Volume 56, Issue 5, Pages 708-716 (December 2014) The Initial Uridine of Primary piRNAs Does Not Create the Tenth Adenine that Is the Hallmark of Secondary piRNAs  Wei Wang, Mayu Yoshikawa, Bo W. Han, Natsuko Izumi, Yukihide Tomari, Zhiping Weng, Phillip D. Zamore  Molecular Cell  Volume 56, Issue 5, Pages 708-716 (December 2014) DOI: 10.1016/j.molcel.2014.10.016 Copyright © 2014 Elsevier Inc. Terms and Conditions

Molecular Cell 2014 56, 708-716DOI: (10.1016/j.molcel.2014.10.016) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 Models for the Origin of the Adenine at the Tenth Position of Ago3 piRNAs (A) Model I posits that g1U causes t1A by Watson and Crick pairing. Model II accommodates the interactions between the 5′ phosphate of guides and the phosphate-binding pocket in Argonaute proteins. (B) Nucleotide composition of g1 from guide piRNAs and t1 (g10) from their targets inferred from Ping-Pong analysis in w1 ovaries. (C) Numbers of cis Ping-Pong pairs. (D) Aub requires g2–g16 complementarity to cleave a target RNA. The frequency of pairing for the individual positions g11–g16 was higher in the biological data than in the shuffled controls (left). When examining the extent of contiguous pairing rather than the frequency of pairing at individual positions, the pairing frequency for the biological data was only higher than the pairing frequency in the shuffled controls when base pairing extended from position g2 to at least g13. Error bars (mean ± 2 × SD, n = 10) indicate the paired frequency for the shuffled controls. See also Figure S1. Molecular Cell 2014 56, 708-716DOI: (10.1016/j.molcel.2014.10.016) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 trans Ping-Pong Analysis Reveals a t1A Preference for Aub (A) Schema for detecting trans-targets. (B) Ping-Pong profile for each guide:target trans pair with at least 16 nt complementarity. See also Figures S2 and S3. Molecular Cell 2014 56, 708-716DOI: (10.1016/j.molcel.2014.10.016) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 The t1A Preference for Aub Is Reinforced in Aub:Ago3ADH and Aub:degradome Guide:Target Pairs (A) Nucleotide composition of position g1 of piRNAs and position t1 of their targets as inferred from piRNAs bound to Ago3ADH or detected directly in the RNA degradome of w1 ovaries. (B) Guide piRNAs and their inferred targets were determined by sequencing the piRNAs in immunoprecipitates of Aub or Ago3ADH. Molecular Cell 2014 56, 708-716DOI: (10.1016/j.molcel.2014.10.016) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 Siwi, the Silkmoth Ortholog of Aub, Prefers t1A Targets (A) FLAG-Siwi or FLAG-BmAgo3 were immunopurified from BmN4 lysates and incubated with target RNAs bearing t1A, U, G, or C but otherwise fully complementary to an abundant g1U or g1C piRNA. Representative data are shown. (B) Quantification of the experiments in (A). Mean ± SD (n = 3) and p values of one-way ANOVA and Tukey’s multiple comparison test are shown. See also Figure S4. Molecular Cell 2014 56, 708-716DOI: (10.1016/j.molcel.2014.10.016) Copyright © 2014 Elsevier Inc. Terms and Conditions