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. 2014 Jul 21;206(2):217–230. doi: 10.1083/jcb.201401002

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© 2014 Huang et al.

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Figure 1. — AGO3 Slicer activity is essential for transposon repression and piRNA biogenesis in ovaries. (A) Structure diagram of the AGO3 protein showing the essential active sites in the MID and PIWI domains. The PAZ and PIWI domains were analyzed by SMART program (http://smart.embl-heidelberg.de/), and the MID domain was identified between PAZ and PIWI domains. The conserved amino acids were identified via alignment of AGO3 with human AGO2 using CLUSTALW. Although the 579Y and 583K sites in the MID domain are responsible for binding to the 5′ terminus of piRNAs, the 643D, 713D, and 842H sites in the PIWI domain constitute the Slicer motif that is essential for the Slicer activity of AGO3 protein. (B) The DDH motif is essential for AGO3 Slicer activity in vitro. The in vitro assay for Slicer activity was performed according to the method described previously (Gunawardane et al., 2007) with minor modifications (see Materials and methods). Only MBP-AGO3^WT, but not MBP itself or MBP-AGO3^DD-AA, could cleave target RNA (Luc passenger siRNA) to release 9-nt products. (C and D) Ovaries from uasp-flag-ago3^WT; nosP-gal4:vp16 and uasp-flag-ago3^DD-AA; nosP-gal4:vp16 (D) or uasp-flag-ago3^YK-AA; nosP-gal4:vp16 (E) females were lysed and the supernatants were immunoprecipitated with anti-Flag antibody. RNAs were extracted from the precipitations and then treated with alkaline phosphatase. RNAs were labeled by ³²P using T4 polynucleotide kinase for detection of piRNAs (top). A small portion of the precipitations was used to perform Western blot assays with anti-Flag antibody to measure the levels of Flag-tagged AGO3 or its mutants in precipitations (bottom). Arrowhead indicates piRNAs. (E) Generation of ago3 mutants. The ago3 gene region contains 7 exons and the second one is biggest and encodes the major part of the PAZ domain. The tilling primers are in the 5′ terminus of this exon and 5′ terminus of the second intron. All alleles obtained are listed on the right. Two null alleles, ago3¹⁷⁷⁷ and ago3⁵⁰²⁷, both result in a stop codon in the second exon. (F) Ovaries from w¹¹¹⁸, uasp-flag-ago3^WT; nosP-gal4:vp16, uasp-flag-ago3^DD-AA; nosP-gal4:vp16, and uasp-flag-ago3^YK-AA; nosP-gal4:vp16 females were used to perform Western blot assays to show the abundance of ectopic Flag-AGO3 proteins. α-Tubulin was used as a loading control. (G) Quantitative PCR assays to detect the fold changes in steady-state RNA levels of Het-A, burdock, and blood transposons in ago3 mutant, uasp-flag-ago3^WT; nosP-gal4:vp16, ago3, and uasp-flag-ago3^DD-AA; nosP-gal4:vp16, ago3 (Normalized to w¹¹¹⁸; n = 3; error bars indicate standard errors).