Extended Data Figure 3. Moonshiner forms an alternative TFIIA-TRF2 complex enriched at piRNA clusters.

a, TRF2 isoform characterization by total wildtype ovary RNAseq (top panel) and LAP-Moon co-IP mass spectrometry (lower panel). The identified TRF2 peptides show that Moonshiner is in complex only with the shorter TRF2 isoform. We therefore investigated specifically this isoform, also known as TRF2S, in the remainder of the paper. b-c, Absolute peptide peak intensities for the main protein interactors identified in Fig. 2b,c. Peak area intensities are displayed as IP values subtracted that of the paired control IP experiment. Based on this, we conclude that TFIIA-S and TRF2 are robust Moonshiner interactors (supportive of an alternative TFIIA-TRF2 complex), while only a small fraction of Moonshiner is bound to Deadlock. Furthermore, the data show that in ovaries, TRF2 interacts predominantly with canonical TFIIA, but also clearly with Moonshiner. Black dots represent individual replicate values. Orange bars show median values. d, Western blot analyses as Extended Data Fig. 2d, but addressing interaction with HA-TRF2 (lower bands likely represent TRF2 decay intermediates). e, Schematic of a developing Drosophila ovariole with germline cells in beige and somatic support cells in green. Confocal images were typically taken from egg-chambers of stage 7 (highlighted by a dashed box). f, Whole egg chamber confocal image stained for DNA (DAPI; blue), LAP-Moonshiner (GFP auto-fluorescence; green), Rhino (magenta), and Deadlock (cyan). The circled nucleus is shown in Fig. 2d. g, Fluorescence images of nurse cell nuclei (depleted for indicated factors using sh-lines) indicating levels and localization of Moonshiner and Rhino (scale bar: 5 µM). h, Western blot showing levels of LAP-Moonshiner in ovaries where the indicated factors were depleted in the germline via sh-lines (ATP synthase serves as loading control).