Piwi-piRNA complexes in the zebrafish germline

Small non-coding RNAs act through diverse RNA interference (RNAi) pathways to transcriptionally or post-transcriptionally silence their targets. piRNAs (Piwi Interacting RNAs) are a distinct class of 30-nucleotide-long RNAs that are only present in the germ line. In zebrafish they bind to the Argonaute related Piwi proteins Ziwi and Zili, which are present in primordial germ cells, as well as male and female mitotic and meiotic germ cells. While Ziwi and Zili both localize to the cytoplasm and nuage, Zili is also found in the nucleus. In addition, Ziwi-piRNA complexes are maternally provided to the embryo. Intriguingly, piRNAs are derived from single-stranded RNA and their biogenesis is independent of Dicer, in contrast to other small RNAs. Furthermore, piRNAs carry a 2’O-methyl modification at their 3' end. Large scale sequencing of piRNA populations has provided insights into piRNA biogenesis and functions. Over 400,000 piRNAs were characterized at various stages of development. Ziwi and Zili bind to piRNAs of opposite polarity derived from the same loci and are involved in piRNA biogenesis. Most piRNAs are derived from repetitive sequences, which are often annotated as transposon. The Piwi-piRNA pathway is involved in silencing of these transposons in the germ line. Perturbation of the piRNA pathway through loss of Ziwi or Zili function results in unrestrained transposon activation and loss of germ cells. While in ziwi mutant zebrafish, germ cells die by apoptosis, zili mutant germ cells are unable to differentiate and are lost much later. In addition, a zili missense allele results in a female specific meiosis I defect, independent of Zili’s role in transposon silencing, significantly extending the role of Piwi proteins beyond the control of transposable elements in vertebrates. Zili interacts with many Tudor domain containing proteins, possibly through its methylated residues. The Tudor domain containing helicase Spindle-E (SpnE) associates with Zili and loss of SpnE function results in loss of piRNAs, showing that SpnE is part of the piRNA pathway. Analysis of spnE mutants indicates a role in transposon control, but also in germ line stem cell maintenance and differentiation. The function of the Piwi-piRNA pathway in zebrafish exceeds that of transposon control. Possibly piRNAs function not only in fertility and germ line maintenance, but also in stem cell biology and chromosome segregation.