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Function of Piwi, a Nuclear Piwi/Argonaute Protein, Is Independent of Its Slicer Activity

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Function of Piwi, a Nuclear Piwi/Argonaute Protein, Is Independent of Its Slicer Activity Function of Piwi, a nuclear Piwi/Argonaute protein, is independent of its slicer activity Nicole Darricarrère, Na Liu, Toshiaki Watanabe, and Haifan Lin1 Yale Stem Cell Center and Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06509 Edited by James A. Birchler, University of Missouri, Columbia, MO, and approved December 11, 2012 (received for review August 1, 2012) The Piwi protein subfamily is essential for Piwi-interacting RNA classical epigenetic phenotypes in Drosophila (15, 17, 18). These (piRNA) biogenesis, transposon silencing, and germ-line develop- observations led us to propose that piRNA molecules can guide ment, all of which have been proposed to require Piwi endonuclease Piwi to specific genomic loci by sequence complementarity, activity, as validated for two cytoplasmic Piwi proteins in mice. where Piwi recruits epigenetic modifiers (17–19). In this epige- However, recent evidence has led to questioning of the generality netic model, the slicer activity of Piwi seems superfluous. Indeed, of this mechanism for the Piwi members that reside in the nucleus. the involvement of the slicer activity of Piwi has been questioned Drosophila offers a distinct opportunity to study the function of recently based on experiments performed in an ovarian somatic nuclear Piwi proteins because, among three Drosophila Piwi pro- cell line (20). However, this analysis was not definitive. It was teins—called Piwi, Aubergine, and Argonaute 3—Piwi is the only based on a single cultured cell type, relied on RNAi that only member of this subfamily that is localized in the nucleus and incompletely reduced endogenous levels of Piwi and tested only expressed in both germ-line and somatic cells in the gonad, where one transposon element. Thus, these results could not predict it is responsible for piRNA biogenesis and regulatory functions es- the consequence of abolishing the slicer function in an organism. sential for fertility. In this study, we demonstrate beyond doubt that Therefore, we sought to test the role of the endonuclease activity the slicer activity of Piwi is not required for any known functions in of Piwi in all known biological processes that involve Piwi fun- vivo. We show that, in transgenic flies with the DDX catalytic triad ction, such as piRNA biogenesis, transposon silencing, germ-line of PIWI mutated, neither primary nor secondary piRNA biogenesis is development, and gametogenesis. detectably affected, transposons remain repressed, and fertility is normal. Our observations demonstrate that the mechanism of Piwi Results BIOCHEMISTRY is independent of its in vitro endonuclease activity. Instead, it is Slicer Mutations Do Not Affect the Stability or Localization of Piwi consistent with the alternative mode of Piwi function as a molecule in Vivo. We replaced the two critical aspartate residues, D614 and involved in the piRNA-directed guidance of epigenetic factors D685, that form the catalytic triad with alanine, as reported in to chromatin. vitro (16) (Fig. 1A). We used FlyC31 site-specific recombination technology to introduce the slicer-deficient Myc–Piwi transgene small RNA | RNase H fold | transposable element | sterility (“DUO,” which means “two” in Latin, herein refers to two-point mutations) or a wild-type Myc–Piwi control transgene (WT) in- he Piwi protein family plays a fundamental role in sexual to a well-characterized genomic site called attP2 to generate – Treproduction. Not only are Piwi proteins highly enriched in transgenic Drosophila strains (21). The WT Myc Piwi gene has the gonads of all animals studied to date, but their reproductive been shown to be fully functional compared with the endogenous phenotypes have been revealed in diverse model organisms, such piwi gene (22). The insertion of the DUO and WT transgenes as Caenorhabditis elegans, Drosophila, zebrafish, and mice (1). precisely at the same site in the genome eliminates potentially fl Piwi proteins are paralogs of Argonaute proteins, sharing the same different in uences of the surrounding genomic context toward domain architecture and the property of binding small noncoding the expression of the inserted transgenes. Additionally, we gen- – erated another DUO Myc–Piwi transgenic line by conventional RNAs (2 6). Argonaute proteins bind to small interfering RNAs – (siRNAs) or microRNAs (miRNAs) and are effectors of RNA P-element transformation, with a WT Myc Piwi transgenic line (G38) previously generated the same way as controls (15). The interference (RNAi) and miRNA pathways; conversely, Piwi pro- – teins bind to Piwi-interacting RNAs (piRNAs) and are involved in expression levels of WT and DUO Myc Piwi proteins in ovaries are very similar among all transgenic lines (Fig. 1B). Likewise, the piRNA pathway in both the germ line and soma during ga- fi – metogenesis (1, 7, 8). the localization pattern of slicer-de cient Myc Piwi (green) both — in the germ-line (red for Vasa, a germ-line marker) and in so- In Drosophila, there are three Piwi proteins Piwi, Aubergine – (Aub), and Argonaute 3 (Ago3) (9)—all of which have been matic cells was indistinguishable from that of WT Myc Piwi (Fig. 1 C–J). Together, these results confirm that these two mutations shown to possess small RNA-dependent endonuclease (slicer) have no effect on the stability or localization of Piwi in vivo. activity by in vitro assays (10, 11), catalyzed by an aspartate cat- alytic triad (DDX) that is positioned within the core of the RNase None of the Essential Functions of Piwi in Somatic and Germ-Line H-fold (12). This activity has been proposed to be essential for the fi Cells Requires its Slicer Activity. To assess the biological role of ampli cation of piRNAs through a mechanism called the ping- the slicer activity of Piwi, we crossed the DUO and WT transgenic pong cycle that occurs in the cytoplasm and uses cRNA targets, such as transposon transcripts, as precursors (13, 14). In addition, the slicer activity has been proposed play a role in silencing target Author contributions: N.D. and H.L. designed research; N.D., N.L., and T.W. performed transposons through cleaving their transcripts (13). research; N.D. and H.L. contributed new reagents/analytic tools; N.D., N.L., T.W., and H.L. Among the three Piwi proteins in Drosophila, only Piwi is analyzed data; and N.D. and H.L. wrote the paper. expressed in both germ-line and somatic cells in the gonad and is The authors declare no conflict of interest. involved in both germ-line and somatic piRNA pathways (15, This article is a PNAS Direct Submission. 16). Piwi is also distinct from the other members of the subfamily Data deposition: The data reported in this paper have been deposited in the Gene in terms of subcellular localization. We showed previously that Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE34032). Piwi is a nuclear protein that associates with an epigenetic factor, 1To whom correspondence should be addressed. E-mail: [email protected]. Heterochromatin Protein 1a, and binds to piRNA-complementary This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. sequences in the genome and that Piwi deficiencies lead to 1073/pnas.1213283110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1213283110 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 myc-tagged Piwi ABno myc control wildtype slicer mutant Myc-tag W[1118] G38 WT WTDUO DUO D614A D685A N-terminal PAZ MID PIWI myc β-Actin 0 0 CEDF0 075 µm µm µm µm 75 75 50 0 0 0 GH0 I J µm µm µm µm 75 75 Myc 75 50 VASA DAPI W[1118] +/+, WT/WT +/+, DUO/DUO +/+, D37/D37 Fig. 1. Slicer-deficient Piwi shows a normal level of expression and pattern of localization. (A) Schematic representation of the domain architecture of PIWI, highlighting the two critical aspartate residues essential for endonuclease activity, which were replaced by alanine in the slicer-deficient transgene. (B) Western blot showing similar levels of WT and DUO Piwi proteins in ovaries. The wild-type nontransgenic line (W1118) is a negative control. Myc–Piwi transgenic line G38 was described (15). Protein levels of two independent site-specific transgenics are shown for wildtype Myc–Piwi at the attP2 site (WT) and + Myc–Piwi slicer mutant (DUO). All transgenes are in the W1118;piwi background. (C–J) Immunofluorescence micrographs of ovarioles from transgenic flies. Myc (green) was costained with Vasa (red) and DAPI (blue). C and G show no Myc staining in Myc-negative W1118 ovaries. D and H show the normal lo- calization of WT Myc–Piwi, whereas E and I show the normal localization of DUO Myc–PIWI (D614A, D685A). F and J show the localization of D37 Myc–PIWI (D614A, D685A). lines into the homozygous piwi1 mutant background as outlined 3A) and normalized to protein loading controls as quantified by in Fig. S1. To our surprise, ovaries from the resulting piwi1/piwi1; Western blotting (Fig. 3 B and C). By comparing the pull-down DUO/DUO genotype are morphologically indistinguishable from efficiency of DUO and WT Myc–Piwi in a wild-type background, piwi1/piwi1;WT/WT and wild-type flies (Fig. 2 A–F), as indicated we confirmed that the slicer point mutations do not impair piRNA by normal 1B1 staining (green) that outlines somatic cells and binding in vivo. Furthermore, the amount of piRNAs relative to highlights spectrosomes and fusomes in the germ line as well as Piwi protein does not differ between slicer-deficient and wild-type Vasa staining (red) that labels germ-line cells. We then quantified strains in the piwi1 background. the fertility of the slicer-deficient flies by counting the number of To further examine whether the slicer mutant Piwi is defective eggs laid per fly per day.
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