Piwi proteins specify an animal-specific subclass of the Argonaute family that, in vertebrates, is specifically expressed in germ cells. We demonstrate that zebrafish Piwi (Ziwi) is expressed in both the male and the female gonad and is a component of a germline-specifying structure called nuage. Loss of Ziwi function results in a progressive loss of germ cells due to apoptosis during larval development. In animals that have reduced Ziwi function, germ cells are maintained but display abnormal levels of apoptosis in adults. In mammals, Piwi proteins associate with approximately 29-nucleotide-long, testis-specific RNA molecules called piRNAs. Here we show that zebrafish piRNAs are present in both ovary and testis. Many of these are derived from transposons, implicating a role for piRNAs in the silencing of repetitive elements in vertebrates. Furthermore, we show that piRNAs are Dicer independent and that their 3' end likely carries a 2'O-Methyl modification.

The molecular mechanism responsible for a decline of stem cell functioning after replicative stress remains unknown. We used mouse embryonic fibroblasts (MEFs) and hematopoietic stem cells (HSCs) to identify genes involved in the process of cellular aging. In proliferating and senescent MEFs one of the most differentially expressed transcripts was Enhancer of zeste homolog 2 (Ezh2), a Polycomb group protein (PcG) involved in histone methylation and deacetylation. Retroviral overexpression of Ezh2 in MEFs resulted in bypassing of the senescence program. More importantly, whereas normal HSCs were rapidly exhausted after serial transplantations, overexpression of Ezh2 completely conserved long-term repopulating potential. Animals that were reconstituted with 3 times serially transplanted control bone marrow cells all died due to hematopoietic failure. In contrast, similarly transplanted Ezh2-overexpressing stem cells restored stem cell quality to normal levels. In a "genetic genomics" screen, we identified novel putative Ezh2 target or partner stem cell genes that are associated with chromatin modification. Our data suggest that stabilization of the chromatin structure preserves HSC potential after replicative stress. (Blood. 2006;

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 0 -O-methyl modification at their 3 0 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 0 -5 0 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.

RNA interference (RNAi)–related pathways affect gene activity by sequence-specific recruitment of Ago proteins to mRNA target molecules. The sequence specificity of this process stems from small RNA (sRNA) co-factors bound by the Ago protein. Stability of sRNA molecules in some pathways is in part regulated by Hen1-mediated methylation of their 3′ ends. Here we describe the effects of the Caenorhabditis elegans HEN1 RNA–methyl-transferase homolog, HENN-1, on the different RNAi pathways in this nematode. We reveal differential effects of HENN-1 on the two pathways that are known to employ methylated sRNA molecules: the 26G and 21U pathways. Surprisingly, in the germline, stability of 21U RNAs, the C. elegans piRNAs, is only mildly affected by loss of methylation; and introduction of artificial 21U target RNA does not further destabilize non-methylated 21U RNAs. In contrast, most 26G RNAs display reduced stability and respond to loss of HENN-1 by displaying increased 3′-uridylation frequencies. Within the 26G RNA class, we find that specifically ERGO-1–bound 26G RNAs are modified by HENN-1, while ALG-3/ALG-4–bound 26G RNAs are not. Global gene expression analysis of henn-1 mutants reveals mild effects, including down-regulation of many germline-expressed genes. Our data suggest that, apart from direct effects of reduced 26G RNA levels of henn-1 on gene expression, most effects on global gene expression are indirect. These studies further refine our understanding of endogenous RNAi in C. elegans and the roles for Hen1 like enzymes in these pathways.