Unknown,Transcriptomics,Genomics,Proteomics

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The Function of Piwi is Independent of Its Slicer Activity

ABSTRACT: In diverse organisms, the highly-conserved Piwi proteins bind to a complex class of small non-coding RNAs called piRNAs, and are essential for germline stem cell maintenance, transposon silencing, fertility, and offspring viability1. By homology with other Argonautes, Piwi proteins have been proposed and later reported to possess endonuclease activity in vitro, effected by its conserved aspartate catalytic triad2,3. Indeed, mutation of these residues in one of the three mouse PIWI proteins, MILI, affects the production of Line 1 piRNAs and spermatogenesis4. In Drosophila, the conserved slicer function has been proposed to be crucial for all Piwi proteins in piRNA amplification and transposon silencing5. Here, we report in vivo evidence that, in contrast to common belief, the catalytic triad of Piwi is not required for the function of PIwi in the germline for stem cell maintenance, fertility, transposon silencing, or piRNA biogenesis. In addition, it is not required for piRNA biogenesis in the soma where Piwi is the sole Piwi-subfamily protein involved in the process. These observations, together with the recent findings on MILI and MIWI2 triad mutant, indicate that even though the slicer function may be required for a subset of piRNA-mediated process, it is overall inconsequential for the function of Piwi proteins Assess the role of the endonuclease activity of Piwi in all known biological processes that involve Piwi function in Drosophila ovary

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Na Liu

PROVIDER: E-GEOD-34032 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Function of Piwi, a nuclear Piwi/Argonaute protein, is independent of its slicer activity.

Darricarrère Nicole N Liu Na N Watanabe Toshiaki T Lin Haifan H

Proceedings of the National Academy of Sciences of the United States of America 20130107 4

The Piwi protein subfamily is essential for Piwi-interacting RNA (piRNA) biogenesis, transposon silencing, and germ-line development, all of which have been proposed to require Piwi endonuclease activity, as validated for two cytoplasmic Piwi proteins in mice. However, recent evidence has led to questioning of the generality of this mechanism for the Piwi members that reside in the nucleus. Drosophila offers a distinct opportunity to study the function of nuclear Piwi proteins because, among thr ...[more]

PMID: 23297219

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Project description:PIWI-clade Argonaute proteins silence transposon expression in animal gonads. Their target specificity is defined by bound ~23-30nt piRNAs that are processed from single-stranded precursor transcripts via two distinct pathways. Primary piRNAs are defined by the endo-nuclease Zucchini, while biogenesis of secondary piRNAs depends on piRNA-guided transcript cleavage and results in piRNA amplification. Here, we analyze the inter-dependencies between these piRNA biogenesis pathways in the developing Drosophila ovary. We show that secondary piRNA-guided target slicing is the predominant mechanism that specifies transcripts—including those from piRNA clusters—as primary piRNA precursors and that defines the spectrum of Piwi-bound piRNAs in germline cells. Post-transcriptional silencing in the cytoplasm therefore enforces nuclear, transcriptional target silencing, which ensures the tight suppression of transposons during oogenesis. As target slicing also defines the nuclear piRNA pool during mouse spermatogenesis, our findings uncover an unexpected conceptual similarity between the mouse and fly piRNA pathways. To understand the hierarchical order of primary versus secondary piRNA biogenesis in Drosophila ovaries, we sequenced piRNAs bound to total-Piwi, germline-Piwi, Aubergine and Argonaute3 from ovaries of germline specific knockdowns of control, piwi, aub, ago3 single knockdowns and aub/ago3 double knockdowns. To determine changes in Transposable Element (TE) transcription or TE RNA steady state in perturbed piRNA pathway conditions, we performed Pol2-ChIP-sequencing and polyA bound RNA-sequencing from ovaries of multiple germline knockdown genotypes. We also sequenced genomic DNA from ovaries of control knockdowns to experimentally estimate the TE copy number in our genetic background. Finally, we used CAP-seq from germline specific Piwi depletions to identify the Transcriptional Start Sites (TSS) in TEs in a deregulated background. Replicates are labeled with R1, R2, R3, R4 where indicated.

Dataset Information

The Function of Piwi is Independent of Its Slicer Activity

Publications

Function of Piwi, a nuclear Piwi/Argonaute protein, is independent of its slicer activity.

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