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. Author manuscript; available in PMC: 2018 Jun 6.
Published in final edited form as: Nature. 2017 Dec 6;552(7684):268–272. doi: 10.1038/nature25018

Figure 2. piRNAs, but not siRNAs, modulate P-element splicing in germ cells.

Figure 2

All analyses were performed in a Harwich background. a, RT-qPCR analysis on piRNA- (piwi, aub, ago3, spnE, and vas) and siRNA-biogenesis (dcr2 and ago2) mutant adult ovaries. Results are presented as means of fold changes in mutants in relation to respective heterozygote ± standard deviation (n>=2 independent biological replicate experiments). b, Ethidium bromide-stained gel displaying RT-PCR reactions with primers flanking the P-element IVS3 intron in piRNA- and siRNA-biogenesis mutants. Size scale in base pairs (bp) is presented for each gel. Experiments were repeated two times with similar results. For gel source data, see Supplementary Figure 1. c, Density plots for normalized strand-specific mRNA steady-state levels (measured by RNA-seq and represented as reads per million, RPM) over consensus P-element sequence (top diagram) in aub/+ heterozygous (yellow, top plot) and aub mutant (blue, bottom plot) adult ovaries. The number and position of split-reads (represented by arcs that connect exons) observed for IVS1, IVS2, and IVS3 splicing junctions is shown below each density plot. Experiments were repeated two times with similar results. d-e, Percentage of splicing for P-element IVS1, IVS2 (d), and IVS3 (e). Splicing was quantified using RNA-seq analysis in aub/+ heterozygous (yellow), aub mutant (blue), piwi/+ heterozygous (beige), and piwi mutant (purple) adult ovaries. Percentage of splicing was calculated as the number of split-reads for each splicing junction normalized to the total number of reads mapping to the same junction. Results are represented as means ± standard deviation (n=2 independent biological replicate experiments). f, Representative confocal projections of RNA-FISH signal (grayscale) showing the accumulation of sense RNA for Burdock and P-element transposons in heterozygous and mutant egg chambers. Bottom panels depict projections of representative nurse cell nuclei (purple dotted line) for the same genotypes. Scale bars, 20 μM. Experiments were repeated two or more times with similar results.