1. Volume 44, Issue 4, Pages 572-584 (November 2011)
Heterotypic piRNA Ping-Pong Requires Qin, a Protein with Both E3 Ligase and Tudor Domains 
Zhao Zhang, Jia Xu, Birgit S. Koppetsch, Jie Wang, Cindy Tipping, Shengmei Ma, Zhiping Weng, William E. Theurkauf, Phillip D. Zamore 
Molecular Cell 
Volume 44, Issue 4, Pages (November 2011)
DOI: /j.molcel
Copyright © 2011 Elsevier Inc. Terms and Conditions

2. Figure 1 The qin Gene and Qin Protein
(A) qin resides on the right arm of chromosome 3, comprising two predicted genes separated by a 51,682 bp intron. qin encodes a protein with E3 ligase and Tudor domains. The blue and red bars indicate the location of probes used in Northern hybridization analyses.
(B) Northern analysis of qin transcripts in ovary RNA using the red probe. Asterisk marks an unidentified transcript.
(C) Agarose gel analysis of the 5′ rapid amplification of cDNA ends (RACE) products from control and gf-1/+; Df/+ ovaries.
(D) Ovary RNA analyzed in parallel by Northern hybridization using the red and blue probes.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions

3. Figure 2 Qin Silences Transposons in Drosophila Ovaries
(A) Northern hybridization for Burdock mRNA.
(B) Quantitative RT-PCR was used to measure transposon expression. Data are normalized to rp49 (RpL32) expression. The bars report mean ± standard deviation for three biological replicates.
(C) Expression of transposons measured using whole genome tiling arrays for qin1/Df ovaries, relative to control (w1). The dashed line indicates equivalent expression in the two genotypes. The 13 transposon families whose expression increased >3-fold among 3 biological replicates (false discovery rate, FDR, < 0.1) in qin mutants, compared to control, are marked in red.
(D) The change in transposon expression, relative to control, was compared for qin and ago3 mutant ovaries.
(E) The change in transposon expression, relative to control, was compared for qin and aub mutant ovaries.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions

4. Figure 3 Qin Colocalizes with Aub and Ago3 in Nuage
(A) Germline γH2Av foci, a hallmark of DNA damage, accumulated in qin and aub mutant ovaries. Stage 2–3 egg chambers are shown.
(B) Stellate silencing in testes requires Qin.
(C) Qin colocalizes with Ago3 and Aub in the nuage.
(D) Enlarged view of the nuage of a single germline nurse cell. At right, quantification of the fluorescence corresponding to Qin::EGFP, Ago3, and Aub along the dash line drawn on the left panel. Scale bars for (A)–(C), 10 μm. Scale bar for (D), 2 μm.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Mutation of qin Decreases the Antisense Character of piRNA Populations
(A) Steady-state protein abundance in control and qin1/Df ovaries, relative to qin/TM6B (red dashed line). Data are mean ± standard deviation for three independent biological samples.
(B) Box plots report the change in abundance of all piRNAs mapping to transposons in control (cn1; ry506), qin/TM6B and qin1/Df ovaries and of the piRNAs associated with immunoprecipitates of each PIWI protein in qin/TM6B and qin1/Df ovaries.
(C) Top: antisense piRNA abundance for each transposon family in qin1/Df ovaries was compared to control. Bottom: box plots report the change in piRNA abundance by family for each of the three groups of transposon families defined in the upper panel.
(D) The piRNA sense fraction for each transposon family was compared between control and qin mutant ovaries.
(E) The sense fraction for each transposon family for Aub- and Ago3-bound piRNAs was compared between qin heterozygous and qin1/Df mutant ovaries.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions

6. Figure 5 Futile Aub:Aub Homotypic Ping-Pong Prevails in qin Mutants
(A) Ping-Pong Z score, measuring the significance of 5′, 10 nt complementarity between piRNAs bound to Aub or Ago3, was calculated for Aub:Aub and Aub:Ago3 pairs for each transposon family for qin/TM6B and qin1/Df ovaries.
(B) Sequence logos (Schneider and Stephens, 1990) for the first 10 nt of Aub- and Ago3-bound total piRNAs; Figure S9A illustrates all 29 nt.
(C) Sequence logos for sense and antisense piRNAs coimmunoprecipitated with Aub and uniquely participating in Aub:Aub Ping-Pong and for piRNAs coimmunoprecipitated with Aub or Ago3 and uniquely participating in Aub:Ago3 Ping-Pong. Insets report the cumulative number of species required to account for a given percent of reads; the total number of reads composing each logo appears above each inset.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions

7. Figure 6 The Association of Ago3 with Aub Requires Qin but not RNA
(A) Strategy for the experiments in (B)–(D).
(B) Binding of Ago3 to Aub does not require RNA.
(C) Treatment of ovary lysate with RNase A degrades both miRNAs and rRNAs as evidenced by the loss of miR-317 and 2S rRNA.
(D) FM-Ago3 associates with Aub in qin/TM6B ovaries; the interaction was reduced in qin1/Df. After probing with anti-FLAG to detect Ago3, anti-Aub was used to measure the efficiency of immunoprecipitation. Figure S9B presents the entire membrane and Figures S9C and S9D present two additional biological replicates.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
Among piRNA Pathway Mutants, qin Has a Unique Effect on the Structure of the Ovarian piRNA Population
For each mutant the figure summarizes the change in the abundance of antisense and of sense piRNAs, relative to cn; ry control ovaries; the fraction of all piRNAs with the sense orientation; and the Ping-Pong Z score. Box plots present the distribution for the 93 transposon families analyzed; outliers are not shown. Numbers report the median value corresponding to the thick vertical line on each box plot.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions