Loss of ribosomal RNA modification causes developmental defects in zebrafish

Non-coding RNAs (ncRNAs) play key roles in diverse cellular activities, and efficient ncRNA function requires extensive posttranscriptional nucleotide modifications. Small nucleolar RNAs (snoRNAs) are a group of ncRNAs that guide the modification of specific nucleotides in ribosomal RNAs (rRNAs) and small nuclear RNAs. To investigate the physiological relevance of rRNA modification in vertebrates, we suppressed the expression of three snoRNAs (U26, U44 and U78), either by disrupting the host gene splicing or by inhibiting the snoRNA precursor processing, and analyzed the consequences of snoRNA loss-of-function in zebrafish. Using a highly sensitive mass spectrometric analysis, we found that decreased snoRNA expression reduces the snoRNA-guided methylation of the target nucleotides. Impaired rRNA modification, even at a single site, led to severe morphological defects and embryonic lethality in zebrafish, which suggests that rRNA modifications play an essential role in vertebrate development. This study highlights the importance of posttranscriptional modifications and their role in ncRNA function in higher eukaryotes

Loss of Ribosomal Protein L11 Affects Zebrafish Embryonic Development through a p53-Dependent Apoptotic Response

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

Involvement of Protein Kinase D1 in Signal Transduction from the Protein Kinase C Pathway to the Tyrosine Kinase Pathway in Response to Gonadotropin-releasing Hormone

The receptor for gonadotropin-releasing hormone (GnRH) belongs to the G protein-coupled receptors (GPCRs), and its stimulation activates extracellular signal-regulated protein kinase (ERK). We found that the transactivation of ErbB4 was involved in GnRH-induced ERK activation in immortalized GnRH neurons (GT1–7 cells). We found also that GnRH induced the cleavage of ErbB4. In the present study, we examined signal transduction for the activation of ERK and the cleavage of ErbB4 after GnRH treatment. Both ERK activation and ErbB4 cleavage were completely inhibited by YM-254890, an inhibitor of G_ proteins. Down-regulation of protein kinase C (PKC) markedly decreased both ERK activation and ErbB4 cleavage. Experiments with two types of PKC inhibitors, Gö 6976 and bisindolylmaleimide I, indicated that novel PKC isoforms but not conventional PKC isoforms were involved in ERK activation and ErbB4 cleavage. Our experiments indicated that the novel PKC isoforms activated protein kinase D (PKD) after GnRH treatment. Knockdown and inhibitor experiments suggested that PKD1 stimulated the phosphorylation of Pyk2 by constitutively activated Src and Fyn for ERK activation. Taken together, it is highly possible that PKD1 plays a critical role in signal transduction from the PKC pathway to the tyrosine kinase pathway. Activation of the tyrosine kinase pathway may be involved in the progression of cancer

The Human Ribosomal Protein Genes: Sequencing and Comparative Analysis of 73 Genes

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 ± 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. [The sequence data described in this paper have been submitted to the DDBJ/EMBL/GenBank databases under accession nos. AB055762–AB055780, AB056456, AB061820–AB061859, AB062066–AB062071, and AB070559.