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Published on the internet 24 AprilNucleic Acids Investigation, 2013, Vol. 41, No. 11 5757?768 doi:ten.1093/nar/gktDe novo piRNA cluster formation within the Drosophila germ line triggered by transgenes containing a transcribed transposon fragmentIvan Olovnikov1,2, Sergei Ryazansky1, Sergey Shpiz1, Sergey Lavrov1, Yuri Abramov1, Chantal Vaury3,four,five, Silke Jensen3,four,5,* and Alla Kalmykova1,*Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia, 2Division of Biology, ??California Institute of Technologies, Pasadena, CA 91125, USA, 3Clermont Universite, Universite d’Auvergne, four Laboratoire du GReD, BP 38, 63001 Clermont-Ferrand, France, INSERM, U 1103, BP 38, 63001 Clermont-Ferrand, France and 5CNRS, UMR 6293, BP 38, 63001 Clermont-Ferrand, FranceReceived December 23, 2012; Revised March six, 2013; Accepted April three,ABSTRACT PIWI-interacting RNAs (piRNAs) offer defence against transposable element (TE) expansion in the germ line of metazoans. piRNAs are processed in the transcripts encoded by specialized heterochromatic clusters enriched in broken copies of transposons. How these regions are recognized as a source of piRNAs continues to be elusive. The aim of this study would be to establish how transgenes that include a fragment of the Long Interspersed Nuclear Elements (LINE)-like I transposon lead to an acquired TE resistance in Drosophila. We show that such transgenes, becoming inserted in distinctive euchromatic regions that generally do not produce tiny RNAs, turn into de novo bidirectional piRNA clusters that silence I-element activity within the germ line.737790-46-4 Order Strikingly, compact RNAs of both polarities are generated in the whole transgene and flanking genomic sequences–not only in the transposon fragment.Price of N-Methylsulfamoyl chloride Chromatin immunoprecipitation analysis shows that in ovaries, the trimethylated histone 3 lysine 9 (H3K9me3) mark associates with transgenes generating piRNAs.PMID:24635174 We show that transgenederived hsp70 piRNAs stimulate in trans cleavage of cognate endogenous transcripts with subsequent processing of the non-homologous components of these transcripts into piRNAs. INTRODUCTION Silencing of transposable elements (TEs) inside the germ cells depends upon a distinct class of 24?0-nt-long RNAs,PIWI-interacting RNAs (piRNAs), linked with PIWI clade Argonaute proteins (1,2). TE repression is provided by the two piRNA classes, the key along with the secondary piRNAs. In fly, most principal piRNAs match defective transposons and derive from the discrete pericentromeric and telomeric heterochromatic loci enriched in damaged repeated sequences, that are called piRNA clusters or piRNA master loci. Main piRNAs are processed in the putative lengthy single-stranded transcripts encoded by these loci and demonstrate a powerful 50 terminal uridine bias (1U bias). Major processing was not too long ago shown to become dependent on the activities of an endoribonuclease Zucchini (3?) and 30 trimmer of but unknown protein identity (6). A subsequent step of piRNA biogenesis, the `ping-pong’ mechanism, is usually a course of action resulting in amplification of antisense piRNAs (1,7). Secondary piRNAs are generated by means of piRNA-guided cleavage of transposon mRNA so that primary antisense piRNA and newly developed sense piRNA have 10-nt overlap. A sense piRNA that arises from this cleavage has adenine in position ten. Inside a equivalent process, sense piRNA can give rise to an antisense piRNA, by interacting having a piRNA cluster transcript. In line with this mode.