title: The capacity of target silencing by Drosophila PIWI and piRNAs.
abstract: Although Piwi proteins and Piwi-interacting RNAs (piRNAs) genetically repress transposable elements (TEs), it is unclear how the highly diverse piRNA populations direct Piwi proteins to silence TE targets without silencing the entire transcriptome. To determine the capacity of piRNA-mediated silencing, we introduced reporter genes into Drosophila OSS cells, which express microRNAs (miRNAs) and piRNAs, and compared the Piwi pathway to the Argonaute pathway in gene regulation. Reporter constructs containing several target sites that were robustly silenced by miRNAs were not silenced to the same degrees by piRNAs. However, another set of reporters we designed to enable a large number of both TE-directed and genic piRNAs to bind were robustly silenced by the PIWI/piRNA complex in OSS cells. These reporters show that a bulk of piRNAs are required to pair to the reporter's transcripts and not the reporter's DNA sequence to engage PIWI-mediated silencing. Following our genome-wide study of PIWI-regulated targets in OSS cells, we assessed candidate gene elements with our reporter platform. These results suggest TE sequences are the most direct of PIWI regulatory targets while coding genes are less directly affected by PIWI targeting. Finally, our study suggests that the PIWI transcriptional silencing mechanism triggers robust chromatin changes on targets with sufficient piRNA binding, and preferentially regulates TE transcripts because protein-coding transcripts lack a threshold of targeting by piRNA populations. This reporter platform will facilitate future dissections of the PIWI-targeting mechanism.
authors.affiliation: Department of Biology and Rosenstiel Basic Medical Science Research Center, Brandeis University, Waltham, Massachusetts 02453, USA.
authors.forename: Christina
authors.initials: C
authors.lastname: Post
authors.affiliation: Department of Biology and Rosenstiel Basic Medical Science Research Center, Brandeis University, Waltham, Massachusetts 02453, USA.
authors.forename: Josef P
authors.initials: JP
authors.lastname: Clark
authors.affiliation: Department of Biology and Rosenstiel Basic Medical Science Research Center, Brandeis University, Waltham, Massachusetts 02453, USA.
authors.forename: Yuliya A
authors.initials: YA
authors.lastname: Sytnikova
authors.affiliation: Department of Biology and Rosenstiel Basic Medical Science Research Center, Brandeis University, Waltham, Massachusetts 02453, USA.
authors.forename: Gung-Wei
authors.initials: GW
authors.lastname: Chirn
authors.affiliation: Department of Biology and Rosenstiel Basic Medical Science Research Center, Brandeis University, Waltham, Massachusetts 02453, USA nlau@brandeis.edu.
authors.forename: Nelson C
authors.initials: NC
authors.lastname: Lau
mesh_terms: D000818:Animals; D060565:Argonaute Proteins / Q000235:genetics*; D004251:DNA Transposable Elements / Q000235:genetics*; D004330:Drosophila / Q000235:genetics; D029721:Drosophila Proteins / Q000235:genetics*; D020868:Gene Silencing; D035683:MicroRNAs / Q000235:genetics; D016366:Open Reading Frames / Q000235:genetics; D016372:RNA, Antisense; D034741:RNA, Small Interfering / Q000235:genetics*
chemical_list: C433807:AGO2 protein, Drosophila; D060565:Argonaute Proteins; D004251:DNA Transposable Elements; D029721:Drosophila Proteins; D035683:MicroRNAs; D016372:RNA, Antisense; D034741:RNA, Small Interfering; C116498:piwi protein, Drosophila
keywords: Piwi; gene silencing; piRNAs