RESUMEN
FEM1A, FEM1B, and FEM1C are evolutionarily-conserved VHL-box proteins, the substrate recognition subunits of CUL2-RING E3 ubiquitin ligase complexes. Here, we report that FEM1 proteins are ancient regulators of Stem-Loop Binding Protein (SLBP), a conserved protein that interacts with the stem loop structure located in the 3' end of canonical histone mRNAs and functions in mRNA cleavage, translation and degradation. SLBP levels are highest during S-phase coinciding with histone synthesis. The ubiquitin ligase complex SCFcyclin F targets SLBP for degradation in G2 phase; however, the regulation of SLBP during other stages of the cell cycle is poorly understood. We provide evidence that FEM1A, FEM1B, and FEM1C interact with and mediate the degradation of SLBP. Cyclin F, FEM1A, FEM1B and FEM1C all interact with a region in SLBP's N-terminus using distinct degrons. An SLBP mutant that is unable to interact with all 4 ligases is expressed at higher levels than wild type SLBP and does not oscillate during the cell cycle. We demonstrate that orthologues of SLBP and FEM1 proteins interact in C. elegans and D. melanogaster, suggesting that the pathway is evolutionarily conserved. Furthermore, we show that FEM1 depletion in C. elegans results in the upregulation of SLBP ortholog CDL-1 in oocytes. Notably, cyclin F is absent in flies and worms, suggesting that FEM1 proteins play an important role in SLBP targeting in lower eukaryotes.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas/metabolismo , Proteolisis , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Secuencias de Aminoácidos , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Línea Celular , Secuencia Conservada , Regulación hacia Abajo , Evolución Molecular , Humanos , Proteínas Nucleares/química , Unión Proteica , Complejos de Ubiquitina-Proteína Ligasa , Factores de Escisión y Poliadenilación de ARNm/químicaRESUMEN
The anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase is known to target the degradation of cyclin B1, which is crucial for mitotic progression in animal cells. In this study, we show that the ubiquitin ligase CRL2ZYG-11 redundantly targets the degradation of cyclin B1 in Caenorhabditis elegans and human cells. In C. elegans, both CRL2ZYG-11 and APC/C are required for proper progression through meiotic divisions. In human cells, inactivation of CRL2ZYG11A/B has minimal effects on mitotic progression when APC/C is active. However, when APC/C is inactivated or cyclin B1 is overexpressed, CRL2ZYG11A/B-mediated degradation of cyclin B1 is required for normal progression through metaphase. Mitotic cells arrested by the spindle assembly checkpoint, which inactivates APC/C, often exit mitosis in a process termed "mitotic slippage," which generates tetraploid cells and limits the effectiveness of antimitotic chemotherapy drugs. We show that ZYG11A/B subunit knockdown, or broad cullin-RING ubiquitin ligase inactivation with the small molecule MLN4924, inhibits mitotic slippage in human cells, suggesting the potential for antimitotic combination therapy.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citología , Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclina B1/metabolismo , Mitosis , Proteolisis , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Animales , Proteína Quinasa CDC2/metabolismo , Caenorhabditis elegans/efectos de los fármacos , Línea Celular Tumoral , Células HEK293 , Humanos , Mitosis/efectos de los fármacos , Nocodazol/farmacología , Unión Proteica/efectos de los fármacos , Proteolisis/efectos de los fármacos , Especificidad por Sustrato/efectos de los fármacos , Imagen de Lapso de TiempoRESUMEN
The mammalian CIP/KIP family of cyclin-dependent kinase (CDK) inhibitors (CKIs) comprises three proteins--p21(Cip1/WAF1), p27(Kip1), and p57(Kip2)--that bind and inhibit cyclin-CDK complexes, which are key regulators of the cell cycle. CIP/KIP CKIs have additional independent functions in regulating transcription, apoptosis and actin cytoskeletal dynamics. These divergent functions are performed in distinct cellular compartments and contribute to the seemingly contradictory observation that the CKIs can both suppress and promote cancer. Multiple ubiquitin ligases (E3s) direct the proteasome-mediated degradation of p21, p27 and p57. This review analyzes recent data highlighting our current understanding of how distinct E3 pathways regulate subpopulations of the CKIs to control their diverse functions.
Asunto(s)
Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/metabolismo , Animales , Apoptosis , Humanos , Especificidad por Sustrato , Transcripción Genética , UbiquitinaciónRESUMEN
Morphogenesis is an important component of animal development. Genetic redundancy has been proposed to be common among morphogenesis genes, posing a challenge to the genetic dissection of morphogenesis mechanisms. Genetic redundancy is more generally a challenge in biology, as large proportions of the genes in diverse organisms have no apparent loss of function phenotypes. Here, we present a screen designed to uncover redundant and partially redundant genes that function in an example of morphogenesis, gastrulation in Caenorhabditis elegans. We performed an RNA interference (RNAi) enhancer screen in a gastrulation-sensitized double-mutant background, targeting genes likely to be expressed in gastrulating cells or their neighbors. Secondary screening identified 16 new genes whose functions contribute to normal gastrulation in a nonsensitized background. We observed that for most new genes found, the closest known homologs were multiple other C. elegans genes, suggesting that some may have derived from rounds of recent gene duplication events. We predict that such genes are more likely than single copy genes to comprise redundant or partially redundant gene families. We explored this prediction for one gene that we identified and confirmed that this gene and five close relatives, which encode predicted substrate recognition subunits (SRSs) for a CUL-2 ubiquitin ligase, do indeed function partially redundantly with each other in gastrulation. Our results implicate new genes in C. elegans gastrulation, and they show that an RNAi-based enhancer screen in C. elegans can be used as an efficient means to identify important but redundant or partially redundant developmental genes.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Gastrulación/genética , Regulación del Desarrollo de la Expresión Génica , Genoma de los Helmintos , Ensayos Analíticos de Alto Rendimiento , Interferencia de ARN , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/embriología , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Duplicación de Gen , Genes de Helminto , Estudios de Asociación Genética , Microscopía Confocal , Familia de Multigenes , Mutación , Fenotipo , Filogenia , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
The Cip/Kip CDK inhibitor (CKI) p21(Cip1/WAF1) has a critical role in the nucleus to limit cell proliferation by inhibiting CDK-cyclin complexes. In contrast, cytoplasmic p21 regulates cell survival and the actin cytoskeleton. These divergent functions for p21 in different cellular compartments suggest the necessity for complex regulation. In this study, we identify the CRL2(LRR-1) ubiquitin ligase as a conserved regulator of Cip/Kip CKIs that promotes the degradation of C. elegans CKI-1 and human p21. The nematode CRL2(LRR-1) complex negatively regulates nuclear CKI-1 levels to ensure G1-phase cell cycle progression in germ cells. In contrast, human CRL2(LRR1) targets cytoplasmic p21, acting as a critical regulator of cell motility that promotes a nonmotile stationary cell state by preventing p21 from inhibiting the Rho/ROCK/LIMK pathway. Inactivation of human CRL2(LRR1) leads to the activation of the actin-depolymerizing protein cofilin, dramatic reorganization of the actin cytoskeleton, and increased cell motility.
Asunto(s)
Actinas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Ciclo Celular/fisiología , Movimiento Celular/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas Represoras/metabolismo , Animales , Caenorhabditis elegans/citología , Proteínas de Caenorhabditis elegans/genética , Núcleo Celular/metabolismo , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Citoesqueleto/metabolismo , Células Germinativas/citología , Células Germinativas/fisiología , Humanos , Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genéticaRESUMEN
In most eukaryotic cells, subsets of microtubules are adapted for specific functions by post-translational modifications (PTMs) of tubulin subunits. Acetylation of the epsilon-amino group of K40 on alpha-tubulin is a conserved PTM on the luminal side of microtubules that was discovered in the flagella of Chlamydomonas reinhardtii. Studies on the significance of microtubule acetylation have been limited by the undefined status of the alpha-tubulin acetyltransferase. Here we show that MEC-17, a protein related to the Gcn5 histone acetyltransferases and required for the function of touch receptor neurons in Caenorhabditis elegans, acts as a K40-specific acetyltransferase for alpha-tubulin. In vitro, MEC-17 exclusively acetylates K40 of alpha-tubulin. Disruption of the Tetrahymena MEC-17 gene phenocopies the K40R alpha-tubulin mutation and makes microtubules more labile. Depletion of MEC-17 in zebrafish produces phenotypes consistent with neuromuscular defects. In C. elegans, MEC-17 and its paralogue W06B11.1 are redundantly required for acetylation of MEC-12 alpha-tubulin, and contribute to the function of touch receptor neurons partly via MEC-12 acetylation and partly via another function, possibly by acetylating another protein. In summary, we identify MEC-17 as an enzyme that acetylates the K40 residue of alpha-tubulin, the only PTM known to occur on the luminal surface of microtubules.
Asunto(s)
Acetiltransferasas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimología , Tubulina (Proteína)/metabolismo , Proteínas de Pez Cebra/metabolismo , Acetilación , Animales , Caenorhabditis elegans/metabolismo , Línea Celular , Dipodomys , Humanos , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Tetrahymena/metabolismo , Tacto , Tubulina (Proteína)/química , Pez Cebra/embriología , Pez Cebra/metabolismoRESUMEN
The faithful replication of genomic DNA is crucial for maintaining genome stability. In eukaryotes, DNA rereplication is prevented by the temporal regulation of replication licensing. Replication-licensing factors are required to form prereplicative complexes during G1 phase, but are inactivated in S phase to prevent rereplication. A vertebrate CUL4 CRL ubiquitin ligase (CRL4) complex containing Cdt2 as the substrate recognition subunit promotes proper DNA replication, in part, by degrading the replication-licensing factor Cdt1 during S phase. We show that the Caenorhabditis elegans CRL4(Cdt2) complex has a conserved role in degrading Cdt1. Furthermore, we show that CRL4(Cdt2) restrains replication licensing in both C. elegans and humans by targeting the degradation of the cyclin-dependent kinase (CDK) inhibitors CKI-1 and p21(Cip1), respectively. Human CRL4(Cdt2) targets the degradation of p21 in S phase, with the in vivo ubiquitylation of p21 by CRL4(Cdt2) dependent on p21 binding to PCNA. Inactivation of Cdt2 induces rereplication, which requires the presence of the CDK inhibitor p21. Strikingly, coinactivation of CRL4(Cdt2) and SCF(Skp2) (which redundantly targets p21 degradation) prevents the nuclear export of the replication-licensing factor Cdc6 during S phase, and the block on nuclear export is dependent on p21. Our work defines the degradation of p21 as a critical aspect of replication licensing in human cells.
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Replicación del ADN , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Secuencia de Bases , Caenorhabditis elegans/metabolismo , Línea Celular , Humanos , Hidrólisis , ARN Interferente Pequeño , UbiquitinaciónRESUMEN
In Caenorhabditis elegans, the Gli-family transcription factor TRA-1 is the terminal effector of the sex-determination pathway. TRA-1 activity inhibits male development and allows female fates. Genetic studies have indicated that TRA-1 is negatively regulated by the fem-1, fem-2, and fem-3 genes. However, the mechanism of this regulation has not been understood. Here, we present data that TRA-1 is regulated by degradation mediated by a CUL-2-based ubiquitin ligase complex that contains FEM-1 as the substrate-recognition subunit, and FEM-2 and FEM-3 as cofactors. CUL-2 physically associates with both FEM-1 and TRA-1 in vivo, and cul-2 mutant males share feminization phenotypes with fem mutants. CUL-2 and the FEM proteins negatively regulate TRA-1 protein levels in C. elegans. When expressed in human cells, the FEM proteins interact with human CUL2 and induce the proteasome-dependent degradation of TRA-1. This work demonstrates that the terminal step in C. elegans sex determination is controlled by ubiquitin-mediated proteolysis.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cullin/metabolismo , Proteínas de Unión al ADN/metabolismo , Procesos de Determinación del Sexo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Ciclosoma-Complejo Promotor de la Anafase , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas Cullin/química , Proteínas Cullin/genética , Trastornos del Desarrollo Sexual , Datos de Secuencia Molecular , Mutación , Fenotipo , Fosfoproteínas Fosfatasas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Especificidad por Sustrato , Ubiquitina/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The cullin CUL-2 is a crucial component of a subclass of multisubunit cullin-RING ubiquitin-ligases. The specificity of CUL-2-based complexes is provided by variable substrate-recognition subunits that bind to specific substrates. In Caenorhabditis elegans, CUL-2 regulates several key processes in cell division and embryonic development, including meiotic progression, anterior-posterior polarity and mitotic chromatin condensation. However, the substrate recognition subunits that work in these CUL-2-dependent processes were unknown. Here, we present evidence that ZYG-11 is the substrate-recognition subunit for a CUL-2-based complex that regulates these functions. We show that ZYG-11 interacts with CUL-2 in vivo and binds to the complex adaptor protein Elongin C using a nematode variant of the VHL-box motif. We show that the ZYG11 gene family encompasses two main branches in metazoa, and provide evidence that members of the extended ZYG11 family in nematodes and humans are conserved components of CUL2-based ubiquitin-ligases.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Proteínas Cullin/genética , Evolución Molecular , Complejos Multiproteicos/genética , Filogenia , Secuencia de Aminoácidos , Animales , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Análisis por Conglomerados , Proteínas Cullin/metabolismo , Elonguina , Humanos , Datos de Secuencia Molecular , Especificidad por Sustrato , Factores de Transcripción/metabolismoRESUMEN
Box C/D RNAs are small, noncoding RNAs that function in RNA modification in eukaryotes and archaea. Here, we report that box C/D RNAs exist in the rare biological form of RNA circles in the hyperthermophilic archaeon Pyrococcus furiosus. Northern analysis of box C/D RNAs reveals two prominent RNA species of different electrophoretic mobilities in total P. furiosus RNA preparations. Together, the results of Northern, ribozyme, RT-PCR, and lariat debranching analyses indicate that the two species are circular and linear RNAs of similar length and abundance. It seems that most, if not all, species of box C/D RNAs exist as circles in P. furiosus. In addition, the circular RNAs are found in complexes with proteins required for box C/D RNA function. Our finding places box C/D RNAs among the extremely few circular RNAs known to exist in nature. Moreover, the unexpected discovery of circular box C/D RNAs points to the existence of a previously unrecognized biogenesis pathway for box C/D RNAs in archaea.
Asunto(s)
Pyrococcus furiosus/genética , ARN de Archaea/genética , ARN/genética , Animales , Secuencia de Bases , Northern Blotting , Western Blotting , Secuencia Conservada , Inmunoprecipitación/métodos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Pyrococcus furiosus/química , ARN de Archaea/química , ARN Catalítico/análisis , ARN Catalítico/genética , ARN Circular , ARN Nucleolar Pequeño/química , ARN Nucleolar Pequeño/genética , Conejos , Proteínas Recombinantes/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleoproteínas Nucleolares Pequeñas/genéticaRESUMEN
The methyltransferase fibrillarin is the catalytic component of ribonucleoprotein complexes that direct site-specific methylation of precursor ribosomal RNA and are critical for ribosome biogenesis in eukaryotes and archaea. Here we report the crystal structure of a fibrillarin ortholog from the hyperthermophilic archaeon Pyrococcus furiosus at 1.97A resolution. Comparisons of the X-ray structures of fibrillarin orthologs from Methanococcus jannashii and Archaeoglobus fulgidus reveal nearly identical backbone configurations for the catalytic C-terminal domain with the exception of a unique loop conformation at the S-adenosyl-l-methionine (AdoMet) binding pocket in P. furiosus. In contrast, the N-terminal domains are divergent which may explain why some forms of fibrillarin apparently homodimerize (M. jannashii) while others are monomeric (P. furiosus and A. fulgidus). Three positively charged amino acids surround the AdoMet-binding site and sequence analysis indicates that this is a conserved feature of both eukaryotic and archaeal fibrillarins. We discuss the possibility that these basic residues of fibrillarin are important for RNA-guided rRNA methylation.