An extended Tudor domain within Vreteno interconnects Gtsf1L and Ago3 for piRNA biogenesis in Bombyx mori.

Piwi-interacting RNAs (piRNAs) direct PIWI proteins to transposons to silence them, thereby preserving genome integrity and fertility. The piRNA population can be expanded in the ping-pong amplification loop. Within this process, piRNA-associated PIWI proteins (piRISC) enter a membraneless organelle called nuage to cleave their target RNA, which is stimulated by Gtsf proteins.

Maelstrom functions in the production of Siwi-piRISC capable of regulating transposons in germ cells.

PIWI-interacting RNAs (piRNAs) bind to PIWI proteins to assemble the piRISC, which represses germline transposons. Maelstrom (Mael) is necessary for piRISC biogenesis in germ cells, but its function remains unclear. Here, we show that Mael interconnects Spindle-E (Spn-E), a key piRISC biogenesis factor, with unloaded Siwi, one of two silkworm PIWI members.

DEAD-box polypeptide 43 facilitates piRNA amplification by actively liberating RNA from Ago3-piRISC.

The piRNA amplification pathway in Bombyx is operated by Ago3 and Siwi in their piRISC form. The DEAD-box protein, Vasa, facilitates Ago3-piRISC production by liberating cleaved RNAs from Siwi-piRISC in an ATP hydrolysis-dependent manner. However, the Vasa-like factor facilitating Siwi-piRISC production along this pathway remains unknown.

Siwi levels reversibly regulate secondary piRISC biogenesis by affecting Ago3 body morphology in Bombyx mori.

Silkworm ovarian germ cells produce the Siwi-piRNA-induced silencing complex (piRISC) through two consecutive mechanisms, the primary pathway and the secondary ping-pong cycle. Primary Siwi-piRISC production occurs on the outer mitochondrial membrane in an Ago3-independent manner, where Tudor domain-containing Papi binds unloaded Siwi via its symmetrical dimethylarginines (sDMAs). Here, we now show that secondary Siwi-piRISC production occurs at the Ago3-positive nuage Ago3 bodies, in an Ago3-dependent manner, where Vreteno (Vret), another Tudor protein, interconnects unloaded Siwi and Ago3-piRISC through their sDMAs.

Use of the CRISPR-Cas9 system for genome editing in cultured Drosophila ovarian somatic cells.

The CRISPR-Cas9 system can be used for genome engineering in many organisms. PIWI-interacting RNAs (piRNAs) play a crucial role in repressing transposons to maintain genome integrity in Drosophila ovaries, and cultured ovarian somatic cells (OSCs) are widely used to elucidate the molecular mechanisms underlying the piRNA pathway. However, the germline-specific piRNA amplification system known as the ping-pong machinery does not occur in OSCs, making them unsuitable for elucidating the underlying mechanisms.

Loss of l(3)mbt leads to acquisition of the ping-pong cycle in Drosophila ovarian somatic cells.

In Drosophila germ cells, PIWI-interacting RNAs (piRNAs) are amplified through a PIWI slicer-dependent feed-forward loop termed the ping-pong cycle, yielding secondary piRNAs. However, the detailed mechanism remains poorly understood, largely because an ex vivo model system amenable to biochemical analyses has not been available. Here, we show that CRISPR-mediated loss of function of lethal (3) malignant brain tumor [l(3)mbt] leads to ectopic activation of the germ-specific ping-pong cycle in ovarian somatic cells.