Modulation of Aub-TDRD interactions elucidates piRNA amplification and germplasm formation

Abstract

Aub guided by piRNAs ensures genome integrity by cleaving retrotransposons, and genome propagation by trapping mRNAs to form the germplasm that instructs germ cell formation. Arginines at the N-terminus of Aub (Aub-NTRs) interact with Tudor and other Tudor domain-containing proteins (TDRDs). Aub-TDRD interactions suppress active retrotransposons via piRNA amplification and form germplasm via generation of Aub-Tudor ribonucleoproteins. Here, we show that Aub-NTRs are dispensable for primary piRNA biogenesis but essential for piRNA amplification and that their symmetric dimethylation is required for germplasm formation and germ cell specification but largely redundant for piRNA amplification.

Figure 1.. Aub, Tud, and Krimp do not localize to nuage in aub^RK.

(A) Schematic representation of wild type (WT) and arginine to lysine (RK) Aub constructs. (B) Western blot detection of immunoprecipitated Aub from ovary lysates of indicated genotypes. aub^WT = aub^QC42/HN2; nos > 3xHA-aub^WT, aub^RK = aub^QC42/HN2; nos > 3xHA-aub^RK. NI, nonimmune serum, sDMA-Aub is detected with SYM11 antibody. (C) Western blot analysis in ovary lysates from indicated genotypes. y w = y¹ w¹, aub = aub^HN2/QC42. Tub serves as loading control. (D) Color-inverted confocal images depicting the localization pattern of indicated proteins (grey) in stage 4–7 egg chambers from indicated genotypes. Scale bar = 5 μm. Source data are available for this figure.

Figure S1.. aub^RK is a hypomorphic mutant.

(A) Drosophila mating scheme for producing offspring that express aub^WT or aub^RK transgenes under nos promoter in aub null background. (B) Western blot detection of HA-tagged Aub^WT or Aub^RK in ovary lysates prepared from select transgenic flies. Tub serves as loading control. (C) Fecundity rate per female in flies aging from 5 to 10 d, from the indicated genotypes. y w = y¹ w¹, aub = aub^QC42/HN2, aub^WT = aub^QC42/HN2; nos > 3xHA-aub^WT, aub^RK = aub^QC42/HN2; nos > 3xHA-aub^RK. (D) Confocal images of Grk localization (green) in stage 10 egg chambers from indicated genotypes. Nuclei are stained with DAPI (blue). Scale bar = 25 μm. (E) Dorsal appendage morphology of embryos laid by mothers of indicated genotype. (F) Color-inverted confocal images of Aub localization (grey) in stage seven egg chambers. Transgenes are expressed by the maternal alpha-tubulin promoter in aub background. Scale bar = 15 μm. (G) Color-inverted images of Aub^RK localization (grey), through HA epitope, in a stage five egg chamber in aub⁺ background. Scale bars = 15 and 5 μm, respectively. (H) Color-inverted confocal images of Aub localization in stage seven egg chambers from indicated genotypes. tud = tud^1/Df(2R)Pu^rP133. Scale bars = 15 and 5 μm, respectively.

Figure S2.. Aub, Tud, and Krimp nuage localization is disrupted in aub^RK, while Ago3 enters the primary piRNA pathway.

(A) Color-inverted confocal images of Aub, Tud, Krimp, Ago3, Qin, Vas, and PIWI localization (grey) in stage 4–7 egg chambers from indicated genotypes. Zoomed-out versions from specimens depicted in main Fig 1D. y w = y¹ w¹, aub = aub^QC42/HN2, aub^WT/RK = aub^QC42/HN2; nos > 3xHA-aub^WT/RK. Scale bar = 15 μm. (B) Genome browser snapshot of normalized piRNA reads from Aub, Ago3, and Piwi libraries of indicated genotypes, mapping to flamenco and traffic jam loci. Plus and minus strand mapping piRNA reads are indicated with blue and green colors, respectively.

Figure 2.. Intact primary piRNA biogenesis and Aub^RK piRNA loading but collapse of heterotypic ping-pong in aub^RK.

(A) Proteins (top) and bound piRNAs (bottom) of immunoprecipitated Aub, Ago3, and Piwi from indicated genotypes. aub^WT,RK = aub^QC42/HN2; nos > 3xHA-aub^WT,RK. NI, nonimmune serum. (B) piRNA length distribution. (C) piRNA nucleotide composition. (D) Relative position of piRNA 5′ ends bound to indicated proteins. (E) Heat map representing z-scores for a 10-nt overlap between Aub–Ago3 transposon aligning piRNA pairs in indicated libraries. piRNA transposons are ranked by mean total piRNA abundance. ppkm, piRNA pairs per kilobase per million. Source data are available for this figure.

Figure 3.. piRNA biogenesis and ping-pong are largely intact in the absence of sDMAs.

(A) Western blot detection of immunoprecipitated Aub from ovary lysates of indicated genotypes. wⁱ = MTD > w^TRiP, csulⁱ¹ = MTD > csul^TRiP1, csulⁱ² = MTD > csul^TRiP2. NI, nonimmune serum, sDMA-Aub is detected with SYM11 antibody. (B) Western blot detection analysis in ovary lysates from indicated genotypes. Tub serves as loading control. (C) Color-inverted confocal images depicting the localization pattern of indicated proteins (grey) in stage 5–8 egg chambers from indicated genotypes. Scale bar = 5 μm. (D) piRNA nucleotide composition. (E) Relative position of piRNA 5′ ends bound to indicated proteins. (F) Heat map representing z-scores for a 10-nt overlap between Aub–Ago3 transposon-aligning piRNA pairs in indicated libraries. piRNA transposons are ranked by mean total piRNA abundance. ppkm, piRNA pairs per kilobase per million. Source data are available for this figure.

Figure S3.. Minimal nuage is formed in csul.

Color-inverted confocal images of Aub, Tud, Krimp, and Ago3 localization (grey) in stage 5–8 egg chambers from indicated genotypes. Zoomed-out versions from specimens depicted in main Fig 3C. wⁱ = MTD > w^TRiP, csulⁱ¹ = MTD > csul^TRiP1, csulⁱ² = MTD > csul^TRiP2. Scale bar = 15 μm.

Figure S4.. Aub^RK reclaims its original localization to germplasm when coexpressed with endogenous wild-type Aub.

(A) Confocal image of Aub^RK localization (green), through HA epitope, at the posterior pole of a stage 10 egg chamber in aub⁺ background. Nuclei are stained with DAPI (blue). Scale bar = 10 μm. (B) Western blot detection of Ago3 from ovary lysate and Ago3-380 immunoprecipitated protein. Antibodies Ago3-380 (upper panel) and Ago3-7B4-C2 (lower panel) were used. NI, nonimmune serum.

Figure 4.. Germplasm does not form when Aub lacks sDMAs or when arginines are replaced with lysines, resulting in sterile offspring.

(A) Confocal images depicting Aub and Tud localization pattern (green) at the posterior pole of stage 10 egg chambers from indicated genotypes. Nuclei are stained with DAPI (blue). y w = y¹ w¹, aub = aub^QC42/HN2, aub^WT,RK = aub^QC42/HN2; nos > 3xHA-aub^WT,RK, csulⁱ¹ = MTD > csul^TRiP1, tud = tud^1/Df(2R)Pu^rP133. Scale bar = 10 μm. (B) Ovaries dissected from female adult offspring of indicated maternal genotypes. csulⁱ² = MTD > csul^TRiP2. Scale bar = 100 μm.