RESUMO
BACKGROUND: The RZZ complex, composed of the proteins Rough-Deal (Rod), Zw10 and Zwilch, plays a central role in the spindle assembly checkpoint (SAC), which assures proper sister chromatid segregation during mitosis. RZZ contributes to the regulation of the spindle assembly checkpoint by helping to recruit Mad1-Mad2 and the microtubule motor dynein to unattached kinetochores. It is an important component of the outer kinetochore and specifically the fibrous corona whose expansion is believed to facilitate microtubule capture. How RZZ carries out its diverse activities is only poorly understood. The C-terminal region of the Rod subunit is relatively well-conserved across metazoan phylogeny, but no function has been attributed to it. RESULTS: To explore the importance of the Rod_C domain in RZZ function in Drosophila, we generated a series of point mutations in a stretch of 200 residues within this domain and we report here their phenotypes. Several of the mutations profoundly disrupt recruitment of RZZ to kinetochores, including one in a temperature-sensitive manner, while still retaining the capacity to assemble into a complex with Zw10 and Zwilch. Others affect aspects of dynein activity or recruitment at the kinetochore. CONCLUSIONS AND SIGNIFICANCE: These results suggest that the Rod_C domain participates in the protein interactions necessary for RZZ recruitment and functionality at kinetochores.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiologia , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Drosophila , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/fisiologia , Animais , Proteínas de Ciclo Celular/genética , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Pontos de Checagem da Fase M do Ciclo Celular , Proteínas Mad2/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Fuso Acromático/metabolismoRESUMO
Proper kinetochore recruitment and regulation of dynein and the Mad1-Mad2 complex requires the Rod-Zw10-Zwilch (RZZ) complex. Here, we describe rod(Z3), a maternal-effect Drosophila mutation changing a single residue in the Rough Deal (Rod) subunit of RZZ. Although the RZZ complex containing this altered subunit (denoted R(Z3)ZZ) is present in early syncytial stage embryos laid by homozygous rod(Z3) mothers, it is not recruited to kinetochores. Consequently, the embryos have no spindle assembly checkpoint (SAC), and syncytial mitoses are profoundly perturbed. The polar body (residual meiotic products) cannot remain in its SAC-dependent metaphase-like state, and decondenses into chromatin. In neuroblasts of homozygous rod(Z3) larvae, R(Z3)ZZ recruitment is only partially reduced, the SAC is functional and mitosis is relatively normal. R(Z3)ZZ nevertheless behaves abnormally: it does not further accumulate on kinetochores when microtubules are depolymerized; it reduces the rate of Mad1 recruitment; and it dominantly interferes with the dynein-mediated streaming of RZZ from attached kinetochores. These results suggest that the mutated residue of rod(Z3) is required for normal RZZ kinetochore recruitment and function and, moreover, that the RZZ recruitment pathway might differ in syncytial stage embryos and post-embryonic somatic cells.
Assuntos
Animais Geneticamente Modificados/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Cinetocoros/fisiologia , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados/embriologia , Animais Geneticamente Modificados/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Feminino , Processamento de Imagem Assistida por Computador , Dados de Sequência Molecular , Neurônios/citologia , Neurônios/metabolismo , Homologia de Sequência de Aminoácidos , Fuso Acromático/fisiologiaRESUMO
Heterochromatic gene silencing relies on combinatorial control by specific histone modifications, the occurrence of transcription, and/or RNA degradation. Once nucleated, heterochromatin propagates within defined chromosomal regions and is maintained throughout cell divisions to warrant proper genome expression and integrity. In the fission yeast Schizosaccharomyces pombe, the Ccr4-Not complex partakes in gene silencing, but its relative contribution to distinct heterochromatin domains and its role in nucleation versus spreading have remained elusive. Here, we unveil major functions for Ccr4-Not in silencing and heterochromatin spreading at the mating type locus and subtelomeres. Mutations of the catalytic subunits Caf1 or Mot2, involved in RNA deadenylation and protein ubiquitinylation, respectively, result in impaired propagation of H3K9me3 and massive accumulation of nucleation-distal heterochromatic transcripts. Both silencing and spreading defects are suppressed upon disruption of the heterochromatin antagonizing factor Epe1. Overall, our results position the Ccr4-Not complex as a critical, dual regulator of heterochromatic gene silencing and spreading.
Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Heterocromatina/genética , Heterocromatina/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Histonas/genética , Histonas/metabolismo , Inativação GênicaRESUMO
Long non-coding RNAs (lncRNAs) contribute to the regulation of gene expression in response to intra- or extracellular signals but the underlying molecular mechanisms remain largely unexplored. Here, we identify an uncharacterized lncRNA as a central player in shaping the meiotic gene expression program in fission yeast. We report that this regulatory RNA, termed mamRNA, scaffolds the antagonistic RNA-binding proteins Mmi1 and Mei2 to ensure their reciprocal inhibition and fine tune meiotic mRNA degradation during mitotic growth. Mechanistically, mamRNA allows Mmi1 to target Mei2 for ubiquitin-mediated downregulation, and conversely enables accumulating Mei2 to impede Mmi1 activity, thereby reinforcing the mitosis to meiosis switch. These regulations also occur within a unique Mmi1-containing nuclear body, positioning mamRNA as a spatially-confined sensor of Mei2 levels. Our results thus provide a mechanistic basis for the mutual control of gametogenesis effectors and further expand our vision of the regulatory potential of lncRNAs.
Assuntos
Regulação Fúngica da Expressão Gênica , Meiose/genética , Mitose/genética , RNA Fúngico/genética , RNA Longo não Codificante/genética , Schizosaccharomyces/genética , Ligação Proteica , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
Selection of the cleavage plane during cytokinesis in dividing cells is linked to the position of the mitotic spindle. A major player in cleavage plane positioning is believed to be the anaphase central spindle and its associated signaling complex called centralspindlin, composed of MgcRacGap and MKLP1. Centralspindlin has the capacity to induce furrowing of the cell cortex by promoting the localized activation of RhoA, which in turn promotes assembly of the contractile ring. We have found a way to induce a cytokinesis-like process in unfertilized Drosophila eggs and very early embryos, when spindle structures are few and located far from invaginating egg cortex. The simple injection of a small molecule inhibitor of Cdk1/Cyclin B (either Roscovitin or RO3306) is sufficient to promote membrane invagination near the site of injection. The furrow generated is in many respects similar to a classical cleavage furrow. Actin, myosin, anillin and MKLP1 are all associated with the forming furrow, which in some cases can entirely circumscribe the unfertilized egg. A similar furrow can also be generated by the localized injection of constitutively active RhoA protein, suggesting that Cdk1 is normally an upstream inhibitor of RhoA activation. We show further that this process apparently is not associated with microtubules. Since simple localized inhibition of Cdk1 is sufficient to induce a furrow, we suggest that in real cytokinesis in normal cells, the localized downregulation of Cdk1 activity at the metaphase-anaphase transition may contribute, along with the spindle, to the positioning of the cleavage furrow.
Assuntos
Proteína Quinase CDC2/antagonistas & inibidores , Ciclina B/antagonistas & inibidores , Citocinese , Drosophila/fisiologia , Óvulo/citologia , Actinas/metabolismo , Animais , Divisão Celular , Células Cultivadas , Proteínas Contráteis/metabolismo , Citocinese/efeitos dos fármacos , Drosophila/embriologia , Proteínas de Drosophila/metabolismo , Embrião não Mamífero/citologia , Proteínas Ativadoras de GTPase , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose , Miosinas/metabolismo , Óvulo/fisiologia , Inibidores de Proteínas Quinases/farmacologia , Purinas/farmacologia , Quinolinas/farmacologia , Roscovitina , Fuso Acromático/metabolismo , Tiazóis/farmacologia , Proteína rhoA de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/farmacologiaRESUMO
In this issue of Structure, Civril et al. (2010) describe an unexpected structural similarity between the kinetochore component Rod-Zw10-Zwilch (RZZ) and the vesicle transport component Nag-Rint1-Zw10 (NRZ).
RESUMO
Mono-ubiquitylation of histone H2B correlates with transcriptional activation and is required for di- and trimethylation at Lys 4 on the histone H3 tail (H3K4) by the SET1/COMPASS methyltransferase complex through a poorly characterized trans-tail pathway. Here we show that mono-ubiquitylation of histone H2B promotes ubiquitylation at Lys 68 and Lys 69 of Swd2, the essential component of SET1/COMPASS in Saccharomyces cerevisiae. We found that Rad6/Bre1 ubiquitylation enzymes responsible for H2B ubiquitylation also participate directly in Swd2 modification. Preventing Swd2 or H2B ubiquitylation did not affect Set1 stability, interaction of Swd2 with Set1 or the ability of Swd2 to interact with chromatin. However, we found that mutation of Lys 68 and Lys 69 of Swd2 markedly reduced trimethylation, and to a lesser extent dimethylation, of H3K4 at the 5'-end of transcribing genes without affecting monomethylation. This effect results from the ability of Swd2 ubiquitylation to control recruitment of Spp1, a COMPASS subunit necessary for trimethylation. Our results further indicate that Swd2 is a major H3-binding component of COMPASS. Swd2 thus represents a key factor that mediates crosstalk between H2B ubiquitylation and H3K4 trimethylation on chromatin.
Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitinação , Cromatina/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Glutationa Transferase/metabolismo , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/isolamento & purificação , Histonas/química , Histonas/genética , Metilação , Modelos Biológicos , Mutação , Plasmídeos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
Plasma membrane transport of single amino-acid methionine in yeast is shown to be mediated by at least seven different permeases whose activities are transcriptionaly and post-transcriptionaly regulated by different ubiquitin-dependent mechanisms. Upon high extracellular methionine exposure, three methionine-permease genes are repressed while four others are induced. SCF(Met30), SCF(Grr1) and Rsp5 ubiquitin ligases are the key actors of the ubiquitin-dependent remodeling of methionine transport. In addition to regulating the activity of Met4, the SCF(Met30) ubiquitin ligase is shown to convey an intracellular signal to a membrane initiated signaling pathway by controlling the nuclear concentration of the Stp1 transcription factor. By coupling intra- and extracellular metabolite sensing, SCF(Met30) thus allows yeast cells to accurately adjust the intermediary sulfur metabolism to the growth conditions. The multiple ubiquitin-dependent mechanisms that function in methionine transport regulation further exemplify the pervasive role of ubiquitin in the adaptation of single-cell organisms to environmental modifications.
Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Metionina/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte , Estabilidade Enzimática , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/genética , Proteínas/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Ligases SKP Culina F-Box/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Compostos de Enxofre/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismoRESUMO
In yeast, the Met4 transcription factor and its cofactors Cbf1, Met28, Met31, and Met32 control the expression of sulfur metabolism and oxidative stress response genes. Met4 activity is tuned to nutrient and oxidative stress conditions by the SCF(Met30) ubiquitin ligase. The mechanism whereby SCF(Met30)-dependent ubiquitylation of Met4 controls Met4 activity remains contentious. Here, we have demonstrated that intracellular cysteine levels dictate the degradation of Met4 in vivo, as shown by the ability of cysteine, but not methionine or S-adenosylmethionine (AdoMet), to trigger Met4 degradation in an str4Delta strain, which lacks the ability to produce cysteine from methionine or AdoMet. Met4 degradation requires its nuclear localization and activity of the 26 S proteasome. Analysis of the regulated degradation of a fully functional Met4-Cbf1 chimera, in which Met4 is fused to the DNA binding domain of Cbf1, demonstrates that elimination of Met4 in vivo can be triggered independently of both its normal protein interactions. Strains that harbor the Met4-Cbf1 fusion as the only source of Cbf1 activity needed for proper kinetochore function exhibit high rates of methionine-dependent chromosomal instability. We suggest that SCF(Met30) activity or Met4 utilization as a substrate may be directly regulated by intracellular cysteine concentrations.