MYCBP is highly expressed in the ovaries

MYCBP was one of the five proteins identified by mass spectrometry following co-immunoprecipitation with Sakura-EGFP from ovary lysates, suggesting a physical interaction between Sakura and MYCBP (Table 1) [8]. We decided to investigate MYCBP since it was predicted to form binary and ternary complexes with Sakura and Otu and structurally resembles Sakura, as we show below (Figs 2C–2E and 3A).

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Table 1. Number of unique peptide counts detected by mass-spec of Sakura-EGFP co-IP samples.

https://doi.org/10.1371/journal.pgen.1011792.t001

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Fig 2. MYCBP interacts with Otu and Sakura.

(A) Co-immunoprecipitation (co-IP) using anti-GFP magnetic beads followed by Western blotting. Ovary lysates expressing MYCBP-EGFP in mycbp^+/+ background and those from w¹¹¹⁸ negative control were analyzed. (B) Co-IP using anti-MYCBP antibodies and ovary lysates from w¹¹¹⁸. IgG was used as a negative control IP. * indicates non-specific bands. (C-E) Structures of (C) MYCBP•Otu(N), (D) MYCBP•MYCBP•Otu(N), and (E) MYCBP•Sakura predicted using Alphafold. (F, G) Schematic diagrams of full-length and fragment constructs of (F) Otu and (G) Sakura used in co-IP assays. The binding assay results from (H) is summarized. (G) Full-length Sakura and Sakura fragments tested in co-immunoprecipitation assays. N: N-terminal, M: middle, C: C-terminal. The co-IP assay results from (H) and (I) are summarized. (H) Co-IP using anti-HA beads followed by Western blotting. S2 cell lysates co-expressing MYCBP-mCherry-3xHA and 3xFLAG-Otu (full-length or fragments) were tested. 3xFLAG-EGFPserved as a negative control. * indicates antibody heavy and light chains. Yellow triangles indicate Otu fragments. (I) Co-IP using anti-FLAG beads followed by Western blotting. S2 cell lysates co-expressing 3xFLAG-EGFP-MYCBP and Sakura-mCherry-3xHA (full-length or fragments) were tested. mCherry-3xHA served as a negative control. Blue triangles indicate Sakura fragments.

https://doi.org/10.1371/journal.pgen.1011792.g002

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Fig 3. Sequential co-immunoprecipitation shows MYCBP, Sakura, and Otu form a ternary complex.

(A) MYCBP•Sakura•Otu structure predicted by AlphaFold. (B) Sequential co-IP followed by Western blotting. S2 cell lysates co-expressing Sakura-EGFP-HRV3Csite-3xFLAG, MYCBP-mCherry-3xHA, and Myc-Otu were used. EGFP-HRV3csite-3xFLAG served as a negative control. The first IP was performed using anti-FLAG beads and eluted using HRV3C protease. Second IP was performed with anti-HA beads. Non-specific bands are marked with *.

https://doi.org/10.1371/journal.pgen.1011792.g003

To examine MYCBP protein expression, we generated polyclonal anti-MYCBP antibodies against the recombinant full-length MYCBP protein. Western blot using this antibody revealed that MYCBP protein is highly expressed in the ovary, including germarium- stage 11 egg chambers, stage 12–13 egg chambers and stage 14 egg chambers (Fig 1C).

MYCBP is highly expressed in germ cells and cytoplasmic

To analyze MYCBP expression and localization, we created transgenic flies expressing a MYCBP-EGFP fusion protein under the control of the mycbp promoter. Oogenesis begins in the germarium, which contains 2–3 GSCs, identifiable by round, unbranched spectrosomes in contact with cap cells (Fig 1A) [5]. In contrast, developing cysts exhibit branched fusomes. We used hu-li tai shao (HTS) antibody to visualize both spectrosomes and fusomes (Fig 1D). Confocal imaging showed that MYCBP-EGFP localizes to the cytoplasm of germ cells, including GSCs, cysts, nurse cells, and developing oocytes (Figs 1D, and S1A and S1B). Within egg chambers, MYCBP-EGFP was highly expressed in germline cells (nurse cells and developing oocytes) and was particularly enriched in developing oocytes, whereas its expression was low in was somatic follicle cells and undetectable in somatic stalk cells (S1A and S1B Fig). Consistently, anti-MYCBP staining of control w¹¹¹⁸ ovaries revealed similar expression and localization patterns (Figs 1E and S1C).

MYCBP forms complexes with Otu and Sakura

To identify MYCBP-interacting proteins, we performed co-immunoprecipitation using anti-GFP beads on ovary lysates from MYCBP-EGFP-expressing flies, followed by mass spectrometry. Ovary lysates from w¹¹¹⁸ flies served as negative controls. Mass spectrometry identified Otu as the top interactor and also detected Sakura (Table 2).

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Table 2. Abundance of Proteins identified by mass-spec of MYCBP-EGFP co-IP samples.

https://doi.org/10.1371/journal.pgen.1011792.t002

Western blotting confirmed that both Otu and Sakura co-immunoprecipitate with MYCBP-EGFP in ovary lysates (Fig 2A). Additionally, endogenous MYCBP co-immunoprecipitated with endogenous Otu and Sakura from wild-type ovary lysates (Fig 2B), confirming these interactions in vivo.

Endogenous MYCBP also co-immunoprecipitated with MYCBP-EGFP in ovary lysates, suggesting an interaction between MYCBP proteins (Fig 2A). N-terminally 3xFLAG-EGFP-tagged MYCBP co-immunoprecipitated with C-terminally mCherry-3xHA tagged MYCBP in S2 cells (S2A Fig), confirming the interaction between two MYCBP proteins.

We predicted the structures of the MYCBP•MYCBP, MYCBP•Otu, MYCBP•MYCBP•Otu, and MYCBP•Sakura complexes using AlphaFold [20; 21], which gave us reasonable predicted structures with sufficient inter-molecular interaction without steric hindrance when using the N-terminal region (1–405aa. Otu(N)), but not the C-terminal region, of Otu (Figs 2C–2E, and S3). In the predicted MYCBP•MYCBP structure, two MYCBP molecule have pseudo-symmetric interaction involving mostly their alpha-helices (S3A Fig). In the predicted structures of both MYCBP•Otu and MYCBP•MYCBP•Otu complexes, Tudor domain of Otu is not involved in the direct interaction with MYCBP. To test these, we created epitope-tagged full-length and truncated Otu—Otu(∆Tudor, corresponding to the endogenous 98 kDa isoform), Otu(N), and Otu(C)—and co-expressed them with epitope-tagged full-length MYCBP in S2 cells. (Fig 2F). Co-immunoprecipitation followed by Western blotting showed that all fragments except Otu(C) interacted with MYCBP (Fig 2H), demonstrating the formation of the MYCBP•Otu and/or MYCBP•MYCBP•Otu complex consistent with the AlphaFold prediction.

AlphaFold predicted a pseudo-symmetric alpha-helical interaction between MYCBP and Sakura, involving the N-terminal and middle regions of Sakura (1–100 aa) (Fig 2E). We generated epitope-tagged full-length and truncated Sakura—Sakura(NM), Sakura(NC), Sakura(MC), Sakura(N), Sakura(M), and Sakura(C) —and tested their ability to bind epitope-tagged full-length MYCBP in S2 cells (Fig 2G). Only full-length Sakura and Sakura(NM) interacted with MYCBP (Fig 2I), supporting the predicted MYCBP•Sakura interaction.

AlphaFold also predicted that MYCBP, Sakura, and Otu could form a ternary complex, MYCBP•Sakura•Otu (Fig 3A). To test this, we performed sequential co-immunoprecipitation from S2 cells co-expressing Sakura-EGFP-HRV3Csite-3xFLAG, MYCBP-mCherry-3xHA, and Myc-Otu (Fig 3B). After FLAG-IP and elution by HRV3C protease cleavage, a second HA-IP detected all three proteins, but not when EGFP-HRV3Csite-3xFLAG was used as a control. Additional co-immunoprecipitation showed that MYCBP-mCherry-3xHA specifically bound Myc-Otu, but not Myc-tagged CycA, Bam, EGFP, or Drosophila ortholog of MYC (dMyc) (S2B and S2C Fig), confirming the specificity of the interaction. We conclude that MYCBP, Sakura, and Otu form a ternary complex.

Additionally, AlphaFold predicted structures of Sakura•Sakura, Sakura•Otu, and Sakura•Sakura•Otu complexes (S2D–S2F Fig) and we confirmed the interaction between two Sakura proteins using S2 cells (S2D Fig) and that between Sakura and Otu [8].

Neither MYCBP nor Sakura affects Otu’s deubiquitinase activity in vitro

Otu possesses deubiquitinase activity [18]. While our previous study showed that Sakura does not influence Otu’s deubiquitinase activity in vitro [8], we hypothesized here that MYCBP may affect Otu’s deubiquitinase activity positively or negatively. Using the same Ub-Rhodamine 110-based assay, we tested whether MYCBP, with or without Sakura, affects Otu’s deubiquitinase activity. The addition of recombinant MYCBP, Sakura, or both had no effect on Otu’s deubiquitinase activity, and neither MYCBP nor Sakura showed deubiquitinase activity (S4 Fig). We concluded that neither MYCBP nor Sakura affects Otu’s deubiquitinase activity in vitro.

mycbp^null mutant flies

To examine MYCBP function in vivo, we generated a mycbp mutant allele (mycbp^null) by CRISPR/Cas9-mediated deletion of 38 nucleotides (nts) within the MYCBP coding region, resulting in a frameshift and premature stop codon (Fig 1B). The predicted truncated protein consists of 37 N-terminal amino acids of MYCBP followed by a 47-aa frameshifted segment. This fragment is unlikely to be functional and no stable protein product was detected (Fig 4A), indicating a null mutation. Homozygous mycbp^null/null flies were viable, demonstrating that MYCBP is not essential for survival.

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Fig 4. mycbp^null female flies are sterile and have rudimentary ovaries.

(A) Western blot of ovary lysates. (B, C) Female fertility assays. (B) The number of eggs laid by test females mated with wild-type (OregonR) males. (C) Hatching rate of the eggs. Mean ± SD (n = 3). P-value < 0.001 (Student’s t-test, unpaired, two-tailed) is indicated by ***. (D) Stereomicroscope images of dissected ovaries. Scale bar: 200 μm.

https://doi.org/10.1371/journal.pgen.1011792.g004

Western blotting confirmed that MYCBP protein is absent in mycbp^null/null ovaries, but present in wild-type (mycbp^+/+) and heterozygous controls (mycbp^null/+) (Fig 4A). No smaller fragments corresponding to truncated MYCBP were detected in either mycbp^null/+ or mycbp^null/null, suggesting that the fragment is unstable or not expressed. We performed mosaic analysis using the FLP-FRT system driven by heat shock-inducible FLP (hs-HLP) to generate control (FRT82B only) and mycbp^null (FRT82B, mycbp^null) germline clones, marked by the absence of GFP, and stained ovaries with anti-MYCBP antibody (S1D Fig). In the same ovarioles of FRT82B, mycbp^null flies, GFP-negative mycbp^null clone cells showed strongly reduced MYCBP staining compared with neighboring GFP-positive control cells; similarly, control clones in FRT82B-only flies displayed normal staining (S1D Fig). These results validate both the mycbp^null allele and the anti-MYCBP antibody.

MYCBP is essential for female fertility

Given MYCBP’s high expression in ovaries (Fig 1C) and its physical interaction with Otu and Sakura (Figs 2 and 3), we hypothesized a role in oogenesis. mycbp^null/null females laid no eggs (Fig 4B and 4C) and their ovaries appeared rudimentary (Fig 4D). Expression of transgenic MYCBP-EGFP in the mycbp^null/null background (mycbp^null/null; mycbp-EGFP) fully rescued both fertility and ovary morphology (Fig 4B–4D). Western blotting confirmed that these rescue flies expressed MYCBP-EGFP at levels comparable to endogenous MYCBP in control flies (Fig 4A).

MYCBP-EGFP expression in the background of mycbp^+/+ was also detected in testes, including germline cells such as GSCs, where it displayed both broad cytoplasmic distribution and punctate cytoplasmic signals in spermatocytes, but was absent from hub cells (S5A Fig). Similarly, endogenous MYCBP expression in a control w¹¹¹⁸ testes showed the same patterm, including punctate localization (S5B Fig). However, mycbp^null/null males were fully fertile (S5C Fig). Therefore, the functional significance of MYCBP expression and localization in testes remains unclear. We conclude that MYCBP is essential for female fertility and normal ovary morphology but is not required for male fertility.

mycbp^null/null ovaries are germless or tumorous

To investigate the ovary phenotype, we analyzed mycbp^null/null flies expressing a Vasa-EGFP reporter, as Vasa is a known germ cell marker. Some mycbp^null/null ovarioles lacked germ cells (“germless”, cyan stars), while others contained germ cells (Fig 5A and 5B). HTS staining revealed overproliferation of GSC-like cells with round spectrosomes in mycbp^null/null ovarioles containing germ cells (Fig 5C, orange stars), indicative of a “tumorous” phenotype as previously described for mutants of bam, otu, sxl, and sakura [9–12,22–26]. Additionally, we observed an excess number of cyst cells with branched fusomes that persisted throughout the ovarioles, suggesting abnormal cyst cell development.

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Fig 5. mycbp^null ovaries are germless and tumorous.

(A, B) Confocal images of ovaries from control (mycbp^null/+) and mycbp^null/null flies expressing Vasa-EGFP. Vasa-EGFP (green) and DAPI (blue). Yellow, orange, and. cyan stars mark normal ovarioles, tumorous, and germless ovarioles, respectively, in Fig 5. (B) Higher-magnification images of germaria. Scale bars: 50 μm (A), 10 μm (B). (C) Confocal images of ovaries from control (mycbp^null/+) and mycbp^null/null flies stained with anti-Hts to label spectrosomes and fusomes. Hts (red) and DAPI (blue). Germaria are outlined. Scale bars: 20 μm. (D) Percentage of normal, germless, and tumorous ovarioles in indicated genotypes (ages 2–5 days). (E) Quantification of GSC-like cells per germarium (ages 2–5 days). (F) Confocal images of ovaries stained with anti-cleaved Caspase-3. Cleaved caspase-3 (red) and DAPI (blue). Scale bars: 50 μm.

https://doi.org/10.1371/journal.pgen.1011792.g005

Within the same ovary, both germless and tumorous ovarioles were observed (Fig 5A–5C). 26% of ovarioles were tumorous, while 74% were germless in 2–5-day-old mycbp^null/null flies (n = 168) while all ovarioles in control (mycbp^+/+ and mycbp^null/+) and mycbp-EGFP rescue flies were normal (Fig 5D). The mean number of GSCs or GSC-like cells in 2–5-day-old mycbp^+/+, mycbp^null/+, and mycbp-EGFP rescue was 2.0 ± 0.6, 2.2 ± 0.6, and 2.1 ± 0.5, respectively while that for mycbp^null/null was 21.7 ± 12.7 (p-value <0.001) (S6 Fig). 95% of mycbp^null/null ovarioles containing germ cells had more than five GSC-like cells (Fig 5E). Tumorous ovarioles displayed markedly elevated cleaved Caspase-3 staining, indicative of apoptosis (Fig 5F). In addition, germaria from tumorous mycbp^null/null ovarioles showed increased levels of the mitotic marker Ser10-phosphorylated Histone H3 (pH3), indicating enhanced mitotic cell proliferation (S7A Fig). A similar increase in pH3 signal was observed in germaria of tumorous sakura^null/null ovarioles (S7B Fig). Together, these findings support the idea that MYCBP is required to regulate GSC survival, proliferation, and differentiation.

Loss of mycbp results in loss of piRNA-mediated transposon silencing

piRNAs are produced in germline cells and somatic follicle cells and transcriptionally and post-transcriptionally silence transposon expression through sequence-complementarity [27,28]. Defects in the piRNA pathway result in oogenesis arrest, germ cell loss, rudimentary ovaries, sterility, upregulation (desilencing) of transposons, increased DNA damage, and apoptotic cell death.

In control ovaries, Vasa-EGFP localized to the perinuclear nuage (Fig 5A and 5B), which is essential for piwi-interacting RNA (piRNA) biogenesis. This localization persisted in mycbp^null/null tumorous ovarioles, suggesting that MYCBP is not required for Vasa localization.

To assess MYCBP’s role in the piRNA pathway, we used the Burdock transposon sensor, which expresses nuclear GFP and β-galactosidase (β-gal) in germ cells but is silenced by piRNAs (Fig 6A) [29]. In germline-specific mycbp RNAi flies (UAS-Dcr2, NGT-Gal4, nos-Gal4-VP16 > mycbp^RNAi), we observed strong reporter expression, showing that loss of mycbp results in loss of piRNA-mediated transposon silencing.

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Fig 6. Loss of mycbp disrupts piRNA-mediated transposon silencing and sxl splicing.

(A) Schematic of the Burdock sensor construct and representative images of sensor expression in ovaries from control (y^RNAi) and mycbp^RNAi flies. The Burdock sensor harbors a nanos promoter, a nuclear localization signal (NLS) appended to GFP and β-gal coding sequences, and a target sequence for Burdock piRNAs in the 3’UTR. RNAi was driven in the germline using UAS-Dcr2, NGT-Gal4, and nos-Gal4-VP16. GFP (green), β-gal (red), and DAPI (blue). Scale bars: 20 μm. 0/3 control samples showed sensor activation; 15/15 mycbp^RNAi samples did. (B) RT-PCR analysis of sxl alternative splicing in ovaries and testes.

https://doi.org/10.1371/journal.pgen.1011792.g006

Sex-specific sxl mRNA alternative splicing is dysregulated in mycbp^null/null ovaries

Sxl is a master regulator of sex determination in Drosophila [30–32]. Sex-specific alternative splicing of sxl transcripts produces distinct mRNA isoforms: the female-specific, which encodes functional Sxl protein, and the male-specific mRNA isoforms, which does not. In the female germline, loss of Sxl function or disruption of female-specific sxl splicing leads to developmental defects, including germline tumors and sterility. Notably, several mutants with tumorous ovariole phenotypes—such as otu and sakura mutants—exhibit aberrant sxl mRNA splicing, resulting in male-specific isoform expression in ovaries and defects in germ cell sexual identity [8,23,14].

As expected, ovaries from control and mycbp-EGFP rescue flies expressed only the female-specific sxl mRNA isoform, while control testes expressed only the male-specific isoform (Fig 6B). In contrast, mycbp^null/null ovaries showed aberrant expression of the male-specific isoform and a reduced level of the female-specific isoform. These results indicate that female-specific sxl splicing is disrupted in the absence of mycbp.

MYCBP is required intrinsically for GSC maintenance and differentiation

To test whether mycbp functions autonomously in the germline, we performed mosaic analysis using the FLP-FRT system driven by heat shock-inducible FLP (hs-HLP) [15,33)]. GSC maintenance was assessed by generating mycbp^null GSC clones and measuring clone loss over time [8,34,35]. At four days post-induction, 30.4% of GSCs were marked (GFP-negative) in controls (FRT82B) and 20.0% were marked in mycbp^null (FRT82B, mycbp^null), establishing initial clone frequencies (Fig 7A). By day 14, marked controle GSCs declined modestly to 19.1% (37.1% loss), whereas marked mycbp^null GSC clones dropped sharply to 4.9% (75.7% loss), indicating that mycbp is intrinsically required for GSC maintenance.

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Fig 7. Germline clonal analysis of mycbp^null.

(A) Percentage of germaria with marked (GFP-negative) GSC clones at 4, 7, and 14 days after clone induction. P-value < 0.001 (Chi-squared test) is indicated by ***. (B) Confocal images of control and mycbp^null clones. GFP (green), Hts (red), and DAPI (blue). Scale bar: 10 μm. (C) Number of marked (GFP-negative) GSC-like cells per germarium containing marked GSCs at 4, 7, and 14 days after clone induction. GSC-like cells containing round spectrosome were identified by anti-Hts staining. P-value < 0.05, < 0.01, and <0.001 indicated by *, **, and *** (Student’s t-test, unpaired, two-tailed).

https://doi.org/10.1371/journal.pgen.1011792.g007

To determine whether mycbp is also intrinsically required for GSC differentiation, we quantified the numbers of marked and unmarked GSC-like cells—germline cells with a round spectrosome—in germaria containing marked GSC clones (Fig 7B). Marked mycbp^null GSC-like cells were significantly more abundant than marked controls at all time points (Fig 7C), and their number increased over time, whereas the number of marked control cells did not (Fig 7C). In contrast, the number of unmarked GSC-like cells in germaria containing marked mycbp^null GSC clones did not differ significantly compared with that in germaria containing marked control GSC clones, and their number did not increase over time (S8 Fig). Thus, in the absence of mycbp, germline cells exhibit uncontrolled proliferation and tumorous phenotypes. These findings demonstrate that mycbp is intrinsically required for both GSCs maintenance and proper differentiation.

Loss of mycbp inhibits Dpp/BMP signaling

The Dpp/BMP signaling pathway plays a central role in regulating GSC self-renewal and differentiation by repressing bam in GSCs, which is de-repressed in daughter cystoblasts (Fig 8A) [5,36]. We hypothesized that the germless and tumorous phenotypes observed in mycbp^null ovaries might result from misregulation of this pathway. To test this, we RNAi-knocked down mycbp in the germline using UAS-Dcr2 and NGT-Gal4 in the flies carrying the bam-GFP reporter [37]. In controls (y^RNAi), Bam-GFP expression was restricted to 8-cell cysts and absent in later stages (Fig 8B). In contrast, mycbp knockdown ovaries exhibited persistent Bam-GFP expression throughout the germarium, including in GSCs (Fig 8B).

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Fig 8. Loss of sakura inhibits Dpp/BMP signaling.

(A) Schematic of Dpp-mediated bam repression via pMad activation. Cap cells secrete diffusible Decapentaplegic (Dpp), which is received by its receptor, a heterodimer of Thick vein (Tkv) and Punt (Put), in GSCs. The activated Dpp signaling eventually phosphorylates Mother-against-dpp (Mad). The phosphorylated Mad (pMad) represses the transcription of bam. The repression of bam in GSCs is crucial for maintaining their stemness. Cystoblasts do not receive Dpp, and Bam expression is crucial for promoting cystoblast differentiation from GSCs. This is the same figure as we used in our previous publication [8]. (B) Confocal images of bam-GFP reporter expression in control (y^RNAi) and mycbp^RNAi ovaries [8]. RNAi was driven in the female germline using UAS-Dcr2 and NGT-Gal4. Bam-GFP (green), Hts (red), and DAPI (blue). Germaria are outlined. Scale bar: 10 μm. (C, D) Confocal images of germaria with control and mycbp^null germline clones stained with (C) anti-Bam and (D) anti-pMad. GFP (green), Bam/pMad (red), and DAPI (blue). Scale bar: 10 μm. (E) Quantification of pMad intensity in GSC clones. Mean ± SD (n = 9 and 13 for FRT82B and FRT82B, mycbp^null, respectively). P-value < 0.001 (Student’s t-test, unpaired, two-tailed) is indicated by ***. (F) Confocal images of germaria with control and mycbp^null germline clones stained with anti-CycA. GFP (green), CycA (red), and DAPI (blue). Scale bar: 10 μm. (G) Quantification of CycA intensity in the germline clones. Mean ± SD (n = 9 and 11 for FRT82B and FRT82B, mycbp^null, respectively). P-value < 0.01 (Student’s t-test, unpaired, two-tailed) is indicated by **.Clones in C, D, and F are GFP-negative.

https://doi.org/10.1371/journal.pgen.1011792.g008

We confirmed this using the FLP-FRT-mediated mycbp^null clones and performed anti-Bam immunostaining. In control clones (FRT82B), Bam expression was confined to 8-cell cysts, whereas in mycbp^null clones (FRT82B, mycbp^null), Bam was aberrantly expressed throughout the germarium, including in GSCs (Fig 8C). This suggests that in the absence of mycbp, Bam expression is no longer repressed by Dpp/BMP signaling in GSCs, resulting in GSC loss.

Dpp/BMP signaling represses bam transcription via the transcription factor Mad, which, when phosphorylated (pMad), translocates into the nucleus to repress bam transcription [5,38,39]. anti-pMad staining revealed significantly reduced pMad in mycbp^null GSC clones compared to control GSCs (Fig 8D and 8E), indicating compromised BMP signaling and transcriptional bam de-respression, though Bam is also known to be regulated post-transcriptionally [40].

Previous studies have shown that ectopic expression of a stable form of CycA leads to germ cell loss [41]. This germ cell loss phenotype is also observed upon ectopic bam expression in GSCs [34,42,43]. It was reported that Bam associates with Otu to promote deubiquitination and stabilization of CycA [18]. Anti-CycA staining showed significantly elevated CycA in mycbp^null clones relative to neighboring wild-type cells in the same germarium (FRT82B, mycbp^null) and control clones in control germarium (FRT82B) (Fig 8F and 8G), suggesting that Bam misexpression in mycbp^null stabilizes CycA.

mycbp is required for oogenesis beyond GSCs and germline cysts

Both mycbp^null/null mutation and mycbp germline RNAi knockdown using UAS-Dcr-2 and NGT-Gal4, which initiates RNAi in germline cells starting from GSCs, resulted in rudimentary ovaries lacking late-stage germline cells (Figs 4D and 9A), preventing assessment of MYCBP’s role beyond the germarium. However, because MYCBP is expressed throughout egg chamber development, (Figs 1C–1E and S1), we hypothesized it may also function in later germline stages.

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Fig 9. mycbp is important for late oogenesis.

(A) Stereomicroscope images of dissected ovaries. Scale bar: 500 μm. y^RNAi served as a control. (B, C) Number of stage 14 oocytes per fly (B) and eggs laid (C) in mycbp RNAi knockdown usingby UAS-Dcr2 and TOsk-Gal4. Mean ± SD (n = 5). P-value < 0.001 (Student’s t-test, unpaired, two-tailed) is indicated by ***. (D) Confocal images of ovaries stained with phalloidin (F-Actin, red), anti-Orb (Green), and DAPI (blue). Yellow arrows indicate normal Orb localization; white arrowheads indicate cytoskeletal disorganization and loss of Orb localization. Scale bar: 100 μm.

https://doi.org/10.1371/journal.pgen.1011792.g009

To test this, we used TOsk-Gal4 (a combination of osk-Gal4 and αTub67C-Gal4) to RNAi-knockdown mycbp in germline cells starting from germarium region 2b onward, thereby sparing GSCs and early cysts [44]. While TOsk-Gal4-driven mycbp RNAi did not affect ovary morphology (Fig 9A), it dramatically reduced stage 14 oocyte production and egg laying compared to control RNAi (y^RNAi) (Fig 9B and 9C), suggesting that mycbp is important for oogenesis beyond the germline cyst stage.

We also analyzed Oo18 RNA-binding protein (Orb), a marker of oocyte identity. In controls, Orb localized to the posterior within stage ~4–8 egg chambers (Fig 9D, yellow arrows). In TOsk-Gal4 > mycbp^RNAi ovaries, Orb localization appeared normal through stage ~6 (Fig 9D, yellow arrows) but was lost by stage ~8 with signs of cytoskeletal disorganization (Fig 9D, white arrowheads). In addition, nurse cell nuclei appeared abnormal in TOsk-Gal4 > mycbp^RNAi ovaries (Fig 9D), suggesting apoptosis, similar to what we observed in mycbp^null mutant ovaries (Fig 5F). These defects likely contribute to the impaired oogenesis observed (Fig 9B and 9C). RNAi efficiency was confirmed by Western blots (Fig 10B and 10C).

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Fig 10. MYCBP is required for Sakura level.

Western blot of ovary lysates. The sakura RNAi #1 (VDRC: v39727) and #2 (VDRC: v103660). Non-specific bands are marked (*). Sakura and alpha-Tubulin images in (E), Sakura, Otu, and alpha-Tubulin images in (F), Otu and alpha-Tubulin images in (H), Sakura, Otu, and alpha-Tubulin images in (I), are from [8]. Results are summarized in (J).

https://doi.org/10.1371/journal.pgen.1011792.g010

Similarly, in TOsk-Gal4 > otu^RNAi ovaries [8], Orb localization and cytoskeletal organization were normal up to approximately stage 6 but became dysregulated by stage ~8 (S9 Fig). We conclude that MYCBP plays essential roles in oogenesis beyond early germline stages, similar to Sakura and Otu [8].

MYCBP is crucial for Sakura levels

Given the formation of the protein complexes among MYCBP, Sakura, and Otu, as well as the shared phenotypes observed in their respective mutants, we investigated whether their protein levels are interdependent. We also assessed Sxl protein levels due to the dysregulation of sxl splicing ovserved in their mutants. We performed Western blots using ovary lysates from genetic mutants (mycbp^null/null, sakura^null/null, and otu^14/14) and from RNAi-mediated knockdowns driven by germline-specific Gal4 drivers. Specifically, we used NGT-Gal4 and nos-Gal4-VP16 to deplete the proteins starting from GSCs, and TOsk-Gal4 to deplete proteins from germarium region 2b onward (Fig 10). otu¹⁴ is a ”differentiated (DIF)” class of allele with defects in egg chamber maturation [14,17,45,46)].

In mycbp^null/null ovaries, Sakura and Otu levels were severely reduced, and Sxl levels were moderately reduced (Fig 10A). In NGT-Gal4 > mycbp^RNAi ovaries, Sakura and Otu levels were markedly reduce, while Sxl levels remain unchanged (Fig 10B). In TOsk-Gal4 > mycbp^RNAi ovaries, Sakura and Sxl levels were reduced, but Otu was not affected (Fig 10C). There findings indicate that MYCBP is essential for Sakura levels and may also influence Otu and Sxl levels in a developmental stage-dependent manner.

In sakura^null/null ovaries, MYCBP, Otu, and Sxl levels were severely reduced (Fig 10D). In NGT-Gal4 > sakura^RNAi ovaries, MYCBP and Otu were strongly reduced, but Sxl levels remained unaffected (Fig 10E). In TOsk-Gal4 > sakura^RNAi ovaries, Sxl was reduced, while MYCBP and Otu levels were unchanged (Fig 10F). There data suggest that Sakura may support the expression or stability of MYCBP, Otu, and Sxl proteins in a stage-specific manner.

In otu^14/14 ovaries, MYCBP, Sakura and Sxl levels were unchanged (Fig 10G). In contrast, in nos-Gal4-VP16 > otu^RNAi ovaries, all three proteins were reduced (Fig 10H), whereas in TOsk-Gal4 > otu^RNAi ovaries, their levels remained unchanged (Fig 10I). There results suggest that Otu may contribute to the expression and/or stability of MYCBP and Sakura proteins under certain developmental conditions. The results are summarized in Fig 10J.

It is important to note that whole ovary lysates include both germline and somatic cells. While Sakura and Otu are expressed exclusively in germ cells in ovaries, MYCBP was enriched in germline cells but was also lowly detectable in somatic follicle cells (Figs 1E, and S1A and S1B) [8]. All Gal4 drivers used (NGT-Gal4, nos-Gal4-VP16 and TOsk-Gal4) are germline-specific. Furthermore, ovaries from mycbp^null/null, sakura^null/null, NGT-Gal4 > mycbp^RNAi, NGT-Gal4 > sakura^RNAi, and nos-Gal4-VP16 > otu^RNAi flies are significantly smaller and degenerated compared to controls (Fig 4D) [8], complicating the interpretation of Western blot results due to tissue loss or altered cell composition. Therefore, we next pursued alternative approaches to more precisely determine their protein level interdependency.

MYCBP is required for Sakura level and Sakura is important for MYCBP level and its posterior localization within egg chambers

We examined how MYCBP and Sakura influence their protein level and localization within egg chambers each other using FLP-FRT-mediated mosaic analysis. In flies expressing Sakura-EGFP, its signal was markedly reduced in mycbp^null clones (FRT82B, mycbp^null) compared to control clones (FRT82B), confirming MYCBP is essential for Sakura protein level (Fig 11A).

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Fig 11. Mutual dependance of MYCBP and Sakura level and localization.

(A) Confocal images of egg chambers with control and mycbp^null germline clones expressing Sakura-EGFP. Marked clones (RFP-negative) are outlined. RFP (red), Sakura-EGFP (green), and DAPI (blue). Scale bar: 20 μm. Yellow arrow: normal Sakura-EGFP posterior enrichment; magenta arrow: reduced Sakura-EGFP signal. Fly genotypes used: hs-flp/w; sakura-EGFP/ + ; FRT82B, ubi-RFP/FRT82B. hs-flp/w; sakura-EGFP/ + ; FRT82B, ubi-RFP/FRT82B, mycbp^null. (B) Confocal images of egg chambers with control and sakura^null germline clones expressing MYCBP-EGFP. Marked clones (RFP-negative) are outlined. RFP (red), MYCBP-EGFP (green), and DAPI (blue). Scale bar: 20 μm. Yellow arrow: normal MYCBP-EGFP posterior enrichment; magenta arrow: reduced levels and loss of posterior localization of MYCBP-EGFP. Fly genotypes used: hs-flp/w; mycbp-EGFP/ + ; FRT82B, ubi-RFP/FRT82B. hs-flp/w; mycbp-EGFP/ + ; FRT82B, ubi-RFP/FRT82B, sakura^null.

https://doi.org/10.1371/journal.pgen.1011792.g011

Conversely, in flies expressing MYCBP-EGFP, its level was decreased and its posterior localization was lost in sakura^null clones (FRT82B, sakura^null) compared in control clones (FRT82B) (Fig 11B), indicating that Sakura is required for MYCBP level and its posterior localization within egg chambers.

MYCBP and Sakura are required for Otu localization to developing oocytes

We examined Otu levels and localization within germaria and egg chambers using otu-EGFP and otu(∆Tudor)-EGFP transgenes [8] in control, mycbp^null, and sakura^null clones. For egg chambers, we additionally stained for Orb, which marks developing oocytes. Otu-EGFP and Otu(∆Tudor)-EGFP levels appeared similar across all genotypes in both the germarium, including in GSCs, and egg chambers (Figs 12 and S10), indicating that MYCBP and Sakura do not affect Otu protein levels. However, Otu-EGFP and Otu(∆Tudor)-EGFP failed to localize to the posterior within the egg chambers in mycbp^null and sakura^null clones (Fig 12). Importantly, Orb remained properly localized in these clones, suggesting that oocyte specification was intact. Thus, MYCBP and Sakura are dispensable for Otu level but are required for its proper localization to developing oocytes.

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Fig 12. Otu localization to the developing oocyte depends on MYCBP and Sakura.

Confocal images of egg chambers with control, mycbp^null, and sakura^null germline clones expressing (A) Otu-EGFP or (B) Otu(ΔTudor)-EGFP. Marked clones (RFP-negative) are outlined. RFP (red), Otu-EGFP or Otu(ΔTudor)-EGFP (green), Orb (white), and DAPI (blue). Scale bar: 20 μm. Yellow arrows: normal posterior enrichment of Otu-EGFP and Otu(ΔTudor)-EGFP; magenta arrows: loss of localization. Orb localization remains intact in mutant clones. Fly genotypes used: hs-flp/w; otu-EGFP/ + ; FRT82B, ubi-RFP/FRT82B. hs-flp/w; otu-EGFP/ + ; FRT82B, ubi-RFP/FRT82B, mycbp^null. hs-flp/w; otu-EGFP/ + ; FRT82B, ubi-RFP/FRT82B, sakura^null. hs-flp/w; otu(ΔTudor)-EGFP/ + ; FRT82B, ubi-RFP/FRT82B. hs-flp/w; otu(ΔTudor)-EGFP/ + ; FRT82B, ubi-RFP/FRT82B, mycbp^null. hs-flp/w; otu(ΔTudor)-EGFP/ + ; FRT82B, ubi-RFP/FRT82B, sakura^null.

https://doi.org/10.1371/journal.pgen.1011792.g012

MYCBP and Sakura are required for Sxl expression in germline cells, including GSCs

We examined Sxl protein levels in germ cells, including GSCs, within the germarium using control, mycbp^null, and sakura^null mosaic clones (Fig 13). In marked control clones (FRT82B), Sxl was highly expressed in GSCs and cystoblasts, as well as in unmarked GSCs and cystoblasts in control germaria (lower germaria in the FRT82B, sakura^null panels) (Fig 13A). In contrast, Sxl levels were markedly reduced in mycbp^null and sakura^null clone cells, both in GSCs (outlined in yellow) and in differentiating germline cells (outlined in white) (Fig 13A and 13B). These results demonstrate that MYCBP and Sakura are required for proper Sxl expression in germline cells, including in GSCs.

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Fig 13. Sxl expression in germaria depends on MYCBP and Sakura.

(A) Confocal images of germaria with control, mycbp^null, and sakura^null germline clones. Marked clones (GFP-negative) are outlined with white and marked GSC clones are outlined with yellow. GFP (green), Sxl (red), and DAPI (blue). Scale bar: 10 μm. Fly genotypes used: hs-flp/w; + ; FRT82B, ubi-RFP/FRT82B. hs-flp/w; + ; FRT82B, ubi-RFP/FRT82B, mycbp^null. hs-flp/w; + ; FRT82B, ubi-RFP/FRT82B, sakura^null. (B) Quantification of Sxl intensity in the GSC clones. Mean ± SD (n = 11). P-value < 0.001 (Student’s t-test, unpaired, two-tailed) is indicated by ***.

https://doi.org/10.1371/journal.pgen.1011792.g013

Fly strains

We generated the mycbp^null strain by introducing indels into the MYCBP coding region using the CRISPR/Cas9 genome editing, as previously reported [8,62–66]. The transgenic mycbp-EGFP strain was established following previously published methods [65,67,68]. A DNA fragment containing the MYCBP coding sequence flanked by ~1.2 kb upstream and ~1 kb downstream genomic sequences was cloned. EGFP was fused in-frame to the C-terminus of the MYCBP coding sequence. The construct was inserted into a pattB plasmid and integrated at the 25C6 landing site using the attP40 fly line and the PhiC31 integrase system (BestGene).

Transgenic otu-EGFP, otu(∆Tudor)-EGFP, and sakura-EGFP strains were previously described [8]. The mycbp-RNAi (VDRC: v41628), y-RNAi (v106068), TOsk-Gal4 (v 314033), Burdock sensor [UAS-Dcr2; NGT-Gal4; nosGal4-VP16, nos > NLS_GF’_lacZ_vas-3’UTR_burdock-target] (v 313217) strains were from Vienna Drosophila Resource Center. UAS-Dcr-2; NGT-Gal4 (BDSC: 25751) and FRT82B/TM6C, Sb (BDSC: 86313) were obtained from the Bloomington Stock Center. The bam-GFP reporter (DGRC: 118177) and vasa-EGFP knocked-in fly (DGRC: 118616) were from Kyoto Drosophila Stock Center. hsFLP; + ; FRT82B, ubi-RFP/TM6B strain was a kind gift from Dr. Wu-Min Deng (Tulane University).

Fertility assay

Fertility assays were performed as described previously [8,63–66,69; 65]. For female fertility, five virgin females of the test genotype were crossed with three wild-type (OregonR) males in cages containing 6-cm grape juice agar plates supplemented with wet yeast paste at 25°C. Plates were replaced daily. Eggs laid on the third plate (from day 3 to day 4) were counted, and hatching was assessed after an additional 24 horus of incubation at 25°C. At least three cages per genotype were analyzed.

For male fertility, a single test male was mated with five wild-type (OregonR) virgin females in a vial at 25°C. After 3 days, the females were transferred to a new vial (vial 1), and then to new vials every 2 days for a total of four vials. Females were removed after 2 days in the fourth vial. Total progeny from all vials were counted. At least five males per genotype were tested.

MYCBP antibody generation

Recombinant full-length MYCBP with a C-terminal HRV3Csite-6xHis tag was expressed in E. coli using a modified pET vector [70] and purified via Ni-sepharose (Cytiva). The His-tag was removed via HRV3C protease cleavage, and the protein was further purified on a HiTrapQ HP column (Cytiva). This antigen was used to generate rabbit polyclonal anti-sera (Pocono Rabbit Farm & Laboratory, Inc.). Rabbit polyclonal anti-MYCBP antibodies were affinity-purified using recombinant MYCBP-HRV3Csite-6xHis protein conjugated to Affigel-15 (Bio-rad).

Immunostaining

Stereomicroscope images of dissected ovaries were taken using a Leica M125 stereomicrocsope. Ovaries from 2- to 5-day-old, yeast-fed females were dissected in 1x PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4) and fixed for 30 minutes at room temperature in a fix buffer (4% formaldehyde, 15 mM PIPES (pH 7.0), 80 mM KCl, 20 mM NaCl₂, 2 mM EDTA, and 0.5 mM EGTA). Samples were washed in PBX (1x PBS + 0.1% Triton X-100) and blocked in a blocking buffer (PBX with 2% donkey serum, 3% BSA [w/v], and 0.02% NAN₃ [w/v]) for 1 hour at room temperature. The ovaries were then incubated with primary antibodies diluted in the blocking buffer overnight at 4°C. Samples were washed three times with PBX and incubated with Alexa Fluor-conjugated secondary antibodies for 2 hours at room temperature, washed again, and mounted in VECTASHIELD PLUS antifade mounting medium with DAPI (H-2000, Vector lab). Confocal images were acquired on a Zeiss LSM700 confocal microscope at the Johns Hopkins University School of Medicine Microscope Facility.

The primary antibodies used for immunostaining were rabbit anti-MYCBP (dilution: 1/100), mouse anti-HTS (1B1) (DSHB, AB_528070, dilution: 1/100), mouse anti-Bam (DSHB, AB_10570327, 1/20), mouse anti-CycA (DSHB, AB_528188, 1/100), mouse anti-Orb (DSHB, AB_528419, 1/100), mouse anti-Sxl M18 (DSHB, AB_528464, 1/20), rabbit anti-pMad (Cell Signaling, Phospho-SMAD1/5 (Ser463/465) mAb #9516, 1/200), and rabbit anti-cleaved caspase-3 (Cell Signaling, Cleaved Caspase-3 (Asp175) #9661, 1/200), rabbit anti-Phospho-Histon H3 (Ser10) (Cell Signaling, #9701, 1/100). Secondary antibodies used were Alexa Fluor 488 Donkey anti-Mouse Igg (ThermoFisher, A21202, 1/100), Alexa Fluor 594 Donkey anti-Mouse Igg (ThermoFisher, A21203, 1/100), Alexa Fluor 594 Donkey anti-Rabbit Igg (ThermoFisher, A21207, 1/100), and Alexa Fluor 647 Donkey anti-mouse Igg (ThermoFisher, A31571, 1/100). Rhodamine phalloidin (ThermoFisher, R415, 1/100) was used to stain F-Actin.

Germline clonal analysis

mycbp^null germline clones were generated using FLP/FRT-mediated recombination [33]. mycbp^null GSC clones were induced by heat-shocking 3-day-old females of the genotype hs-flp/w; + ; FRT82B, ubi-GFP/FRT82B, mycbp^null at 37°C for 1 hour, twice daily with an 8-hour interval. Controls were hs-flp/w; + ; FRT82B, ubi-GFP/FRT82B. Ovaries were dissected 4, 7, and 14 days post-heat shock.

To generate mycbp^null mutant clones in the presence of transgenic reports, files of following genotypes were used. hs-flp/w; otu-EGFP/ + ; FRT82B, ubi-RFP/FRT82B, mycbp^null, hs-flp/w; otu(ΔTudor)-EGFP/ + ; FRT82B, ubi-RFP/FRT82B, mycbp^null and hs-flp/w; sakura-EGFP/ + ; FRT82B, ubi-RFP/FRT82B, mycbp^null. Control genotypes lacking the mycbp^null allele were also analyzed. Flies were dissected 3–5 days after clone induction.

Western blot

Lysates of hand-dissected ovaries and tissues were prepared by homogenizing in RIPA buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% [v/v] IGEPAL CA-630, 0.1% [w/v] sodium dodecyl sulfate (SDS), 0.5% [w/v] sodium deoxycholate, 1 mM ethylenediaminetetraacetic acid (EDTA), 5 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride (PMSF)) [8,63,66,68]. Homogenates were centrifuged at 21,000g at 4°C for 10 min, and the protein concentrations of the supernatant were determined using the BCA protein assay kit (Pierce). Fifteen μg of total protein was loaded per lane for Western blot.

The sources and dilutions of the primary antibodies were as below. Rabbit anti-MYCBP (1/10000, generated in this study), Rabbit anti-Sakura (1/10000, [8]), rabbit anti-Otu (1/10000, [8]), mouse anti-Sxl M18 (1/1000, DSHB, AB_528464), mouse anti-Sxl M114 (1/1000, DSHB, AB_528463), rabbit anti-alpha-Tubulin [EP1332Y] (1/10000, Abcam, ab52866), mouse anti-alpha-Tubulin [12G10] (1/10000, DSHB, AB_1157911), mouse anti-FLAG (1/10000, Sigma, F1804), mouse anti-HA (1/10000, Sigma, H3663), and mouse anti-GFP [GF28R] (1/3000, Invitrogen, 14-6674-82). IRDye 800CW goat anti-mouse IgG (LiCor), IRDye 800CW goat anti-rabbit IgG (LiCor), IRDye 680RD goat anti-mouse (LiCor), and IgG IRDye 680RD goat anti-rabbit (LiCor) were used as secondary antibodies. The membranes were scanned using the Li-Cor Odyssey CLx Imaging System.

Mass spectrometry

Immunoprecipitation of MYCBP-EGFP protein was performed using the GFP-Trap Magnetic Agarose Kit (Proteintech, gtmak-20) on dissected ovaries from flies harboring mycbp-EGFP transgene, with w1118 flies as controls. Ovaries were homogenized in 200 μL ice-cold lysis buffer (10 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5 mM EDTA, 0.05% [v/v] IGEPAL CA-630) containing 1 × protease inhibitor cocktail (100 × protease inhibitor cocktail contains 120 mg/ml 1 mM 4-(2-aminoethyl) benzene sulfonyl fluoride hydrochloride (AEBSF), 1 mg/ml aprotinin, 7 mg/ml bestatin, 1.8 mg/ml E-64, and 2.4 mg/ml leupeptin). After homogenization, the tubes were placed on ice for 30 minutes, and the homogenates were extensively pipetted every 10 minutes. The lysates were then centrifuged at 17,000x g for 10 minutes at 4°C. The supernatants were transferred to pre-chilled tubes, and 300 μL dilution buffer (10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA) supplemented with 1x protease inhibitor cocktail were added. The diluted lysates were then added to the GFP-trap magnetic beads in 1.5 mL tubes and rotated for 1 hour at 4°C. After separating the beads with a magnetic tube rack, the beads were washed three times with 500 μL wash buffer (10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.05% [v/v] IGEPAL CA-630). Proteins were eluted with 40 μL acidic elution buffer (200 mM glycine pH 2.5) followed by immediate neutralization with 5 uL neutralization buffer (1 M Tris pH 10.4).

As a quality control before mass spectrometry, ~ 5 μL of the samples were mixed with an equal volume of 2 × SDS-PAGE loading buffer (80 mM Tris-HCl [pH 6.8], 2% [w/v] SDS, 10% [v/v] glycerol, 0.0006% [w/v] bromophenol blue, 2% [v/v] 2-mercaptoethanol), heated at 95 °C for 3 min, and run on 4–20% Mini-PROTEAN TGX Precast Protein Gels (Biorad, #4561094). Silver staining was then performed by using Pierce Silver Stain Kit (ThermoFisher, 24612) to assess the quality of the immunoprecipitated protein samples. Mass spectrometry was conducted at the Mass Spectrometry Core at the Department of Biological Chemistry, Johns Hopkins School of Medicine, as previously described [8,65].

Co-immunoprecipitation

For endogenous MYCBP co-IP, ovaries from wilt-type (w¹¹¹⁸) flies were homogenized in ice-cold lysis buffer (10 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5 mM EDTA, 0.05% [v/v] IGEPAL CA-630) containing 1 × protease inhibitor cocktail. Lysates were centrifuged at 21,000g at 4°C for 10 min, and supernatants were used for immunoprecipitation. Four μg of rabbit anti-MYCBP and control rabbit IgG (Cell Signaling, #2729) were incubated with 50 μL of Dynabeads Protein G (ThermoFisher, 10004D) for 20 min at room temperature. The beads were washed once with PBST (1x PBS with 0.1% Tween-20), then incubated with lysates at room temperature for 30 min, followed by three washes with PBST. Proteins were eluted with 2x SDS-PAGE loading buffer (80 mM Tris-HCl [pH 6.8], 2% [w/v] SDS, 10% [v/v] glycerol, 0.0006% [w/v] bromophenol blue, 2% [v/v] 2-mercaptoethanol) and heated at 70°C for 10 min. After bead separation using a magnetic tube rack, the eluted proteins in 2x SDS-PAGE loading buffer were heated again at 95°C for 3 min.

For transient expression in S2 cells, a total of 1 μg plasmid DNA (pAc5.1/V5-HisB. Invitrogen) was transfected into cells in 6-well plates using Effectene (Qiagen, 301425). After 3 days, cells were harvested and lysed in ice-cold lysis buffer with 1 × protease inhibitor cocktail, and centrifuged at 17,000x g for 10 min at 4°C. For HA-IP, supernatants were incubated with 25 μL (0.25 mg) of Pierce Anti-HA Magnetic Beads (ThermoFisher, 88837) at room temperature for 30 min. The beads were then washed three times with TBST (1x TBS [50 mM Tris/HCl and 150 mM NaCl, pH 7.6] with 0.05% Tween-20). For FLAG-IP, 2 μg of mouse anti-FLAG [Sigma, F1804] was pre-bound to 50 μL of Dynabeads Protein G (ThermoFisher, 10004D) for 10 min at room temperature. The beads were washed once with PBST. The S2 cell lysate supernatant was incubated with the washed beads at room temperature for 15 min. The beads were washed three times with PBST. In both anti-HA and anti-FLAG immunoprecipitations, proteins were eluted with 2 × SDS-PAGE loading buffer. For anti-HA, the beads in 2x SDS-PAGE loading buffer were heated at 95°C for 7 min. For anti-FLAG, the beads were heated at 70°C for 10 min, then were separated using a magnetic tube rack. The eluted proteins in 2x SDS-PAGE loading buffer were heated again at 95°C for 3 min.

Sequential co-immunoprecipitation

S2 cells were transfected with the Sakura-EGFP-HRV3Csite-3xFLAG, EGFP-HRV3Csite-3xFLAG, MYCB-mCherry-3xHA, and Myc-Otu plasmid constructs (pAc5.1/V5-HisB. Invitrogen) with the Effectene. A total of 1 μg plasmids were transfected in each well of 6-well plates. After 3 days, cells were harvested, lysed and centrifuged as described above.

For the first IP, 2 μg of mouse anti-FLAG [Sigma, F1804] was pre-bound to 50 μL of Dynabeads Protein G and was incubated with lysate supernatant at room temperature for 15 min. Beads were washed three times with PBST, then proteins were eluted by incubation with 100 μL cleavage buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 5% glycerol, 2 mM EDTA) containing 25 nM GST-HRV3C protease at 4°C for 6 hours. 20 μL of the eluate was mixed with 2x SDS-PAGE loading buffer and heated at 95°C for Western blot analysis.

The remaining ~80 μL eluate was subjected to a second IP using 25 μL (0.25 mg) of Pierce Anti-HA Magnetic Beads (ThermoFisher, 88837) pre-equilibrated in lysis buffer. After a 30 min incubation at room temperature, beads were washed three times with TBST and proteins were eluted by heating at 95°C for 7 min in 2 × SDS-PAGE loading buffer.

RT-PCR

Total RNAs from ovaries and testes and were prepared using miRVana (Thermo Fisher Scientific), followed by DNase treatment with Turbo DNase (Thermo Fisher Scientific). cDNA was synthesized from 1 μg of RNA using SuperScript VILO MasterMix (Thermo Fisher Scientific). To examine sxl alternative splicing, PCR was performed using GoTaq Green Master Mix (Promega) with primers sxl-F (5′-CTCACCTTCGATCGAGGGTGTA-3′) and sxl-R (5′-GATGGCAGAGAATGGGAC-3′) followed by agarose gel electrophoresis and SYBR Safe staining.

In vitro deubiquitination assay

Purified Otu, luciferase, Sakura [8], and MCYBP proteins (latter purified as for antibody generation) were mixed with Ub-Rhodamine 110 (Ubiquitin-Proteasome Biotechnologies, M3020) in a 30 μL in reaction (20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 5 mM MgCl₂, 2 mM DTT). Reactions were performed for 60 min in a black 384-well low-volume plate and fluorescence signals were measured using SpectraMax i3x Multi-Mode microplate reader (excitation/emission: 485/20 nm and 530/20 nm).