The critical role of protein arginine methyltransferase prmt8 in zebrafish embryonic and neural development is non-redundant with its paralogue prmt1.

Protein arginine methyltransferase (PRMT) 1 is the most conserved and widely distributed PRMT in eukaryotes. PRMT8 is a vertebrate-restricted paralogue of PRMT1 with an extra N-terminal sequence and brain-specific expression. We use zebrafish (Danio rerio) as a vertebrate model to study PRMT8 function and putative redundancy with PRMT1. The transcripts of zebrafish prmt8 were specifically expressed in adult zebrafish brain and ubiquitously expressed from zygotic to early segmentation stage before the neuronal development. Whole-mount in situ hybridization revealed ubiquitous prmt8 expression pattern during early embryonic stages, similar to that of prmt1. Knockdown of prmt8 with antisense morpholino oligonucleotide phenocopied prmt1-knockdown, with convergence/extension defects at gastrulation. Other abnormalities observed later include short body axis, curled tails, small and malformed brain and eyes. Catalytically inactive prmt8 failed to complement the morphants, indicating the importance of methyltransferase activity. Full-length prmt8 but not prmt1 cRNA can rescue the phenotypic changes. Nevertheless, cRNA encoding Prmt1 fused with the N-terminus of Prmt8 can rescue the prmt8 morphants. In contrast, N-terminus- deleted but not full-length prmt8 cRNA can rescue the prmt1 morphants as efficiently as prmt1 cRNA. Abnormal brain morphologies illustrated with brain markers and loss of fluorescent neurons in a transgenic fish upon prmt8 knockdown confirm the critical roles of prmt8 in neural development. In summery, our study is the first report showing the expression and function of prmt8 in early zebrafish embryogenesis. Our results indicate that prmt8 may play important roles non-overlapping with prmt1 in embryonic and neural development depending on its specific N-terminus.

The predominant protein arginine methyltransferase PRMT1 is critical for zebrafish convergence and extension during gastrulation.

Protein arginine methyltransferase (PRMT)1 is the predominant type I methyltransferase in mammals. In the present study, we used zebrafish (Danio rerio) as the model system to elucidate PRMT1 expression and function during embryogenesis. Zebrafish prmt1 transcripts were detected from the zygote period to the early larva stage. Knockdown of prmt1 by antisense morpholino oligo (AMO) resulted in delayed growth, shortened body-length, curled tails and cardiac edema. PRMT1 protein level, type I protein arginine methyltransferase activity, specific asymmetric protein arginine methylation and histone H4 R3 methylation all decreased in the AMO-injected morphants. The morphants showed defective convergence and extension and the abnormalities were more severe at the posterior than the anterior parts. Cell migration defects suggested by the phenotypes were not only observed in the morphant embryos, but also in a cellular prmt1 small-interfering RNA knockdown model. Rescue of the phenotypes by co-injection of wild-type but not catalytic defective prmt1 mRNA confirmed the specificity of the AMO and the requirement of methyltransferase activity in early development. The results obtained in the present study demonstrate a direct link of early development with protein arginine methylation catalyzed by PRMT1.

Low RET mutation frequency and polymorphism analysis of the RET and EDNRB genes in patients with Hirschsprung disease in Taiwan.

Hirschsprung disease (HSCR), or congenital intestinal aganglionosis, is a relatively common disorder characterized by the absence of ganglion cells in the nerve plexuses of the lower digestive tract, resulting in intestinal obstruction in neonates. Mutations in genes of the RET receptor tyrosine kinase and endothelin receptor B (EDNRB) signaling pathways have been shown to be associated in HSCR patients. In this study, we collected genomic DNA samples from 55 HSCR patients in central Taiwan and analyzed the coding regions of the RET and EDNRB genes by PCR amplification and DNA sequencing. In the 55 patients, an A to G transition was detected in two (identical twin brothers). The mutation was at the end of RET exon 19 at codon 1062 (Y1062C), a reported critical site for the signaling pathways. Single nucleotide polymorphisms (SNP) in exons 2, 7, 11, 13, and 15 of RET and exon 4 of EDNRB in the HSCR patients or controls were detected. The differences between patients and controls in allele distribution of the five RET polymorphic sites were statistically significant. The most frequent genotype encompassing exons 2 and 13 SNPs (the polymorphic sites with the highest percentage of heterozygotes) was AA/GG in patients, which was different from the AG/GT in the normal controls. Transmission disequilibrium was observed in exons 2, 7, and 13, indicating nonrandom association of the susceptibility alleles with the disease in the patients. This study represents the first comprehensive genetic analysis of HSCR disease in Taiwan.

Identification and phylogenetic analyses of the protein arginine methyltransferase gene family in fish and ascidians.

Protein arginine methyltransferases (PRMT) involved in the regulations of signal transduction, protein subcellular localization, and transcription have been mostly studied in mammals and yeast. In this study orthologues of eight human PRMT genes (PRMT1-7 and HRMT1L3) were identified in both puffer fish Fugu rubripes and zebrafish Danio rerio. The fish PRMT genes appear to be conserved with their mammalian orthologues at the levels of amino acid sequences as well as genomic structures. All vertebrate PRMT genes contain 10-16 coding exons except PRMT6 that contains only one coding exon. Western blot analyses of zebrafish tissue extracts confirmed the expression of some PRMT proteins in zebra fish. We further identified six PRMT members (PRMT1, 3-7) in an invertebrate chordate Ciona intestinalis. Genomic structures of the PRMT orthologues are no more conserved in the ascidians, as PRMT3 and PRMT5 contain only one coding exon while PRMT6 contains six exons. PRMT2 and HRMT1L3 that are missing in Ciona appear to be vertebrate-specific. HRMT1L3 is a PRMT1 paralogue with highly conserved sequences and exact exon junctions, whereas the PRMT2 orthologues are very diverged. Different PRMT orthologues are likely to evolve at different rates and the PRMT1 orthologues appear to be most conserved through evolution. Furthermore, phylogenetic analyses using the core regions of various PRMT genes show that PRMT5 with the type II PRMT activity is separated in one branch. All other PRMT genes including PRMT1, 2, 3, 4, 6, 7 and HRMT1L3 clustered in the other branch, probably represent the genes for the type I activity.