Small regulatory RNAs guide Argonaute (Ago) proteins in a sequence-specific manner to their targets and thereby play important roles in eukaryotic gene silencing1. Of the three small RNA classes, microRNAs and siRNAs are processed from double-stranded precursors into defined 21- to 23-mers by Dicer, an endoribonuclease with intrinsic ruler function. piRNAs—the 22-30 nt long guides for PIWI-clade Ago proteins that silence transposons in animal gonads—are generated Dicer-independently from single-stranded precursors2,3. piRNA 5' ends are defined either by Zucchini, a mitochondria-anchored endonuclease4,5, or by piRNA-guided target cleavage6,7. Formation of piRNA 3' ends is poorly understood. Here, we find that two genetically and mechanistically distinct pathways generate piRNA 3' ends in Drosophila. The initiating nucleases are either Zucchini or the PIWI-clade proteins Aubergine (Aub)/Ago3. While Zucchini-mediated cleavages directly define mature piRNA 3' ends8,9, Aub/Ago3-mediated cleavages liberate pre-piRNAs that require extensive resection by the 3'-to-5' exoribonuclease Nibbler/Mut-710–13. The relative activity of these two pathways dictates the extent to which piRNAs are fueled into cytoplasmic or nuclear PIWI-clade proteins and thereby sets the balance between post-transcriptional and transcriptional silencing. Strikingly, loss of both Zucchini and Nibbler reveals a minimal, Argonaute-driven small RNA biogenesis pathway where piRNA 5' and 3' ends are directly produced by closely spaced Aub/Ago3-mediated cleavage events. Our data establish a coherent blueprint for piRNA biogenesis, and set the stage for the mechanistic dissection of the processes that govern piRNA 3' end formation.

The primary embryonic axes in flies, frogs and fish are formed through translational regulation of localized transcripts before fertilization 1 . In Drosophila, the axes are established through the transport and translational regulation of gurken (grk) and bicoid (bcd) messenger RNA (mRNA) in the oocyte and embryo 1 . bcd and grk mRNA are both translationally silent while being localized within the oocyte along microtubules by cytoplasmic Dynein 1-4 . Once localized, grk is translated at the dorsoanterior of the oocyte to send a TGF-alpha signal to the overlying somatic cells 5 . In contrast, bcd is translationally repressed in the oocyte until its activation in early embryos to form an anteroposterior morphogenetic gradient 6 . How this differential translational regulation is achieved is not fully understood. Here, we address this question using ultrastructural analysis, super-resolution microscopy and live cell imaging. We show that grk and bcd ribonucleoprotein (RNP) complexes associate with electron dense bodies that lack ribosomes and contain translational repressors, characteristic of Processing bodies (P bodies), which are regions of cytoplasm where translational decisions are made. Endogenous grk mRNA forms dynamic RNP particles that become docked and translated at the periphery of P bodies, where we show that the translational activator Orb/CEPB and the anchoring factor Squid (Sqd) are also enriched. In contrast, an excess of grk mRNA becomes localized inside the P bodies, where endogenous bcd mRNA is localized and translationally repressed. Interestingly, bcd mRNA dissociates from P bodies in embryos following egg activation, when it is known to become translationally active. We propose a general principle of translational regulation during axis specification involving remodeling of transport RNPs and dynamic partitioning of different transcripts between the translationally active edge of P bodies and their silent core.

Splicing of pre-mRNAs results in the deposition of the exon junction complex (EJC) upstream of exon-exon boundaries. The EJC plays crucial post-splicing roles in export, translation, localization, and nonsense-mediated decay of mRNAs. It also aids faithful splicing of pre-mRNAs containing large introns, albeit via an unknown mechanism. Here, we show that the core EJC plus the accessory factors RnpS1 and Acinus aid in definition and efficient splicing of neighboring introns. This requires prior deposition of the EJC in close proximity to either an upstream or downstream splicing event. If present in isolation, EJC-dependent introns are splicing-defective also in wild-type cells. Interestingly, the most affected intron belongs to the piwi locus, which explains the reported transposon desilencing in EJC-depleted Drosophila ovaries. Based on a transcriptome-wide analysis, we propose that the dependency of splicing on the EJC is exploited as a means to control the temporal order of splicing events.[Keywords: Piwi-piRNA pathway; SR proteins Acinus and RnpS1; exon junction complex; intron definition; splicing; transposon silencing] Supplemental material is available for this article. Expression of protein-coding genes involves a series of interlinked and interdependent molecular events, including nuclear steps such as pre-mRNA transcription, intron removal via splicing, 59 and 39 modifications, and export, followed by cytoplasmic events such as mRNA translation and degradation. A central factor connecting nuclear premRNA maturation to mRNA fate is the exon junction complex (EJC), a multisubunit protein complex that is deposited ;24 nucleotides (nt) upstream of individual exon-exon boundaries after splicing (Le Hir et al. 2000a).Assembly of the EJC on an RNA depends on tight RNA binding of a trimeric nuclear complex of the sequenceindependent DEAD-box RNA clamp eIF4AIII and a heterodimer of Mago and