Volume 47, Issue 6, Pages 970-979 (September 2012) A Role for Fkbp6 and the Chaperone Machinery in piRNA Amplification and Transposon Silencing  Jordi Xiol, Elisa Cora, Rubina Koglgruber, Shinichiro Chuma, Sailakshmi Subramanian, Mihoko Hosokawa, Michael Reuter, Zhaolin Yang, Philipp Berninger, Andres Palencia, Vladimir Benes, Josef Penninger, Ravi Sachidanandam, Ramesh S. Pillai  Molecular Cell  Volume 47, Issue 6, Pages 970-979 (September 2012) DOI: 10.1016/j.molcel.2012.07.019 Copyright © 2012 Elsevier Inc. Terms and Conditions

Molecular Cell 2012 47, 970-979DOI: (10.1016/j.molcel.2012.07.019) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 L1 Retrotransposon Derepression in Fkbp6 Mutant Mice (A) Detection of Fkbp6 in Tdrd1 immunoprecipitates from mouse testes lysates. (B) Immunofluorescence detection of indicated proteins on embryonic day 18 (E18) testis cryosections. Fkbp6 is cytosolic and not enriched in pi-bodies (arrowheads) containing Mili and Tdrd1. Scale bar is indicated. (C and D) Northern analysis (C) and quantitative-reverse transcription PCR detection (D) for transposon mRNAs in testis total RNA from indicated genotypes. Loading control (ethidium bromide for rRNA) is shown. The number in brackets identifies the biological replicates. Each bar represents individual biological replicate and standard deviation (SD) between technical replicates within a biological sample is indicated. (E) Detection of L1ORF1p in Fkbp6 mutant E18 testis. (F) Methylation-sensitive Southern blot analysis for L1 elements using testis genomic DNA. A methylation-sensitive restriction fragment in the Fkbp6 knockout mutant is indicated (arrow). HpaII (H) and MspI (M) are the methylation-sensitive and -insensitive restriction enzymes used, respectively. Molecular Cell 2012 47, 970-979DOI: (10.1016/j.molcel.2012.07.019) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 Fkbp6 Is Required for Biogenesis of Miwi2 piRNAs (A) Association of piRNAs with Piwi proteins examined by immunoprecipitation and 5′ end labeling from new born pups (P0). RNA markers (nucleotides, nt) are shown. (B–D) Genome annotations (B), length distribution (C), and mapping to transposon consensus of reads (D) in testes total small RNA libraries from P0 animals. (E) Distribution of read lengths of Mili-associated piRNAs from P0 animals is plotted. (F) Genome annotation profile of reads is shown. (G) The nucleotide preference at indicated positions of the piRNA is analyzed. (H) Distribution of Mili reads on LINE1 consensus and two prepachytene piRNA clusters. (I) Prepachytene piRNAs are still associated with Mili in the Fkbp6 mutant 10-day-old (P10) animals. (J) Detection of indicated proteins in E18.5 fetal testes from indicated genotypes. Scale bar is shown. Molecular Cell 2012 47, 970-979DOI: (10.1016/j.molcel.2012.07.019) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 Fkbp6 Is Inactive as an Isomerase but Associates with Hsp90 via Its TPR Domain (A) Prolyl isomerization assay with recombinant full length Fkbp6 or its FK domain alone. Positive controls (Fkbp5, -3, and -12) and negative controls (-, buffer only and GST) are indicated. The standard deviation (SD) between technical replicates is indicated. (B) Isothermal calorimetry (ITC) measurements of binding between Fkbp12 or Fkbp6 (FK domain only) and the small molecule inhibitor FK-506. High affinity observed with Fkbp12 is reported in Kd. (C) Surface model of FK domains of human Fkbp12 and human Fkbp6 generated from crystallographic data. (D) Alignment of residues forming the FK domain from demonstrated active or inactive isomerases. Identity (dark red) or biochemical conservation (light red) is indicated by different background colors. The asterisk indicates one of the key residues (W60) in the active isomerases. (E) Sequence alignment of TPR domains from indicated proteins. Highly conserved residues are shaded in red. The residue K254 (arrowhead) in mouse Fkbp6 when mutated abolishes interaction with Hsp90. (F) Fkbp6 interacts with Hsp90 in mouse testes extract. (G) HA-Fkbp6 coprecipitates endogenous Hsp90 from HEK293T cells by interaction via its TPR domain. (H) Direct interaction between Bombyx Shutdown (BmShu) and BmHsp90 is demonstrated using recombinant proteins. Molecular Cell 2012 47, 970-979DOI: (10.1016/j.molcel.2012.07.019) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 PingPong Byproducts Accumulate in Ago3 Complexes upon Hsp90 Inhibition (A) BmN4 cells treated with DMSO or Geldanamycin (GA) (incubation time in days) were subject to immunoprecipitation (IP) with Piwi antibodies. Bound RNAs were detected by 5′ end labeling, revealing 16 nt RNAs in Ago3 complexes. Note that RNA markers generated by alkaline hydrolysis (Alk.) migrate one nucleotide (nt) faster than the actual size. (B) Density of normalized reads from indicated libraries mapping to Bombyx transposon consensus. (C) Heatmap showing overall strand-bias of reads from indicated libraries on 345 Bombyx transposon consensus sequences. Classification of transposons into groups is based on that proposed by (Li et al., 2009). (D) Correlation plot showing the distance between the 5′ end of Ago3 piRNAs and 3′ ends of 16-mer reads (byproducts) from the opposite strand. A model showing the distance relationships between the Ping-pong partner piRNAs and 16-mer sequences. (E) Appearance of 16-mer species is abolished by a catalytic triad mutation (ADH) in Ago3. (F) Correlation plot showing distance between 5′ ends of Siwi piRNAs and 16-mers (on the same strand) over indicated transposon consensus. (G) Correlation plot showing distance between 5′ ends of Ago3-associated short reads (12–20 nt) from untreated BmN4 cells and Ago3 piRNAs (on the opposite strand) over the indicated transposon consensus. The peak at position 9 indicates a 10 bp overlap, potentially arising from degraded piRNAs. The peak at 25 is indicative of short reads whose 5′ ends are 26 nt away from the 5′ end of Ago3 piRNAs. This peak derives exclusively from reads that are 16 nt in length (bottom panel), indicating that these are potentially from the 16-mer byproducts generated during secondary biogenesis in untreated cells. (H) Localization of endogenous Piwi proteins in BmN4 cells and their coloclaization with HA-tagged Bombyx Vasa. (I) Arginine (R)→Lysine (K) mutation of Ago3 affects its granule accumulation in BmN4 cells. (J) Association of piRNAs with HA-tagged Piwi proteins and their R→K mutant versions in BmN4 cells. (K) The mislocalized Ago3R→K mutant fails to accumulate 16-mers upon Hsp90 inhibition (GA). (L) 5′ end labeling of RNAs with or without prior dephosphorylation is shown. (M) A model showing the generation of 16-mers and implication of a 5′ nuclease activity (5′Nuc) in the Ping-pong cycle. Molecular Cell 2012 47, 970-979DOI: (10.1016/j.molcel.2012.07.019) Copyright © 2012 Elsevier Inc. Terms and Conditions