtubule binding by Ska, rather than acting in force production for chromosome movement, may instead serve to promote PP1 recruitment to kinetochores fully attached to spindle microtubules at metaphase.


Assuntos
Anáfase , Proteínas Cromossômicas não Histona/metabolismo , Cinetocoros/metabolismo , Metáfase , Proteína Fosfatase 1/metabolismo , Células HeLa , Humanos , Domínios Proteicos
11.
Methods Mol Biol ; 1170: 549-62, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24906336

RESUMO

Live-cell fluorescence microscopy is a powerful tool for characterizing aberrant mitotic phenotypes resulting from exposure to chemical inhibitors or after depletion of protein targets by RNA interference or other methods. Live imaging of cultured cells during mitotic progression presents challenges in maintaining optimal health of cells while achieving the temporal and spatial resolution to accomplish the goals of the study. We describe herein strategies to monitor and analyze mammalian cell mitosis with standard, inverted, fluorescence microscopy systems that are widely available.


Assuntos
Microscopia de Fluorescência/métodos , Mitose , Técnicas de Cultura de Células/métodos , Sobrevivência Celular , Proteínas de Fluorescência Verde/análise , Células HeLa , Histonas/análise , Humanos , Imagem Óptica/métodos , Proteínas Recombinantes de Fusão/análise
12.
Mol Biol Cell ; 25(5): 594-605, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24403607

RESUMO

The spindle and kinetochore-associated (Ska) protein complex is a heterotrimeric complex required for timely anaphase onset. The major phenotypes seen after small interfering RNA-mediated depletion of Ska are transient alignment defects followed by metaphase arrest that ultimately results in cohesion fatigue. We find that cells depleted of Ska3 arrest at metaphase with only partial degradation of cyclin B1 and securin. In cells arrested with microtubule drugs, Ska3-depleted cells exhibit slower mitotic exit when the spindle checkpoint is silenced by inhibition of the checkpoint kinase, Mps1, or when cells are forced to exit mitosis downstream of checkpoint silencing by inactivation of Cdk1. These results suggest that in addition to a role in fostering kinetochore-microtubule attachment and chromosome alignment, the Ska complex has functions in promoting anaphase onset. We find that both Ska3 and microtubules promote chromosome association of the anaphase-promoting complex/cyclosome (APC/C). Chromosome-bound APC/C shows significantly stronger ubiquitylation activity than cytoplasmic APC/C. Forced localization of Ska complex to kinetochores, independent of microtubules, results in enhanced accumulation of APC/C on chromosomes and accelerated cyclin B1 degradation during induced mitotic exit. We propose that a Ska-microtubule-kinetochore association promotes APC/C localization to chromosomes, thereby enhancing anaphase onset and mitotic exit.


Assuntos
Ciclossomo-Complexo Promotor de Anáfase/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Mitose/fisiologia , Anáfase/efeitos dos fármacos , Anáfase/genética , Anáfase/fisiologia , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular , Cromossomos Humanos/efeitos dos fármacos , Cromossomos Humanos/metabolismo , Ciclina B1/metabolismo , Células HeLa , Humanos , Metáfase/efeitos dos fármacos , Metáfase/genética , Metáfase/fisiologia , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose/efeitos dos fármacos , Mitose/genética , Modelos Biológicos , Nocodazol/farmacologia , Moduladores de Tubulina/farmacologia
13.
Mol Biol Cell ; 22(8): 1191-206, 2011 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-21325631

RESUMO

Mitosis requires precise coordination of multiple global reorganizations of the nucleus and cytoplasm. Cyclin-dependent kinase 1 (Cdk1) is the primary upstream kinase that directs mitotic progression by phosphorylation of a large number of substrate proteins. Cdk1 activation reaches the peak level due to positive feedback mechanisms. By inhibiting Cdk chemically, we showed that, in prometaphase, when Cdk1 substrates approach the peak of their phosphorylation, cells become capable of proper M-to-G1 transition. We interfered with the molecular components of the Cdk1-activating feedback system through use of chemical inhibitors of Wee1 and Myt1 kinases and Cdc25 phosphatases. Inhibition of Wee1 and Myt1 at the end of the S phase led to rapid Cdk1 activation and morphologically normal mitotic entry, even in the absence of G2. Dampening Cdc25 phosphatases simultaneously with Wee1 and Myt1 inhibition prevented Cdk1/cyclin B kinase activation and full substrate phosphorylation and induced a mitotic "collapse," a terminal state characterized by the dephosphorylation of mitotic substrates without cyclin B proteolysis. This was blocked by the PP1/PP2A phosphatase inhibitor, okadaic acid. These findings suggest that the positive feedback in Cdk activation serves to overcome the activity of Cdk-opposing phosphatases and thus sustains forward progression in mitosis.


Assuntos
Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Mitose , Proteínas Nucleares/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas de Xenopus/antagonistas & inibidores , Fosfatases cdc25/antagonistas & inibidores , Animais , Proteína Quinase CDC2/antagonistas & inibidores , Proteína Quinase CDC2/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Ciclina B/genética , Ciclina B/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Retroalimentação Fisiológica/efeitos dos fármacos , Feminino , Fase G2/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Proteínas de Membrana , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Fosforilação/efeitos dos fármacos , Prometáfase/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteína Fosfatase 2/antagonistas & inibidores , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Fase S/efeitos dos fármacos , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis , Fosfatases cdc25/genética , Fosfatases cdc25/metabolismo
14.
Curr Biol ; 21(12): 1018-24, 2011 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-21658943

RESUMO

BACKGROUND: Chromosome instability is thought to be a major contributor to cancer malignancy and birth defects. For balanced chromosome segregation in mitosis, kinetochores on sister chromatids bind and pull on microtubules emanating from opposite spindle poles. This tension contributes to the correction of improper kinetochore attachments and is opposed by the cohesin complex that holds the sister chromatids together. Normally, within minutes of alignment at the metaphase plate, chromatid cohesion is released, allowing each cohort of chromatids to move synchronously to opposite poles in anaphase, an event closely coordinated with mitotic exit. RESULTS: Here we show that during experimentally induced metaphase delay, spindle pulling forces can cause asynchronous chromatid separation, a phenomenon we term "cohesion fatigue." Cohesion fatigue is not blocked by inhibition of Plk1, a kinase essential for the "prophase pathway" of cohesin release from chromosomes, or by depletion of separase, the protease that normally drives chromatid separation at anaphase. Cohesion fatigue is inhibited by drug-induced depolymerization of mitotic spindle microtubules and by experimentally increasing the levels of cohesin on mitotic chromosomes. In cells undergoing cohesion fatigue, cohesin proteins remain associated with the separated chromatids. CONCLUSION: In cells arrested at metaphase, pulling forces originating from kinetochore-microtubule interactions can, with time, rupture normal sister chromatid cohesion. This cohesion fatigue, resulting in unscheduled chromatid separation in cells delayed at metaphase, constitutes a previously overlooked source for chromosome instability in mitosis and meiosis.


Assuntos
Proteínas de Ciclo Celular/fisiologia , Cromátides , Proteínas Cromossômicas não Histona/fisiologia , Metáfase , Células HeLa , Humanos , Coesinas
15.
Curr Biol ; 19(17): 1467-72, 2009 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-19646878

RESUMO

The mitotic spindle checkpoint monitors proper bipolar attachment of chromosomes to the mitotic spindle. Previously, depletion of the novel kinetochore complex Ska1/Ska2 was found to induce a metaphase delay. By using bioinformatics, we identified C13orf3, predicted to associate with kinetochores. Recently, three laboratories independently indentified C13orf3 as an additional Ska complex component, and therefore we adopted the name Ska3. We found that cells depleted of Ska3 by RNAi achieve metaphase alignment but fail to silence the spindle checkpoint or enter anaphase. After hours of metaphase arrest, chromatids separate but retain robust kinetochore-microtubule attachments. Ska3-depleted cells accumulate high levels of the checkpoint protein Bub1 at kinetochores. Ska3 protein accumulation at kinetochores in prometaphase is dependent on Sgo1 protein. Sgo1, which accumulates at the centromeres earlier, in prophase, is not dependent on Ska3. Sgo1-depleted cells show a stronger premature chromatid separation phenotype than those depleted of Ska3. We hypothesize that Ska3 functions to coordinate checkpoint signaling from the microtubule binding sites within a kinetochore by laterally linking the individual binding sites. We suggest that this network plays a major role in silencing the spindle checkpoint when chromosomes are aligned at metaphase to allow timely anaphase onset and mitotic exit.


Assuntos
Cromossomos Humanos , Proteínas Associadas aos Microtúbulos/fisiologia , Mitose/fisiologia , Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Biologia Computacional , Células HeLa , Humanos , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose/genética , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestrutura , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Fuso Acromático/metabolismo
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