RESUMEN
Members of the ELAV family of proteins contain three RNA recognition motifs (RRMs), which are highly conserved. ELAV, a Drosophila melanogaster member of this family, provides a vital function and exhibits a predominantly nuclear localization. To investigate if the RNA-binding property of each of the ELAV RRMs is required for ELAV's in vivo function, amino acid residues critical in RNA binding for each RRM were individually mutated. A stringent genetic complementation test revealed that when the mutant protein was the sole source of ELAV, RNA-binding ability of each RRM was essential to ELAV function. To assess the degree to which each domain was specific for ELAV function and which domains perhaps performed a function common to related ELAV proteins, we substituted an ELAV RRM with the corresponding RRM from RBP9, the D. melanogaster protein most homologous to ELAV; HuD, a human ELAV family protein; and SXL, which, although evolutionarily related, is not an ELAV family member. This analysis revealed that RRM3 replacements were fully functional, but RRM1 and RRM2 replacements were largely nonfunctional. Under less stringent conditions RRM1 and RRM2 replacements from SXL and RRM1 replacement from RBP9 were able to provide supplemental function in the presence of a mutant hypomorphic ELAV protein.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster/genética , Hormonas de Insectos/genética , Mutación , Proteínas de Unión al ARN , ARN/metabolismo , Ribonucleoproteínas/genética , Proteínas Ribosómicas/genética , Proteínas de Saccharomyces cerevisiae , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Aminoácidos/química , Animales , Drosophila melanogaster/química , Proteínas ELAV , Prueba de Complementación Genética , Immunoblotting , Inmunohistoquímica , Datos de Secuencia Molecular , Mutagénesis , Proteínas del Tejido Nervioso/genética , Plásmidos/genética , Reacción en Cadena de la Polimerasa , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ribonucleoproteínas/química , Ribonucleoproteínas/fisiología , Ribonucleósido Difosfato Reductasa , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Transgenes , Proteínas Supresoras de TumorRESUMEN
The neuron specific Drosophila ELAV protein belongs to the ELAV family of RNA binding proteins which are characterized by three highly conserved RNA recognition motifs, an N-terminal domain, and a hinge region between the second and third RNA recognition motifs. Despite their highly conserved RNA recognition motifs the ELAV family members are a group of proteins with diverse posttranscriptional functions including splicing regulation, mRNA stability and translatability and have a variety of subcellular localizations. The role of the ELAV hinge in localization and function was examined using transgenes encoding ELAV hinge deletions, in vivo. Subcellular localization of the hinge mutant proteins revealed that residues between amino acids 333-374 are necessary for nuclear localization. This delineated sequence has no significant homology to classical nuclear localization sequences, but it is similar to the recently characterized nucleocytoplasmic shuttling sequence, the HNS, from a human ELAV family member, HuR. This defined sequence, however, was insufficient for nuclear localization as tested using hinge-GFP fusion proteins. Functional assays revealed that mutant proteins that fail to localize to the nucleus are unable to provide ELAV vital function, but their function is significantly restored when translocated into the nucleus by a heterologous nuclear localization sequence tag.
Asunto(s)
Núcleo Celular/metabolismo , Drosophila melanogaster/metabolismo , Ribonucleoproteínas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN , Proteínas ELAV , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Mutagénesis Sitio-Dirigida , Ribonucleoproteínas/genética , Ribonucleoproteínas/fisiología , Eliminación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The subcellular distribution of the Drosophila nervous system-specific RNA binding domain-containing protein ELAV was investigated using ELAV-specific antibodies and scanning confocal laser microscopy. ELAV is predominantly localized within the nucleus where it concentrates within discrete domains we describe as dots and webs. To characterize these discrete domains an analysis of Drosophila coiled bodies was initiated. The polyclonal antibody R288 raised against human coilin was used to identify coiled bodies in cells of the Drosophila larval central nervous system. Double-labeling immunohistochemistry showed that, similar to vertebrate and plant systems, small nuclear ribonucleoproteins are enriched within these structures. Further analysis of ELAV revealed that subnuclear domains enriched with this molecule localize within and close to coiled bodies and close to subnuclear domains enriched with splicing factors. A preliminary analysis aimed at defining a region within ELAV that may mediate a molecular or functional interaction important for its subnuclear localization revealed that deletion of the ELAV alanine/glutamine-rich amino-terminal auxiliary domain has no discernible effect on localization and that proteins produced from elav lethal alleles distribute normally.
Asunto(s)
Núcleo Celular/fisiología , Drosophila melanogaster/fisiología , Neuronas/metabolismo , Ribonucleoproteínas/metabolismo , Animales , Sitios de Unión , Núcleo Celular/ultraestructura , Sistema Nervioso Central/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Proteínas ELAV , Genes Letales , Humanos , Larva , Ratones , Mutación , Proteínas Nucleares/inmunología , Proteínas Nucleares/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/inmunología , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Fracciones SubcelularesRESUMEN
We report here that disruption of a recently discovered kinesin-like protein in Drosophila melanogaster, KLP61F, results in a mitotic mutation lethal to the organism. We show that in the absence of KLP61F function, spindle poles fail to separate, resulting in the formation of monopolar mitotic spindles. The resulting phenotype of metaphase arrest with polyploid cells is reminiscent of that seen in the fungal bimC and cut7 mutations, where it has also been shown that spindle pole bodies are not segregated. KLP61F is specifically expressed in proliferating tissues during embryonic and larval development, consistent with a primary role in cell division. The structural and functional homology of the KLP61F, bimC, cut7, and Eg5 kinesin-like proteins demonstrates the existence of a conserved family of kinesin-like molecules important for spindle pole separation and mitotic spindle dynamics.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster/citología , Cinesinas/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Mitosis/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Embrión no Mamífero/citología , Genes Fúngicos , Genes Letales , Cinesinas/genética , Larva , Metafase , Proteínas Asociadas a Microtúbulos/química , Datos de Secuencia Molecular , Mutagénesis Insercional , Estructura Secundaria de Proteína , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Huso Acromático/ultraestructuraRESUMEN
We isolated a cDNA for p20K, a secreted protein preferentially synthesized in nonproliferating cells. p20K mRNA and protein levels declined rapidly following treatment with various mitogens. DNA sequence analysis of the p20K cDNA predicted a novel protein distantly related to alpha 2 mu-globulin and plasma retinol-binding protein.
Asunto(s)
Proteínas Sanguíneas/genética , Factor de Crecimiento Epidérmico/farmacología , Insulina/farmacología , Proteínas de los Retroviridae/farmacología , Secuencia de Aminoácidos , Animales , Proteínas Aviares , Secuencia de Bases , Proteínas Sanguíneas/metabolismo , Células Cultivadas , ADN/genética , Regulación de la Expresión Génica , Interfase , Lipocalinas , Datos de Secuencia Molecular , Proteína Oncogénica pp60(v-src) , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Chicken embryo fibroblasts (CEF) infected with a temperature-sensitive Rous sarcoma virus (RSV) mutant, tsNY72-4, express a set of pp60v-src-induced RNAs soon after shift to the permissive temperature. By subtractive and differential screening, we have cloned 12 of these sequences, 2 of which were c-fos and krox-24. Serum induced all the v-src-inducible genes tested, suggesting that these genes serve roles in normal cell division and are not specific to transformation per se. Significantly, however, v-src produced prolonged, and in some cases kinetically complex, patterns of induction compared to serum. For most of the clones, phorbol 12-tetradecanoate 13-acetate (TPA) induced mRNAs with kinetics similar to that of serum. However, one clone (CEF-4) was expressed in a biphasic manner. Another (CEF-10) was repressed by TPA at 1 hr, after which this mRNA was permanently induced. The pattern of repression-induction of CEF-10 mRNA is the inverse of protein kinase C (PKC) activity in the cell, suggesting that PKC actively represses this gene. In vivo expression of CEF-10 mRNA is restricted predominantly to the lung. A full-length CEF-10 cDNA encodes a 41-kDa protein that has an amino-terminal signal peptide for secretion, contains a markedly high number of cysteine residues, and shows no sequence similarity to known proteins.