During oogenesis, self-renewal and differentiation of germline stem cells (GSCs) must be tightly regulated. The Drosophila female germline serves as an excellent model for studying these regulatory mechanisms. This study reports that a previously uncharacterized gene CG14545, which was named sakura (alternatively bourbon), is essential for oogenesis and female fertility in Drosophila. Sakura is predominantly expressed in the ovaries, particularly in the germline cells, including GSCs. sakura null mutant female flies display rudimentary ovaries with germline-less and tumorous phenotypes, fail to produce eggs, and are completely sterile. The germline-specific depletion of sakura impairs Dpp/BMP signaling, leading to aberrant bag-of-marbles (bam) expression, resulting in faulty differentiation and loss of GSCs. sakura is also necessary for normal levels of piwi-interacting RNAs (piRNAs) levels and for female-specific splicing of sex-lethal (sxl), a master regulator of sex identity determination. This study identified Ovarian Tumor (Otu) as a protein binding partner of Sakura and found that loss of otu phenocopies loss of sakura in ovaries. Thus, this study identified Sakura as a crucial factor for GSC renewal and differentiation and oogenesis, and it is proposed that Sakura and Otu function together in these processes (Azlan, 2025a).

This study identified Sakura, encoded by a previously uncharacterized gene CG14545, as an essential factor for oogenesis and female fertility. Sakura is specifically expressed in germline cells in the ovary, including GSCs, is localized to the cytoplasm, and is enriched in the developing oocytes. sakura homozygous null mutant flies are viable but completely female-sterile and male-fertile. Loss of sakura, either through null mutation or germline RNAi including in GSCs, results in rudimentary ovaries exhibiting germless and tumorous phenotypes (Azlan, 2025a).

The tumorous phenotype associated with the loss of sakura is characterized by an excess of GSC-like cells, which feature round spectrosomes, as well as cyst cells with branched fusomes. The increased number of GSC-like cells in the loss of sakura suggests dysregulation of GSC self-renewal and differentiation. Meanwhile, the presence of excess cyst cells with branched fusomes indicates abnormal differentiation and division of cysts. Differentiation of cystoblasts typically involves four mitotic divisions with incomplete cytokinesis, leading to 16 cyst cells interconnected by branched fusomes. The degree of fusome branching serves as a marker for the stages of cyst cell division, with increased branching from 2-cell to 16-cell cysts. Notably, fusomes begin to degenerate and disappear after 16 cell cysts as the germline cyst enters the meiotic zone or region 2 of the germarium. The persistence of cyst cells with branched fusomes in sakura ^null/null suggests that sakura is crucial for the proper division and differentiation of cysts, and it is speculated to be required for germline cysts to enter meiotic division (Azlan, 2025a).

Mosaic analysis of sakura^null indicates that sakura is intrinsically required for the establishment of GSCs in the ovary. When mutant clones were induced in the PGC stage, significantly fewer sakura^null marked GSCs were observed in adult ovaries, suggesting that many sakura^null PGCs fail to survive or differentiate into GSCs. Furthermore, induction of sakura^null clones in adult ovaries led to a more rapid decline in marked sakura^null GSCs compared to controls, indicating that sakura is also intrinsically required for GSC maintenance. Over time, germaria containing sakura^null GSC clones became increasingly tumorous, with an expanding population of sakura^null GSC-like cells. In contrast, the number of GSC-like cells across the entire and all ovarioles, regardless of whether GSCs in the niche remain, declined over time in sakura^null ovaries. These results suggest that sakura^null GSCs initially undergo aberrant proliferation, leading to tumor formation, but ultimately sakura^null GSCs and GSC-like cells are lost through a cell death mechanism, as evidenced by the elevated levels of cleaved Caspase-3 in sakura^null ovaries (Azlan, 2025a).

Dpp/BMP signaling governs GSC self-renewal and cystoblast differentiation by repressing bam in GSCs and de-repressing it in daughter cystoblasts. This process is mediated by the transcription factor Mad, which, when phosphorylated (pMad), translocates into the nucleus to repress bam transcription. The current findings indicate that Bam expression is not limited to 8 cell cysts but continues throughout the germarium in ovaries lacking sakura function. The misexpression of Bam in GSCs likely results from reduced levels of pMad in the GSCs. Given the low pMad levels observed upon loss of sakura, it is proposed that bam misregulation in GSCs occurs primarily at the transcriptional level, although Bam is also known to be subject to post-transcriptional regulation. The continued expression of Bam throughout the germarium, well beyond the GSC niche, suggests a failure to terminate bam expression at the 16 cell cyst stage - a developmental transition when bam is normally turned off. Thus, sakura mutants likely exhibit two distinct molecular defects contributing to bam overexpression: (1) impaired Dpp/BMP signaling within the GSC niche, and (2) a failure to downregulate bam at 16 cell stage. Given the limited space within the GSC niche, the latter defect may be the predominant contributor to the observed bam overexpression. The molecular mechanism that silences bam at the 16 cell cyst stage remains poorly understood, and how sakura loss perturbs this regulation is unclear. The sakura mutant may, therefore, serve as a valuable model to investigate the mechanism that normally suppresses bam expression at the 16 cell stage (Azlan, 2025a).

Transposons are mobile genetic elements that, if not silenced, can generate DNA damage and genomic instability. piRNAs derived from transposons and other repeats can target and silence transposon RNAs to preserve genome integrity in germ cells. Loss of piRNAs leads to transposon derepression, resulting in increased DNA damage, which subsequently triggers cell death. Genetic damage in germ cells can cause developmental defects and diseases that may be inherited by the next generation. Thus, the elimination of defective germ cells is crucial for maintaining germline integrity of a species. This study found that loss of sakura results in reduced piRNA levels and loss of piRNA-mediated transposon silencing in the germline. The observed apoptosis, indicated by elevated cleaved Caspase-3 levels in sakura mutant ovaries, suggests that desilencing of transposons due to reduced piRNA levels likely results in increased DNA damage, triggering cell death. It is speculated that the germless phenotype in sakura mutants may partly arise from an apoptotic germline elimination program activated to maintain germline integrity (Azlan, 2025a).

In addition to its expression in GSCs and cysts in the germarium, Sakura is also expressed in germline cells in later-stage egg chambers and is required at this stage for proper oogenesis. When Sakura is depleted from region 2b of the germarium onward -leaving GSCs and early cyst cells intact-females have significantly fewer stage 14 oocytes and lay significantly fewer eggs. The failure to produce stage 14 mature oocytes likely stems from cytoskeletal disorganization that disrupts oocyte development, as evidenced by mislocalization of Orb. Notably, piRNA pathway mutants also exhibit similar Orb mislocalization. Therefore, the reduced piRNA levels may contribute to the oocyte development defects, including Orb mislocalization, in sakura mutants (Azlan, 2025a).

Sakura does not possess any known protein domains. To infer its function, Otu, a protein known to be crucial for oogenesis, was identifued as a protein partner of Sakura. Mutations in otu gene lead to a range of ovarian phenotypes, including germ cell loss, tumorous egg chambers filled with undifferentiated germ cells, defects in germline sexual identity determination, abnormalities in nurse cell chromosome structure, and defects in oocyte determination. This study showed that germline depletion of Otu via RNAi phenocopies loss of sakura. Similar to Sakura, loss of otu inhibits Dpp/BMP signaling, resulting in low pMad levels in GSCs and Bam de-repression. Additionally, loss of otu also results in the loss of piRNA-mediated silencing, paralleling the effects of sakura loss. Furthermore, germline knockdown of otu yielded a similar ratio of germless ovaries as seen in sakura-RNAi, and simultaneous knockdown of both otu and sakura exacerbated the germless ovary phenotype. These observations raise the possibility that Sakura and Otu function together to regulate germ cell maintenance in the ovaries and are involved in Dpp/BMP signaling to balance stem cell renewal and differentiation (Azlan, 2025a).

Otu possesses deubiquitinase activity, catalyzed by its N-terminal Otu domain. In germ cells, Otu interacts with Bam, forming a deubiquitinase complex that deubiquitinates and thereby stabilizes CycA, promoting GSC differentiation. The predicted structure of Sakura and Otu complex suggests that the Mid domain of Sakura directly contacts the Otu domain of Otu. Sakura lacks deubiquitinase activity and it does not directly affect Otu's deubiquitinase rate in in vitro assays using Ub-Rhodamine 110 as a substrate. This does not preclude the possibility that Sakura may still influence Otu's deubiquitinase activity, potentially guiding Otu to its substrate and determining substrate specificity. In vitro assays may not have detected such specificity changes (Azlan, 2025a).

Otu is an RNA-binding protein whose deubiquitinase activity is enhanced by RNA binding. Bam, along with other proteins such as Bgcn, Mei-P26, and Sxl, binds nanos mRNA -a key stem cell maintenance factor - and represses its translation once germ cells have exited the GSC niche. Bam and Otu form a protein complex. Future studies should explore whether Sakura modulates Otu's RNA-binding properties and its interaction with other proteins. Although Sakura is exclusively expressed in ovaries, particularly in germline cells including GSCs, Otu is broadly expressed in various tissues, including testes and gut. This restricted expression pattern suggests that Sakura may serve as a female germline-specific cofactor that enhances or modifies Otu's molecular functions. Identifying Otu's RNA targets and deubiquitinase substrates beyond CycA, as well as determining how Sakura binding influences these activities, will be key to elucidating their roles in oogenesis. One intriguing possibility is that Sakura regulates Otu's activity toward piRNA pathway components, thereby contributing to proper piRNA biogenesis and function. Additionally, the Sakura-Otu complex may directly regulate essential post-transcriptional processes such as sxl alternative splicing and translational control of other oogenic RNAs (Azlan, 2025a).

The self-renewal and differentiation of germline stem cells (GSCs) are tightly regulated during oogenesis. The Drosophila female germline provides a powerful model to study these regulatory mechanisms. Sakura (also known as Bourbon/CG14545) has been identified as a crucial factor for maintenance and differentiation of GSCs and oogenesis, and Sakura binds to Ovarian Tumor (Otu), another essential regulator of these processes. This study identified MYCBP (c-Myc binding protein) as an additional essential component of this regulatory network. MYCBP physically associates with itself, Sakura, and Otu, forming binary and ternary complexes including a MYCBP*Sakura*Otu complex. MYCBP is highly expressed in the ovary, and mycbp null mutant females exhibit rudimentary ovaries with germline-less and tumorous ovarioles, fail to produce eggs, and are completely sterile. Germline-specific depletion of mycbp disrupts Dpp/BMP signaling, causing aberrant expression of bag-of-marbles (bam) and leading to defective differentiation and GSC loss. In addition, mycbp is required for female-specific splicing of sex-lethal (sxl), a master regulator of sex identity determination. These phenotypes closely resemble those observed in sakura and otu mutants. Together, the findings reveal that MYCBP functions in concert with Sakura and Otu to coordinate self-renewal and differentiation of GSCs and oogenesis in Drosophila (Azlan, 2025b).

Previous work showed that Sakura and Otu form a protein complex. This study identified MYCBP, encoded by the previously uncharacterized gene CG17202, as a binding partner of both Otu and Sakura. The data support that MYCBP binds with itself, Sakura, and Otu, forming binary and ternary complexes, including the MYCBP-Sakura-Otu ternary complex. Structural predictions suggest that MYCBP and Sakura resemble each other and engage in a pseudo-symmetric interaction. Mutations in mycbp, otu, and sakura result in strikingly similar phenotypes, and all three proteins are highly expressed in germline cells of the ovary, localize to the cytoplasm, and are enriched in developing oocytes. These observations strongly indicate that MYCBP, Sakura, and Otu function cooperatively in the germline during oogenesis. It is proposed that the MYCBP-Sakura-Otu complex regulates Dpp/BMP signaling and the expression of Bam, CycA, and Sxl in GSCs, including playing a crucial role in female-specific sxl splicing, thereby controlling GSC maintenance, proliferation, and differentiation. Additionally, it is proposed that formation of the MYCBP-Sakura-Otu complex is important for the enrichment of Otu in developing oocytes within egg chambers, which is essential for proper oogenesis. MYCBP-Sakura-Otu complex may bind and regulate target RNAs and deubiquitinate target proteins (Azlan, 2025b).

Since there are multiple protein association states that can be possibly formed among MYCBP, Sakura, and Otu, including MYCBP alone, Sakura alone, Otu alone, MYCBP-MYCBP, Sakura-Sakura, MYCBP-Sakura, MYCBP-Otu, MYCBP-MYCBP-Otu, Sakura-Otu, Sakura-Sakura-Otu, and MYCBP-Sakura-Otu, the relative expression levels among the three proteins and potential regulatory mechanisms for their interaction may determine the relative abundance of these multiple protein complexes, which could be critically important for GSC maintenance and differentiation and oogenesis (Azlan, 2025b).

Both tumorous and germless ovarioles were observed in the mycbp, otu, and sakura mutant ovaries. While upregulation of Bam and CycA and activation of the apoptotic pathway may underlie the germless phenotypes, the mechanism leading to tumorous ovarioles remains unclear. The relative balance of the multiple protein complexes formed by MYCBP, Sakura, and Otu may be differently disrupted among ovarioles within the same mutant, resulting in both tumorous and germless phenotypes. Furthermore, tumorous ovarioles may eventually become germless due to germline apoptosis. The precise mechanism by which these two distinct phenotypes arise within the same mutant ovaries warrants future investigation (Azlan, 2025b).

In mycbp^null germline clone cells, Sakura protein levels were severely reduced, and Otu lost its localization to developing oocytes, despite unchanged Otu protein levels and normal posterior localization of Orb. These results indicate that MYCBP is required for Sakura protein expression and/or stability, as well as for proper Otu localization. Similarly, in sakura^null germline clones, MYCBP levels were reduced, and both MYCBP and Otu lost their posterior localization, again without affecting Otu levels or Orb localization, indicating that Sakura is crucial for MYCBP protein expression and/or stability, as well as for proper MYCBP and Otu localization to developing oocytes. These mutual dependencies of protein expression/stability and oocyte localization among MYCBP, Sakura, and Otu further support the model that they function as protein complexes (Azlan, 2025b).

Although MYCBP and Sakura did not directly affect Otu's deubiquitinase activity in vitro using Ub-Rhodamine 110 as a model substrate, this does not rule out the possibility that they influence Otu's enzymatic activity in vivo. For instance, they may modulate Otu's substrate specificity. Previous work has shown that Otu also interacts with Bam-primarily through its Otu domain-to form a deubiquitinase complex that deubiquitinates and thereby stabilizes CycA, promoting GSC differentiation. It is possible that MYCBP and Sakura regulate the interaction between Otu and Bam and/or modulate the enzymatic activity of the Otu-Bam complex. For example, binding of MYCBP and/or Sukura to Otu may be mutually exclusive with Bam binding. Alternatively, MYCBP, Sakura, Otu, and Bam might form a ternary complex, while this study found that MYCBP does not bind Bam. Further studies are required to elucidate whether and how MYCBP and Sakura influence Otu's protein interactions and enzymatic function (Azlan, 2025b).

Otu also functions as an RNA-binding protein, and its deubiquitinase activity is enhanced by RNA binding. Sxl controls both alternative mRNA splicing and translation of downstream targets, and promotes its own expression via a positive autoregulatory loop. Female-specific splicing of sxl mRNA is disrupted in mycbp, sakura and otu mutant ovaries, leading to production of the male-specific isoform. Consistent with these findings, Sxl protein expression was found in germline cells in germaria including GSCs depends on MYCBP and Sakura. However precise mechanism how MYCBP, Sakura, and Otu play an essential role in Sxl expression remains unknown. Bam, together with Bgcn, Mei-P26, and Sxl, binds nanos mRNA-a key stem cell maintenance-and represses its translation after germ cells exit the niche. Identifying the RNA targets and deubiquitinase substrates of Otu beyond CycA and how MYCBP and Sakura regulate these Otu's activities will be critical to understanding their roles in oogenesis and other developmental processes. MYCBP-Sakura-Otu complex may bind directly to RNAs and regulate post-transcriptional processes such as sxl alternative splicing and translational control of oogenic RNAs (Azlan, 2025b).

Sakura is exclusively expressed in female germline cells including GSCs, and MYCBP is also highly expressed in these cells. However, MYCBP is additionally expressed at lower levels in somatic follicle cells in egg chambers and in other tissues, including testes, and Otu is broadly expressed in various tissues such as the gut and testis as well as in female germline cells in ovaries. These differential expression patterns suggest that Otu may have tissue-specific functions depending on the presence or absence of MYCBP and Sakura (Azlan, 2025b).

Transposons pose significant threat to genomic stability by inducing DNA damage if not properly silenced. piRNAs suppress transposons through transcriptional and post-transcriptional silencing mechanisms, thus preserving genome integrity. Loss of piRNA function results in transposon derepression, increased DNA damage, germ cell apoptosis, arrested oogenesis, and sterility. Because damaged germ cells can transmit harmful mutations to the next generation, selective elimination of defective germ cells is critical for maintaining germline integrity of a species.This study found that loss of function of mycbp, sakura or otu impairs piRNA-mediated transposon silencing and causes apoptosis. Thus, the germless phenotypes may arise, at least in part, from activation of a transposon-induced apoptotic elimination program. It will be important to investigate whether MYCBP, Sakura, and Otu's have any direct roles in the piRNA pathway (Azlan, 2025b).

MYCBP and Otu are conserved through human (human MYCBP, also known as AMY-1, and OTUD4) while Sakura is not. Human MYCBP was suggested to bind via its C-termina region to the N-terminal region of C-MYC and stimulate the activation of E-box-dependent transcription by C-MYC. However, this study showed that Drosophila MYCBP does not bind Drosophila ortholog of MYC (dMyc). Large scale protein interaction studies indicated that human MYCBP and OTUD4 associate. Alphafold suggested that human MYCBP and OTUD4 form complexes, MYCBP-OTUD4 and/or MYCBP-MYCBP-OTUD4, via the N-termina region of OTUD4, suggesting the evolutionary conserved interaction between MYCBP ortholog and Otu ortholog (Azlan, 2025b).

After these studies were published in which Sakura was identified, characterized by Buszczak group 2025, referring to Sakura as Bourbon. Consistent with both previous and current findings from this lab, the Buszczak group concluded that Sakura, MYCBP, and Otu form a ternary complex and function together to regulate germline differentiation, including promoting Sxl expression. While the overall conclusions align, there are notable differences as well. Mercer proposed that Sakura stabilizes Otu and facilitates physical interactions between Otu and MYCBP. Their conclusions were based on observations that Otu-GFP levels in the germarium driven by nos-Gal4 > UASp-otu::GFP were markedly reduced in sakura mutants, and that MYCBP and Otu failed to in S2 cells unless Sakura was co-expressed. While observed a reduction of endogenous Otu was also observed in whole ovary lysates of sakura and mycbp null mutants and NGT-Gal4 driven RNAi by Western blot, interpretation of these data is complicated by the severe ovarian degeneration in these mutants. In contrast, the current mosaic clone analyses-using Otu-EGFP transgene expressed from the otu promoter-clearly demonstrated that Otu protein levels were not reduced in mycbp^null or sakura^null germline cells within germaria or egg chambers, although Otu enrichment in developing oocytes was lost. Furthermore, this study showed that MYCBP and Otu physically associate in S2 cells without Sakura, unlike the findings reported in Mercer. This discrepancy may reflect differences in epitope-tagging strategies. The current results indicate that neither Sakura nor MYCBP is required for Otu stability of for the physical interaction between Otu and MYCBP or Sakura (Azlan, 2025b).

In summary, this study identifies and characterizes evolutionary conserved MYCBP as a novel and essential regulator of Drosophila oogenesis. Together with Sakura and Otu, MYCBP likely orchestrates germline cell fate decision, maintenance, and differentiation (Azlan, 2025b).