Hen1 is required for oocyte development and piRNA stability in zebrafish
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PROVIDER: S-EPMC3252586 | biostudies-literature | 2012 Jan
REPOSITORIES: biostudies-literature
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Project description:Piwi-interacting RNAs (piRNAs) are germ line-specific small RNA molecules that have a function in genome defence and germ cell development. They associate with a specific class of Argonaute proteins, named Piwi, and function through an RNA interference-like mechanism. piRNAs carry a 2'-O-methyl modification at their 3' end, which is added by the Hen1 enzyme. We show that zebrafish hen1 is specifically expressed in germ cells and is essential for maintaining a female germ line, whereas it is dispensable in the testis. Hen1 protein localizes to nuage through its C-terminal domain, but is not required for nuage formation. In hen1 mutant testes, piRNAs become uridylated and adenylated. Uridylation frequency is highest on retro-transposon-derived piRNAs and is accompanied by decreased piRNA levels and mild derepression of transposon transcripts. Altogether, our data suggest the existence of a uridylation-mediated 3'-5' exonuclease activity acting on piRNAs in zebrafish germ cells, which is counteracted by nuage-bound Hen1 protein. This system discriminates between piRNA targets and is required for ovary development and fully efficient transposon silencing.
| S-EPMC2982757 | biostudies-literature
Project description:Small non-coding RNAs (sRNAs) such as microRNAs (miRNAs), small interfering RNAs (siRNAs) and piwi-interacting RNAs (piRNAs) regulate the levels of endogenous, viral and transposable element RNA in plants (excluding piRNAs) and animals. These pathways are explored mainly in bilaterian animals, such as vertebrates, arthropods and nematodes, where siRNAs and piRNAs, but not miRNAs bind their targets with a perfect match and mediate the cleavage of the target RNA. Methylation of the 3' ends of piRNAs and siRNAs by the methyltransferase HEN1 protects these sRNAs from degradation. There is a noticeable selection in bilaterian animals against miRNA-mRNA perfect matching, as it leads to the degradation of miRNAs. Cnidarians (sea anemones, corals, hydroids and jellyfish), are separated from bilaterians by more than 600 million years. As opposed to bilaterians, cnidarian miRNAs frequently bind their targets with a nearly perfect match. Knowing that an ortholog of HEN1 is widely expressed in the sea anemone Nematostella vectensis, we tested in this work whether it mediates the stabilization of its sRNAs. We show that the knockdown of HEN1 in Nematostella results in a developmental arrest. Small RNA sequencing revealed that the levels of both miRNAs and piRNAs drop dramatically in the morphant animals. Moreover, knockdown experiments of Nematostella Dicer1 and PIWI2, homologs of major bilaterian biogenesis components of miRNAs and piRNAs, respectively, resulted in developmental arrest similar to HEN1 morphants. Our findings suggest that HEN1 mediated methylation of sRNAs reflects the ancestral state, where miRNAs were also methylated. Thus, we provide the first evidence of a methylation mechanism that stabilizes miRNAs in animals, and highlight the importance of post-transcriptional regulation in non-bilaterian animals.
| S-EPMC6114907 | biostudies-literature
Project description:Knockdown of Dicer and PIWI2 in Nematostella results in a developmental arrest. Small RNA sequencing revealed that the levels of both miRNAs and piRNAs drop dramatically in the morphant animals.
2019-01-01 | GSE115184 | GEO
Project description:knockdown of HEN1 in Nematostella results in a developmental arrest. Small RNA sequencing revealed that the levels of both miRNAs and piRNAs drop dramatically in the morphant animals.
2019-01-01 | GSE110148 | GEO
Project description:In eukaryotes, aberrant expression of transposable elements (TEs) is detrimental to the host genome. Piwi-interacting RNAs (piRNAs) of ∼23 to 30 nucleotides bound to PIWI clade Argonaute proteins silence transposons in a manner that is strictly dependent on their sequence complementarity. Hence, a key goal in understanding piRNA pathways is to determine mechanisms that modulate piRNA sequences. Here, we identify a protein-protein interaction between the 3'-to-5' exoribonuclease Nibbler (Nbr) and Piwi that links Nbr activity with piRNA pathways. We show that there is a delicate balance in the interplay between Nbr and Hen1, a methyltransferase involved in 2'-O-methylation at the 3' terminal nucleotides of piRNAs, thus connecting two genes with opposing activities in the biogenesis of piRNA 3' ends. With age, piRNAs become shorter and fewer in number, which is coupled with the derepression of select TEs. We demonstrate that activities of Nbr and Hen1 inherently contribute to TE silencing and age-dependent profiles of piRNAs. We propose that antagonistic roles of Nbr and Hen1 define a mechanism to modulate piRNA 3' ends.
| S-EPMC4760310 | biostudies-literature
Project description:small RNA libraries from wild-type and Hen1 mutant testes were made with either polyA tailing (VASAGFPHen1minus/plus) or adapter ligation (Hen1Testis and WTTestis) and sequenced on an Illumina GAII platform.
2011-11-09 | GSE33582 | GEO
Project description:small RNA libraries from wild-type and Hen1 mutant testes were made with either polyA tailing (VASAGFPHen1minus/plus) or adapter ligation (Hen1Testis and WTTestis) and sequenced on an Illumina GAII platform. RNA was isolated from total testis tissue of both Hen1 wildtype and Hen1 mutant animals. After size selection from gel, the small RNA libraries wre made.
2011-11-09 | E-GEOD-33582 | biostudies-arrayexpress
Project description:BackgroundThe MVD gene mutations are identified in porokeratosis, which is considered a skin-specific autoinflammatory keratinization disease. However, the biological function of MVD gene remains largely unknown. Therefore, we analyzed the function of mvda gene, orthologous to the human MVD gene, in developing zebrafish.MethodsMorpholino antisense oligonucleotide technique was used to generate mvda loss-of-function phenotypes. Knockdown of mvda was confirmed by RT-PCR and Sanger sequencing. Scanning and transmission electron microscopy were performed to analyze the morphology of the epidermis. Angiogenesis study was presented using the Tg(fli1a:EGFP)y1 transgenic strain. In addition, acridine orange staining was used to examine the apoptotic cells in vivo.ResultsAs expected, the mvda morphants showed abnormal morphology of the epidermis. Moreover, we observed ectopic sprouts in trunk angiogenesis and impaired formation of the caudal vein plexus in the mvda-deficient zebrafish. Besides, increased apoptosis was found throughout the tail, heart, and eyes in mvda zebrafish morphants.ConclusionsThese findings indicated the essential role of mvda in the early development of zebrafish. This was the first in vivo knockdown study of the zebrafish mvda gene, which might offer insight into the biological function of the human MVD gene.
| S-EPMC8164810 | biostudies-literature
Project description:Mild mutations in BRCA2 (FANCD1) cause Fanconi anemia (FA) when homozygous, while severe mutations cause common cancers including breast, ovarian, and prostate cancers when heterozygous. Here we report a zebrafish brca2 insertional mutant that shares phenotypes with human patients and identifies a novel brca2 function in oogenesis. Experiments showed that mutant embryos and mutant cells in culture experienced genome instability, as do cells in FA patients. In wild-type zebrafish, meiotic cells expressed brca2; and, unexpectedly, transcripts in oocytes localized asymmetrically to the animal pole. In juvenile brca2 mutants, oocytes failed to progress through meiosis, leading to female-to-male sex reversal. Adult mutants became sterile males due to the meiotic arrest of spermatocytes, which then died by apoptosis, followed by neoplastic proliferation of gonad somatic cells that was similar to neoplasia observed in ageing dead end (dnd)-knockdown males, which lack germ cells. The construction of animals doubly mutant for brca2 and the apoptotic gene tp53 (p53) rescued brca2-dependent sex reversal. Double mutants developed oocytes and became sterile females that produced only aberrant embryos and showed elevated risk for invasive ovarian tumors. Oocytes in double-mutant females showed normal localization of brca2 and pou5f1 transcripts to the animal pole and vasa transcripts to the vegetal pole, but had a polarized rather than symmetrical nucleus with the distribution of nucleoli and chromosomes to opposite nuclear poles; this result revealed a novel role for Brca2 in establishing or maintaining oocyte nuclear architecture. Mutating tp53 did not rescue the infertility phenotype in brca2 mutant males, suggesting that brca2 plays an essential role in zebrafish spermatogenesis. Overall, this work verified zebrafish as a model for the role of Brca2 in human disease and uncovered a novel function of Brca2 in vertebrate oocyte nuclear architecture.
| S-EPMC3069109 | biostudies-literature
Project description:Transposable elements (TEs) are mobile genetic elements that can have many deleterious effects on the fitness of their host. The germline-specific PIWI pathway guards the genome against TEs, deriving its specificity from sequence complementarity between PIWI-bound small RNAs (piRNAs) and the TEs. The piRNAs are derived from so-called piRNA clusters. Recent studies have demonstrated that the piRNA repertoire can be adjusted to accommodate recent TE invasions by capturing invading TEs in piRNA loci. Thus far, no information concerning piRNA divergence is available from vertebrates. We present piRNA analyses of two relatively divergent zebrafish strains. We find that significant differences in the piRNA populations have accumulated, most notably among active class I TEs. This divergence can be split into differences in piRNA abundance per element and differences in sense/antisense polarity ratios. In crosses between animals of the different strains, many of these differences are resolved in the progeny. However, some differences remain, often leaning to the maternally contributed piRNA population. These differences can be detected at least two generations later. Our data illustrate, for the first time, the fluidity of piRNA populations in vertebrates and how the established diversity is transmitted to future generations.
| S-EPMC3677245 | biostudies-literature