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Series GSE102013 Query DataSets for GSE102013
Status Public on Aug 16, 2017
Title Recruitment of Armitage and Yb to a transcript triggers its phased processing into primary piRNAs in Drosophila ovaries
Organism Drosophila melanogaster
Experiment type Non-coding RNA profiling by high throughput sequencing
Summary Small RNAs called PIWI -interacting RNAs (piRNAs) are essential for transposon control and fertility in animals. Primary processing is the small RNA biogenesis pathway that uses long single-stranded RNA precursors to generate millions of individual piRNAs, but the molecular mechanisms that identify a transcript as a precursor are poorly understood. Here we demonstrate that artificial tethering of the piRNA biogenesis factor, Armi, to a transcript is sufficient to direct it into primary processing in Drosophila ovaries and in an ovarian cell culture model. In the fly ovarian somatic follicle cells, the transcript becomes cleaved in a stepwise manner, with a 5ʹ→3ʹ directionality, liberating U1-containing ~24 nt piRNAs that are loaded into Piwi. Although uridines are preferred for generation of piRNA 5ʹ ends, processing takes place even in their absence, albeit at a lower efficiency. We show that recombinant Armi has 5ʹ→3ʹ helicase activity, and mutations that abolish it reduce piRNA processing. Another somatic piRNA pathway factor Yb, and an interactor of Armi, is also able to trigger piRNA biogenesis when tethered to a transcript. Tethering-mediated primary piRNA biogenesis is also functional in the fly ovarian germline and loads all the three PIWI proteins present in this environment. Our study finds a broad correlation between piRNA processing and localization of the tethered factors to the cytoplasmic perinuclear ribonucleoprotein granules called germline nuage or somatic Yb bodies. We conclude that transcripts bound by Armi and Yb are identified as piRNA precursors, resulting in localization to cytoplasmic processing granules and their subsequent engagement by the resident piRNA biogenesis machinery.
 
Overall design Different proteins were tested in their ability to trigger piRNA production from the reporter transcript upon their tethering to the transcript. Transgenic fly lines and ovarian somatic cell (OSC) culture were used. Immunopurified piRNAs were sequenced and those originating from the reporter transcript were identified.
 
Contributor(s) Raman Pandey R, Homolka D, Chen K, Sachidanandam R, Fauvarque M, Pillai RS
Citation(s) 28827804
Submission date Jul 28, 2017
Last update date Jul 25, 2021
Contact name Ramesh Pillai
E-mail(s) ramesh.pillai@unige.ch
Organization name University of Geneva
Department Department of Molecular Biology
Street address 30, Quai Ernest-Ansermet
City Gneveva
ZIP/Postal code CH-1211
Country Switzerland
 
Platforms (1)
GPL13304 Illumina HiSeq 2000 (Drosophila melanogaster)
Samples (108)
GSM2721796 RR242;protein: none;reporter 1347:Luc-LacZ;OSC;PIWI IP
GSM2721797 RR243;protein: none;reporter 1347:Luc-LacZ;OSC;PIWI IP
GSM2721798 RR248;protein: none;reporter 1347:Luc-LacZ;OSC;PIWI IP
Relations
BioProject PRJNA396206
SRA SRP113774

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE102013_RAW.tar 14.2 Mb (http)(custom) TAR (of TXT)
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
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