Summary piRNAs silence transposons and maintain genome integrity during germ-line development. In Drosophila, transposon-rich heterochromatic clusters encode piRNAs either on both genomic strands (dual-strand clusters) or predominantly one genomic strand (uni-strand clusters). Primary piRNAs derived from these clusters are proposed to drive a ping-pong amplification cycle catalyzed by proteins that localize to the perinuclear nuage. We show that the HP1 homologue Rhino is required for nuage organization, transposon silencing, and ping-pong amplification of piRNAs. rhi mutations virtually eliminate piRNAs from the dual-strand clusters and block production of putative precursor RNAs from both strands of the major 42AB dual-strand cluster, but do not block production of transcripts or piRNAs from the uni-strand clusters. Furthermore, Rhino protein associates with the 42AB dual-strand cluster, but does not bind to uni-strand cluster 2 or flamenco. Rhino thus appears to promote transcription of dual-strand clusters, leading to production of piRNAs that drive the ping-pong amplification cycle.

With more chromosomes than any other sequenced genome, the macronuclear genome of Oxytricha trifallax has a unique and complex architecture, including alternative fragmentation and predominantly single-gene chromosomes.

Summary Transposons evolve rapidly and can mobilize and trigger genetic instability. piRNAs silence these genome pathogens, but it is unclear how the piRNA pathway adapts to invasion of new transposons. In Drosophila, piRNAs are encoded by heterochromatic clusters and maternally deposited in the embryo. Paternally inherited P-element transposons thus escape silencing and trigger a hybrid sterility syndrome termed P-M hybrid dysgenesis. We show that P-M hybrid dysgenesis activates both P-elements and resident transposons, and disrupts the piRNA biogenesis machinery. As dysgenic hybrids age, however, fertility is restored, P-elements are silenced, and P-element piRNAs are produced de novo. In addition, the piRNA biogenesis machinery assembles and resident elements are silenced. Significantly, resident transposons insert into piRNA clusters, and these new insertions are transmitted to progeny, produce novel piRNAs, and are associated with reduced transposition. P-element invasion thus triggers heritable changes in genome structure that appear to enhance transposon silencing.

RNA interference (RNAi) has become a research tool to control gene expression in various organisms and holds potential as a new therapeutic strategy. The mechanism of small interfering RNA (siRNA)-mediated RNAi involves target mRNA cleavage and destruction in the cytoplasm. We investigated siRNA-mediated induction of RNAi in the nucleus of human cells. Notably, we observed highly efficient knockdown of small nuclear RNA 7SK by siRNA. siRNA- and microRNA-programmed RNA-induced silencing complexes (RISCs) were present in both cytoplasmic and nuclear compartments and specifically cleaved their perfectly matched target RNA with markedly high efficiencies. Our results provide the first evidence that human RISCs programmed with siRNA are present in the nucleus and can knock down target RNA levels. These studies reveal new roles for the RNAi machinery in modulating post-transcriptional gene expression in the nucleus.

Transposons are prominent features of most eukaryotic genomes and mobilization of these elements triggers genetic instability. Transposon silencing is particularly critical in the germline, which maintains the heritable genetic complement. Piwi-interacting RNAs (piRNAs) have emerged as central players in transposon silencing and genome maintenance during germline development. In particular, research on Drosophila oogenesis has provided critical insights into piRNA biogenesis and transposon silencing. In this system, the ability to place piRNA mutant phenotypes within a well-defined developmental framework has been instrumental in elucidating the molecular mechanisms underlying the connection between piRNAs and transposon control.