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

Tetsutaro Sumiyoshi,Hitomi Yamamoto,Mikiko C Siomi,Kaoru Sato,Haruhiko Siomi,Yuka W Iwasaki

doi:10.1101/gad.283929.116

Tetsutaro Sumiyoshi, Hitomi Yamamoto + Show 4 more

Open Access

https://doi.org/10.1101/gad.283929.116

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Journal: Genes & development	Publication Date: Jul 15, 2016
Citations: 58	License type: cc-by

Affiliation: The University of Tokyo, Keio University

Abstract

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. Perinuclear foci resembling nuage, the ping-pong center, appeared following l(3)mbt mutation. This activation of the ping-pong machinery in cultured cells will greatly facilitate elucidation of the mechanism underlying secondary piRNA biogenesis in Drosophila.

Highlights

These findings of l(3)mbt mutations resulting in ectopic acquisition of germline traits in the brain prompted us to examine whether depletion of l(3)mbt in ovarian somatic cell (OSC) initiates the ping-pong cycle and causes the accumulation of secondary PIWI-interacting RNAs (piRNAs) in cells
We found that both RNAi- and CRISPRmediated loss of function of l(3)mbt lead to ectopic expression of Aub, AGO3, and Vasa—the core factors necessary for operating the germ-specific ping-pong cycle—in OSCs
Ago3 and vasa were significantly up-regulated after l(3)mbt siRNA treatment, while changes in the level of piwi mRNA were negligible (Supplemental Fig. S1B)

Summary

RESEARCH COMMUNICATION

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. the detailed mechanism remains poorly understood, largely because an ex vivo model system amenable to biochemical analyses has not been available. Perinuclear foci resembling nuage, the ping-pong center, appeared following l(3)mbt mutation This activation of the ping-pong machinery in cultured cells will greatly facilitate elucidation of the mechanism underlying secondary piRNA biogenesis in Drosophila. A few small RNAs in l(3)mbt mutant brain tumors were annotated as piRNAs (Janic et al 2010) These findings of l(3)mbt mutations resulting in ectopic acquisition of germline traits in the brain prompted us to examine whether depletion of l(3)mbt in OSCs initiates the ping-pong cycle and causes the accumulation of secondary piRNAs in cells.

Results and Discussion

Materials and methods

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