RESUMEN
Ribosome is responsible for protein synthesis in all organisms and ribosomal proteins (RPs) play important roles in the formation of a functional ribosome. L11 was recently shown to regulate p53 activity through a direct binding with MDM2 and abrogating the MDM2-induced p53 degradation in response to ribosomal stress. However, the studies were performed in cell lines and the significance of this tumor suppressor function of L11 has yet to be explored in animal models. To investigate the effects of the deletion of L11 and its physiological relevance to p53 activity, we knocked down the rpl11 gene in zebrafish and analyzed the p53 response. Contrary to the cell line-based results, our data indicate that an L11 deficiency in a model organism activates the p53 pathway. The L11-deficient embryos (morphants) displayed developmental abnormalities primarily in the brain, leading to embryonic lethality within 6-7 days post fertilization. Extensive apoptosis was observed in the head region of the morphants, thus correlating the morphological defects with apparent cell death. A decrease in total abundance of genes involved in neural patterning of the brain was observed in the morphants, suggesting a reduction in neural progenitor cells. Upregulation of the genes involved in the p53 pathway were observed in the morphants. Simultaneous knockdown of the p53 gene rescued the developmental defects and apoptosis in the morphants. These results suggest that ribosomal dysfunction due to the loss of L11 activates a p53-dependent checkpoint response to prevent improper embryonic development.
Asunto(s)
Apoptosis/genética , Proteínas Ribosómicas/genética , Proteína p53 Supresora de Tumor/genética , Proteínas de Pez Cebra/genética , Pez Cebra/embriología , Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Embrión no Mamífero/metabolismo , Desarrollo Embrionario/genética , Neuronas/metabolismo , ARN Mensajero/metabolismo , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
Ribosomes are responsible for protein synthesis in all cells. Ribosomal protein S19 (RPS19) is one of the 79 ribosomal proteins (RPs) in vertebrates. Heterozygous mutations in RPS19 have been identified in 25% of patients with Diamond-Blackfan anemia (DBA), but the relationship between RPS19 mutations and the pure red-cell aplasia of DBA is unclear. In this study, we developed an RPS19-deficient zebrafish by knocking down rps19 using a Morpholino antisense oligo. The RPS19-deficient animals showed a dramatic decrease in blood cells as well as deformities in the head and tail regions at early developmental stages. These phenotypes were rescued by injection of zebrafish rps19 mRNA, but not by injection of rps19 mRNAs with mutations that have been identified in DBA patients. Our results indicate that rps19 is essential for hematopoietic differentiation during early embryogenesis. The effects were specific to rps19, but knocking down the genes for three other RPs, rpl35, rpl35a and rplp2, produced similar phenotypes, suggesting that these genes might have a common function in zebrafish erythropoiesis. The RPS19-deficient zebrafish will provide a valuable tool for investigating the molecular mechanisms of DBA development in humans.
Asunto(s)
Anemia de Diamond-Blackfan/sangre , Anemia de Diamond-Blackfan/embriología , Proteínas Ribosómicas/deficiencia , Proteínas de Pez Cebra/deficiencia , Proteínas de Pez Cebra/genética , Pez Cebra/sangre , Pez Cebra/embriología , Anemia de Diamond-Blackfan/genética , Animales , Secuencia de Bases , Cartilla de ADN/genética , Modelos Animales de Enfermedad , Eritropoyesis/genética , Marcación de Gen , Humanos , Mutación , Fenotipo , ARN sin Sentido/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteínas Ribosómicas/sangre , Proteínas Ribosómicas/genética , Pez Cebra/genética , Proteínas de Pez Cebra/sangreRESUMEN
Many nucleotides in rRNAs are modified. We devised a method to locate 2(')-O-methyl nucleotide residues using a conventional DNA sequencer. We found 38 2(')-O-methyl nucleotides in the 26S rRNA of Caenorhabditis elegans using this method. Fourteen of the 38 residues are conserved in both human and yeast rRNAs and 14 residues are conserved in either human or yeast rRNA. The remaining 10 nucleotides are uniquely methylated in C. elegans 26S rRNA. We searched the C. elegans genomic sequence for small nucleolar RNAs (snoRNAs), which guide the methylation of ribose residues, and predicted 18 snoRNA sequences that are expected to guide the methylation of some of these nucleotide residues.
Asunto(s)
ARN Ribosómico/química , ARN Nucleolar Pequeño/química , Animales , Secuencia de Bases , Caenorhabditis elegans , Metilación de ADN , Cartilla de ADN/farmacología , ADN Complementario/metabolismo , Exones , Genes Fúngicos , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Nucleótidos/química , ARN/metabolismo , ARN Ribosómico/genética , ARN Nucleolar Pequeño/metabolismo , Ribosa/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The ribosome, as a catalyst for protein synthesis, is universal and essential for all organisms. Here we describe the structure of the genes encoding human ribosomal proteins (RPs) and compare this class of genes among several eukaryotes. Using genomic and full-length cDNA sequences, we characterized 73 RP genes and found that (1) transcription starts at a C residue within a characteristic oligopyrimidine tract; (2) the promoter region is GC rich, but often has a TATA box or similar sequence element; (3) the genes are small (4.4 kb), but have as many as 5.6 exons on average; (4) the initiator ATG is in the first or second exon and is within plus minus 5 bp of the first intron boundaries in about half of cases; and (5) 5'- and 3'-UTRs are significantly smaller (42 bp and 56 bp, respectively) than the genome average. Comparison of RP genes from humans, Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae revealed the coding sequences to be highly conserved (63% homology on average), although gene size and the number of exons vary. The positions of the introns are also conserved among these species as follows: 44% of human introns are present at the same position in either D. melanogaster or C. elegans, suggesting RP genes are highly suitable for studying the evolution of introns.