RESUMEN
Recent studies indicate that the protein affected in spinal muscular atrophy, SMN, plays a role in the assembly of a number of macromolecular complexes that function in the nucleus, interacting with its partner proteins via their arginine- and glycine-rich domains.
Asunto(s)
Atrofia Muscular Espinal/etiología , Proteínas del Tejido Nervioso/metabolismo , Cuerpos Enrollados/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Unión Proteica , Empalme del ARN , Proteínas de Unión al ARN , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Ribosomas/metabolismo , Proteínas del Complejo SMN , Empalmosomas/metabolismo , Transcripción GenéticaRESUMEN
Disruption of the survival motor neuron (SMN) gene leads to selective loss of spinal motor neurons, resulting in the fatal human neurodegenerative disorder spinal muscular atrophy (SMA). SMN has been shown to function in spliceosomal small nuclear ribonucleoprotein (snRNP) biogenesis and pre-mRNA splicing. We have demonstrated that SMN also interacts with fibrillarin, a highly conserved nucleolar protein that is associated with all Box C/D small nucleolar RNAs and functions in processing and modification of rRNA. Fibrillarin and SMN co-immunoprecipitate from HeLa cell extracts indicating that the proteins exist as a complex in vivo. Furthermore, in vitro binding studies indicate that the interaction between SMN and fibrillarin is direct and salt-stable. We show that the glycine/arginine-rich domain of fibrillarin is necessary and sufficient for SMN binding and that the region of SMN encoded by exon 3, including the Tudor domain, mediates the binding of fibrillarin. Tudor domain missense mutations, including one found in an SMA patient, impair the interaction between SMN and fibrillarin (as well as the common snRNP protein SmB). Our results suggest a function for SMN in small nucleolar RNP biogenesis (akin to its known role as an snRNP assembly factor) and reveal a potential link between small nucleolar RNP biogenesis and SMA.
Asunto(s)
Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Secuencia de Aminoácidos , Animales , Arginina/química , Nucléolo Celular/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , ADN Complementario/metabolismo , Exones , Biblioteca de Genes , Glicina/química , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutación Missense , Proteínas del Tejido Nervioso/genética , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Proteínas de Unión al ARN , Proteínas del Complejo SMN , Técnicas del Sistema de Dos Híbridos , XenopusRESUMEN
U2 small nuclear (sn)RNA contains a large number of posttranscriptionally modified nucleotides, including a 5' trimethylated guanosine cap, 13 pseudouridines, and 10 2'-O-methylated residues. Using Xenopus oocytes, we demonstrated previously that at least some of these modified nucleotides are essential for biogenesis of a functional snRNP. Here we address the subcellular site of U2 internal modification. Upon injection into the cytoplasm of oocytes, G-capped U2 that is transported to the nucleus becomes modified, whereas A-capped U2 that remains in the cytoplasm is not modified. Furthermore, by injecting U2 RNA into isolated nuclei or enucleated oocytes, we observe that U2 internal modifications occur exclusively in the nucleus. Analysis of the intranuclear localization of fluorescently labeled RNAs shows that injected wild-type U2 becomes localized to nucleoli and Cajal bodies. Both internal modification and nucleolar localization of U2 are dependent on the Sm binding site. An Sm-mutant U2 is targeted only to Cajal bodies. The Sm binding site can be replaced by a nucleolar localization signal derived from small nucleolar RNAs (the box C/D motif), resulting in rescue of internal modification as well as nucleolar localization. Analysis of additional chimeric U2 RNAs reveals a correlation between internal modification and nucleolar localization. Together, our results suggest that U2 internal modification occurs within the nucleolus.
Asunto(s)
Autoantígenos/metabolismo , Núcleo Celular/metabolismo , Oocitos/fisiología , Procesamiento Postranscripcional del ARN , ARN Nuclear Pequeño/metabolismo , Ribonucleoproteínas Nucleares Pequeñas , Transporte Activo de Núcleo Celular , Animales , Autoantígenos/genética , Autorradiografía , Microinyecciones , Conformación de Ácido Nucleico , Oocitos/citología , Unión Proteica , ARN Nuclear Pequeño/química , ARN Nuclear Pequeño/genética , Xenopus laevis , Proteínas Nucleares snRNPRESUMEN
Telomerase RNA is an essential component of the ribonucleoprotein enzyme involved in telomere length maintenance, a process implicated in cellular senescence and cancer. Vertebrate telomerase RNAs contain a box H/ACA snoRNA motif that is not required for telomerase activity in vitro but is essential in vivo. Using the Xenopus oocyte system, we have found that the box H/ACA motif functions in the subcellular localization of telomerase RNA. We have characterized the transport and biogenesis of telomerase RNA by injecting labeled wild-type and variant RNAs into Xenopus oocytes and assaying nucleocytoplasmic distribution, intranuclear localization, modification, and protein binding. Although yeast telomerase RNA shares characteristics of spliceosomal snRNAs, we show that human telomerase RNA is not associated with Sm proteins or efficiently imported into the nucleus. In contrast, the transport properties of vertebrate telomerase RNA resemble those of snoRNAs; telomerase RNA is retained in the nucleus and targeted to nucleoli. Furthermore, both nuclear retention and nucleolar localization depend on the box H/ACA motif. Our findings suggest that the H/ACA motif confers functional localization of vertebrate telomerase RNAs to the nucleus, the compartment where telomeres are synthesized. We have also found that telomerase RNA localizes to Cajal bodies, intranuclear structures where it is thought that assembly of various cellular RNPs takes place. Our results identify the Cajal body as a potential site of telomerase RNP biogenesis.
Asunto(s)
Núcleo Celular/metabolismo , ARN Nucleolar Pequeño/metabolismo , ARN/metabolismo , Telomerasa/metabolismo , Transporte Activo de Núcleo Celular , Animales , Compartimento Celular , Nucléolo Celular/metabolismo , Cuerpos Enrollados/metabolismo , Humanos , Modelos Moleculares , Conformación de Ácido Nucleico , XenopusRESUMEN
The 5'-cap structure of most spliceosomal small nuclear RNAs (snRNAs) and certain small nucleolar RNAs (snoRNAs) undergoes hypermethylation from a 7-methylguanosine to a 2,2, 7-trimethylguanosine structure. 5'-Cap hypermethylation of snRNAs is dependent upon a conserved sequence element known as the Sm site common to most snRNAs. Here we have performed a mutational analysis of U3 and U14 to determine the cis-acting sequences required for 5'-cap hypermethylation of Box C/D snoRNAs. We have found that both the conserved sequence elements Box C (termed C' in U3) and Box D are necessary for cap hypermethylation. Furthermore, the terminal stem structure that is formed by sequences that flank Box C (C' in U3) and Box D is also required. However, mutation of other conserved sequences has no effect on hypermethylation of the cap. Finally, the analysis of fragments of U3 and U14 RNAs indicates that the Box C/D motif, including Box C (C' in U3), Box D and the terminal stem, is capable of directing cap hypermethylation. Thus, the Box C/D motif, which is important for snoRNA processing, stability, nuclear retention, protein binding, nucleolar localization and function, is also necessary and sufficient for cap hypermethylation of these RNAs.
Asunto(s)
Metilación de ADN , Caperuzas de ARN/metabolismo , ARN Nucleolar Pequeño/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Animales , Núcleo Celular/metabolismo , Femenino , Modelos Moleculares , Mutación , Oocitos , Caperuzas de ARN/genética , Estabilidad del ARN , ARN Nucleolar Pequeño/química , ARN Nucleolar Pequeño/genética , XenopusRESUMEN
U3 small nucleolar RNA (snoRNA) is a member of the Box C/D family of snoRNAs which functions in ribosomal RNA processing. U3-55k is a protein that has been found to interact with U3 but not other members of the Box C/D snoRNA family. We have found that interaction of the U3-55k protein with U3 RNA in vivo is mediated by the conserved Box B/C motif which is unique to U3 snoRNA. Mutation of Box B and Box C, but not of other conserved sequence elements, disrupted interaction of U3-55k with U3 RNA. Furthermore, a fragment of U3 containing only these two conserved elements was bound by U3-55k in vivo. RNA binding assays performed in vitro indicate that Box C may be the primary determinant of the interaction. We have cloned the cDNA encoding the Xenopus laevis U3-55k protein and find strong homology to the human sequence, including six WD repeats. Deletion of WD repeats or sequences near the C-terminus of U3-55k resulted in loss of association with U3 RNA and also loss of localization of U3-55k to the nucleolus, suggesting that protein-protein interactions contribute to the localization and RNA binding of U3-55k in vivo.
Asunto(s)
ARN Nucleolar Pequeño/metabolismo , Ribonucleoproteínas Nucleolares Pequeñas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Clonación Molecular , Humanos , Datos de Secuencia Molecular , Unión Proteica , ARN Nucleolar Pequeño/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Ribonucleoproteínas Nucleolares Pequeñas/química , Homología de Secuencia de Aminoácido , Xenopus laevisRESUMEN
The two major families of small nucleolar RNAs (snoRNAs), Box C/D and Box H/ACA, are generated in the nucleoplasm and transported to the nucleolus where they function in rRNA processing and modification. We have investigated the sequences involved in the intranuclear transport of Box H/ACA snoRNAs by assaying the localization of injected fluorescent RNAs in Xenopus oocyte nuclear spreads. Our analysis of U17, U64 and U65 has revealed that disruption of either of the conserved sequence elements, Box H or Box ACA, eliminates nucleolar localization. In addition, the stem present at the base of the 3' hairpin is required for efficient nucleolar localization of U65. Fragments or rearrangements of U65 that consist of Box H and Box ACA flanking either the 5' or 3' hairpin are targeted to the nucleolus. The targeting is dependent on the presence of the Box sequences, but not on their orientation. Our results indicate that in each of the two major families of snoRNAs, a motif composed of the signature conserved sequences and an adjacent structural element that tethers the sequence elements directs the nucleolar localization of the RNAs. We demonstrate that telomerase RNA is also targeted to the nucleolus by a Box ACA-dependent mechanism.
Asunto(s)
ARN Nucleolar Pequeño/genética , ARN Nucleolar Pequeño/metabolismo , Animales , Secuencia de Bases , Nucléolo Celular/metabolismo , Secuencia Conservada , Femenino , Humanos , Técnicas In Vitro , Datos de Secuencia Molecular , Mutación , Señales de Localización Nuclear/genética , Conformación de Ácido Nucleico , Oocitos/metabolismo , ARN Nucleolar Pequeño/química , Telomerasa/genética , XenopusRESUMEN
To identify genomic regions required for establishment and patterning of the epidermis, we screened 58 deficiencies that collectively delete at least approximately 67% of the Caenorhabditis elegans genome. The epidermal pattern of deficiency homozygous embryos was analyzed by examining expression of a marker specific for one of the three major epidermal cell types, the seam cells. The organization of the epidermis and internal organs was also analyzed using a monoclonal antibody specific for epithelial adherens junctions. While seven deficiencies had no apparent effect on seam cell production, 21 were found to result in subnormal, and five in excess numbers of these cells. An additional 23 deficiencies blocked expression of the seam cell marker, in some cases without preventing cell proliferation. Two deficiencies result in multinucleate seam cells. Deficiencies were also identified that result in subnormal numbers of epidermal cells, hyperfusion of epidermal cells into a large syncytium, or aberrant epidermal differentiation. Finally, analysis of internal epithelia revealed deficiencies that cause defects in formation of internal organs, including circularization of the intestine and bifurcation of the pharynx lumen. This study reveals that many regions of the C. elegans genome are required zygotically for patterning of the epidermis and other epithelia.
Asunto(s)
Tipificación del Cuerpo , Caenorhabditis elegans/genética , Epidermis/embriología , Cigoto , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/embriología , Diferenciación Celular , Células Epidérmicas , Genes Letales , Homocigoto , Faringe/embriología , FenotipoRESUMEN
In the early Caenorhabditis elegans embryo most of the ectoderm arises from the AB blastomere, one of the six founder cells. We report that nonequivalent blastomeres are generated at the third division round in the AB lineage. Each AB granddaughter divides to produce one cell that has the potential to make abundant epidermis and one that instead produces primarily nervous system. This unequal distribution of the potential to make epidermis occurs in an AB granddaughter that is isolated by laser-ablation of all other cells or during the development of an isolated AB blastomere in culture. The fidelity of this event is normally masked by a signal from the MS founder cell, which induces mesoderm in particular AB descendants. When MS induction is prevented by laser cell-ablation or by a mutation in the glp-1 gene, the epidermal fate map of the AB great granddaughters becomes left-right symmetrical. Cell lineage analyses demonstrate that, in fact, the AB lineage becomes entirely left-right symmetrical in the absence of MS induction. This accounts for the extra epidermal cells previously observed in a glp-1 mutant. Our results suggest that epidermal differentiation in the nematode may be controlled by a cell-autonomous mechanism that differentially allocates epidermal potential during AB development and that MS induction generates the left-right asymmetry in the fates of AB descendants in part by overriding this potential.