Inhibition of Hsp90 reduces Siwi- and BmAgo3-bound piRNAs in vivo. (A) Abundance of 1U (but not 10A) and 10A (but not 1U) piRNAs in the total small RNA library with DMSO or 17-AAG treatment. Reads were normalized to 5S ribosomal RNA fragments. 1U piRNAs and 10A piRNAs were reduced by ∼60% and ∼40%, respectively, by 17-AAG. (B) The 1U/10A composition of Siwi- and BmAgo3-bound piRNAs was essentially unaffected by 17-AAG treatment. (C) Comparison of the 1U/10A ratio of piRNAs for each transposon with DMSO (x-axis) or 17-AAG treatment (y-axis). (Red circles) Siwi- and (blue circles) BmAgo3-bound piRNAs; the color density reflects the P-value of a Fisher’s exact test for 2 × 2 contingency tables consisting of the numbers of 1U and 10A piRNA reads under DMSO and 17-AAG treatment conditions. The change was small overall, but statistically significant changes of individual transposon-derived piRNAs pointed to loosening of the specific 1U and 10A biases of Siwi- and BmAgo3-bound piRNAs, respectively. (D) The length distribution of Siwi-bound piRNAs (27–28 nt) and BmAgo3-bound piRNAs (26–27 nt) was essentially unaffected by 17-AAG. (E) Length distribution and the abundance of piRNAs in the total small RNA library with DMSO or 17-AAG treatment. Reads were normalized to 5S ribosomal RNA fragments. The shift in the length distribution indicates that the reduction of Siwi-bound piRNAs was more pronounced than BmAgo3-bound piRNAs.
