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1.
Genes Dev ; 30(19): 2213-2225, 2016 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-27798844

RESUMEN

Caspases are key components of apoptotic pathways. Regulation of caspases occurs at several levels, including transcription, proteolytic processing, inhibition of enzymatic function, and protein degradation. In contrast, little is known about the extent of post-transcriptional control of caspases. Here, we describe four conserved RNA-binding proteins (RBPs)-PUF-8, MEX-3, GLD-1, and CGH-1-that sequentially repress the CED-3 caspase in distinct regions of the Caenorhabditis elegans germline. We demonstrate that GLD-1 represses ced-3 mRNA translation via two binding sites in its 3' untranslated region (UTR), thereby ensuring a dual control of unwanted cell death: at the level of p53/CEP-1 and at the executioner caspase level. Moreover, we identified seven RBPs that regulate human caspase-3 expression and/or activation, including human PUF-8, GLD-1, and CGH-1 homologs PUM1, QKI, and DDX6. Given the presence of unusually long executioner caspase 3' UTRs in many metazoans, translational control of executioner caspases by RBPs might be a strategy used widely across the animal kingdom to control apoptosis.


Asunto(s)
Apoptosis/genética , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/genética , Caspasas/genética , Caspasas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Unión al ARN/metabolismo , Regiones no Traducidas 3'/genética , Animales , Sitios de Unión , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Células Germinativas/citología , Células HeLa , Humanos , Procesamiento Postranscripcional del ARN
2.
Genome Res ; 25(11): 1680-91, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26232411

RESUMEN

In animals, microRNAs frequently form families with related sequences. The functional relevance of miRNA families and the relative contribution of family members to target repression have remained, however, largely unexplored. Here, we used the Caenorhabditis elegans miR-58 miRNA family, composed primarily of the four highly abundant members miR-58.1, miR-80, miR-81, and miR-82, as a model to investigate the redundancy of miRNA family members and their impact on target expression in an in vivo setting. We found that miR-58 family members repress largely overlapping sets of targets in a predominantly additive fashion. Progressive deletions of miR-58 family members lead to cumulative up-regulation of target protein and RNA levels. Phenotypic defects could only be observed in the family quadruple mutant, which also showed the strongest change in target protein levels. Interestingly, although the seed sequences of miR-80 and miR-58.1 differ in a single nucleotide, predicted canonical miR-80 targets were efficiently up-regulated in the mir-58.1 single mutant, indicating functional redundancy of distinct members of this miRNA family. At the aggregate level, target binding leads mainly to mRNA degradation, although we also observed some degree of translational inhibition, particularly in the single miR-58 family mutants. These results provide a framework for understanding how miRNA family members interact to regulate target mRNAs.


Asunto(s)
Caenorhabditis elegans/genética , MicroARNs/genética , Estabilidad del ARN/genética , ARN Mensajero/genética , Regulación hacia Arriba , Animales , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Represión Epigenética , MicroARNs/metabolismo , Proteómica , ARN Mensajero/metabolismo , Análisis de Secuencia de ARN , Transcriptoma
3.
RNA ; 19(10): 1317-26, 2013 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-23974436

RESUMEN

To understand the function of the hundreds of RNA-binding proteins (RBPs) that are encoded in animal genomes it is important to identify their target RNAs. Although it is generally accepted that the binding specificity of an RBP is well described in terms of the nucleotide sequence of its binding sites, other factors such as the structural accessibility of binding sites or their clustering, to enable binding of RBP multimers, are also believed to play a role. Here we focus on GLD-1, a translational regulator of Caenorhabditis elegans, whose binding specificity and targets have been studied with a variety of methods such as CLIP (cross-linking and immunoprecipitation), RIP-Chip (microarray measurement of RNAs associated with an immunoprecipitated protein), profiling of polysome-associated mRNAs and biophysical determination of binding affinities of GLD-1 for short nucleotide sequences. We show that a simple biophysical model explains the binding of GLD-1 to mRNA targets to a large extent, and that taking into account the accessibility of putative target sites significantly improves the prediction of GLD-1 binding, particularly due to a more accurate prediction of binding in transcript coding regions. Relating GLD-1 binding to translational repression and stabilization of its target transcripts we find that binding sites along the entire transcripts contribute to functional responses, and that CDS-located sites contribute most to translational repression. Finally, biophysical measurements of GLD-1 affinity for a small number of oligonucleotides appear to allow an accurate reconstruction of the sequence specificity of the protein. This approach can be applied to uncover the specificity and function of other RBPs.


Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Regulación de la Expresión Génica , Modelos Teóricos , Sistemas de Lectura Abierta/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , Animales , Sitios de Unión , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Inmunoprecipitación , Unión Proteica , ARN Mensajero/química , ARN Mensajero/metabolismo
4.
Proteomics ; 12(3): 340-5, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22290800

RESUMEN

The community working on model organisms is growing steadily and the number of model organisms for which proteome data are being generated is continuously increasing. To standardize efforts and to make optimal use of proteomics data acquired from model organisms, a new Human Proteome Organisation (HUPO) initiative on model organism proteomes (iMOP) was approved at the HUPO Ninth Annual World Congress in Sydney, 2010. iMOP will seek to stimulate scientific exchange and disseminate HUPO best practices. The needs of model organism researchers for central databases will be better represented, catalyzing the integration of proteomics and organism-specific databases. Full details of iMOP activities, members, tools and resources can be found at our website http://www.imop.uzh.ch/ and new members are invited to join us.


Asunto(s)
Arabidopsis/química , Modelos Animales , Proteoma , Animales , Animales de Laboratorio , Bases de Datos de Proteínas , Humanos
5.
Cell Death Differ ; 26(10): 2157-2178, 2019 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-30728462

RESUMEN

Post-transcriptional control of mRNAs by RNA-binding proteins (RBPs) has a prominent role in the regulation of gene expression. RBPs interact with mRNAs to control their biogenesis, splicing, transport, localization, translation, and stability. Defects in such regulation can lead to a wide range of human diseases from neurological disorders to cancer. Many RBPs are conserved between Caenorhabditis elegans and humans, and several are known to regulate apoptosis in the adult C. elegans germ line. How these RBPs control apoptosis is, however, largely unknown. Here, we identify mina-1(C41G7.3) in a RNA interference-based screen as a novel regulator of apoptosis, which is exclusively expressed in the adult germ line. The absence of MINA-1 causes a dramatic increase in germ cell apoptosis, a reduction in brood size, and an impaired P granules organization and structure. In vivo crosslinking immunoprecipitation experiments revealed that MINA-1 binds a set of mRNAs coding for RBPs associated with germ cell development. Additionally, a system-wide analysis of a mina-1 deletion mutant compared with wild type, including quantitative proteome and transcriptome data, hints to a post-transcriptional regulatory RBP network driven by MINA-1 during germ cell development in C. elegans. In particular, we found that the germline-specific Argonaute WAGO-4 protein levels are increased in mina-1 mutant background. Phenotypic analysis of double mutant mina-1;wago-4 revealed that contemporary loss of MINA-1 and WAGO-4 strongly rescues the phenotypes observed in mina-1 mutant background. To strengthen this functional interaction, we found that upregulation of WAGO-4 in mina-1 mutant animals causes hypersensitivity to exogenous RNAi. Our comprehensive experimental approach allowed us to describe a phenocritical interaction between two RBPs controlling germ cell apoptosis and exogenous RNAi. These findings broaden our understanding of how RBPs can orchestrate different cellular events such as differentiation and death in C. elegans.


Asunto(s)
Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Interferencia de ARN , Animales , Células Germinativas
6.
Nat Cell Biol ; 16(8): 812-20, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-25064737

RESUMEN

Microtubule-targeting chemotherapeutics induce apoptosis in cancer cells by promoting the phosphorylation and degradation of the anti-apoptotic BCL-2 family member MCL1. The signalling cascade linking microtubule disruption to MCL1 degradation remains however to be defined. Here, we establish an in vivo screening strategy in Caenorhabditis elegans to uncover genes involved in chemotherapy-induced apoptosis. Using an RNAi-based screen, we identify three genes required for vincristine-induced apoptosis. We show that the DEP domain protein LET-99 acts upstream of the heterotrimeric G protein alpha subunit GPA-11 to control activation of the stress kinase JNK-1. The human homologue of LET-99, DEPDC1, similarly regulates vincristine-induced cell death by promoting JNK-dependent degradation of the BCL-2 family protein MCL1. Collectively, these data uncover an evolutionarily conserved mediator of anti-tubulin drug-induced apoptosis and suggest that DEPDC1 levels could be an additional determinant for therapy response upstream of MCL1.


Asunto(s)
Apoptosis/efectos de los fármacos , Apoptosis/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Moduladores de Tubulina/farmacología , Animales , Apoptosis/fisiología , Caenorhabditis elegans/citología , Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/genética , Evolución Molecular , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Genes de Helminto/efectos de los fármacos , Células HeLa , Humanos , Sistema de Señalización de MAP Quinasas , Células MCF-7 , Microtúbulos/genética , Microtúbulos/metabolismo , Mutación , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Fosforilación/efectos de los fármacos , Proteolisis/efectos de los fármacos , Interferencia de ARN , Proteínas Represoras/genética , Transducción de Señal/genética , Vincristina/farmacología
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