RESUMO
CeR-2 RNA is one of the newly identified Caenorhabditis elegans noncoding RNAs (ncRNAs). The characterization of CeR-2 by RNomic studies has failed to classify it into any known ncRNA family. In this study, we examined the spatiotemporal expression patterns of CeR-2 to gain insight into its function. CeR-2 is expressed in most cells from the early embryo to adult stages. The subcellular localization of this RNA is analogous to that of fibrillarin, a major protein of the nucleolus. It was observed that knockdown of C/D small nucleolar ribonucleoproteins (snoRNPs), but not of H/ACA snoRNPs, resulted in the aberrant nucleolar localization of CeR-2 RNA. A mutant worm with a reduced amount of cellular CeR-2 RNA showed changes in its pre-rRNA processing pattern compared with that of the wild-type strain N2. These results suggest that CeR-2 RNA is a C/D snoRNA involved in the processing of rRNAs.
Assuntos
Caenorhabditis elegans/genética , Processamento Pós-Transcricional do RNA , RNA Ribossômico/metabolismo , RNA Nucleolar Pequeno/metabolismo , Animais , Sequência de Bases , Caenorhabditis elegans/metabolismo , Dados de Sequência Molecular , Mutação , Precursores de RNA/metabolismo , RNA Nucleolar Pequeno/química , RNA Nucleolar Pequeno/isolamento & purificação , Ribonucleoproteínas Nucleolares Pequenas/genética , Alinhamento de SequênciaRESUMO
U3 small nucleolar RNA (snoRNA) is one of the members of the box C/D class of snoRNA and is essential for ribosomal RNA (rRNA) processing to generate 18S rRNA in the nucleolus. Although U3 snoRNA is abundant, and is well conserved from yeast to mammals, the genes encoding U3 snoRNA in C. elegans have long remained unidentified. A recent RNomics study in C. elegans predicted five distinct U3 snoRNA genes. However, characterization of these candidates for U3 snoRNA has yet to be performed. In this study, we isolated and characterized four candidate RNAs for U3 snoRNA from the immunoprecipitated RNAs of C. elegans using an antibody against the 2,2,7-trimethylguanosine (TMG) cap. The sequences were identical to the predicted U3 sequences in the RNomics study. Here, we show the several lines of evidence that the isolated RNAs are the true U3 snoRNAs of C. elegans. Moreover, we report the novel expression pattern of U3 snoRNA and fibrillarin, which is an essential component of U3 small nucleolar ribonucleoprotein complex, during early embryo development of C. elegans. To our knowledge, this is the first observation of the inconsistent localization U3 snoRNA and fibrillarin during early embryogenesis, providing novel insight into the mechanisms of nucleologenesis and ribosome production during early embryogenesis.
Assuntos
Proteínas de Caenorhabditis elegans/análise , Caenorhabditis elegans/embriologia , Proteínas Cromossômicas não Histona/análise , RNA Nucleolar Pequeno/análise , RNA Nucleolar Pequeno/química , Animais , Sequência de Bases , Caenorhabditis elegans/química , Caenorhabditis elegans/genética , Embrião não Mamífero/química , Dados de Sequência Molecular , RNA de Helmintos/análise , RNA de Helmintos/química , RNA de Helmintos/isolamento & purificação , RNA Nucleolar Pequeno/isolamento & purificaçãoRESUMO
C. elegans small RNAs (<50 nt) were separated by two-dimensional gel electrophoresis (2D-PAGE). cDNAs were prepared from the RNAs extracted from randomly chosen 2D-PAGE spots. Although many cDNA sequences corresponded to parts of known RNAs, twelve novel small RNA candidates were identified: eleven from 2D-PAGE spots of the mixed-stage worm RNA preparation and one from those of the embryonic RNA preparation. These are encoded in the intergenic regions, in the introns of protein-coding genes, in the anti-sense strand of protein-coding sequences and repetitive sequence regions of the genome. None of them showed a characteristic structure of miRNAs, suggesting that they are candidates of other or new classes of RNAs.