
00genesis otu bam MYCBP

<P>Azlan, A., Fukunaga, R. (2025b). MYCBP interacts with Sakura and Otu and is essential for germline stem cell renewal and differentiation and oogenesis. PLoS Genet, 21(12):e1011792 PubMed ID: <a href="https://pubmed.ncbi.nlm.nih.gov/41325460">41325460</A>

<a name="AzlanB"><P><B><DT>MYCBP interacts with Sakura and Otu and is essential for germline stem cell renewal and differentiation and oogenesis<DD></B>

  <P>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.  <a href="../sturtevant/bourbon.htm">Sakura</a> (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 <a href="https://flybase.org/reports/FBgn0038043">MYCBP (c-Myc binding protein)</a> 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 <i>mycbp</i> null mutant females exhibit rudimentary ovaries with germline-less and tumorous ovarioles, fail to produce eggs, and are completely sterile. Germline-specific depletion of <i>mycbp</i> disrupts Dpp/BMP signaling, causing aberrant expression of <i>bag-of-marbles (bam)</i> and leading to defective differentiation and GSC loss. In addition, <i>mycbp</i> is required for female-specific splicing of <i>sex-lethal (sxl)</i>, a master regulator of sex identity determination. These phenotypes closely resemble those observed in <i>sakura</i> and <i>otu</i> 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).

<P>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 <i>mycbp, otu</i>, and <i>sakura</i> 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 <i>sxl</i> 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).

<P>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).

<P>Both tumorous and germless ovarioles were observed in the <i>mycbp, otu</i>, and <i>sakura</i> 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).

<P>In <l>mycbp<sup>null</sup></i> 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 <i>sakura<sup>null</sup></i> 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).

<P>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).

<P>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 <i>sxl</i> mRNA is disrupted in <i>mycbp</i>, <i>sakura</i> and <i>otu</i> 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 <i>nanos</i> 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 <i>sxl</i> alternative splicing and translational control of oogenic RNAs (Azlan, 2025b).

<P>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).

<P>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 <i>mycbp, sakura</i> or <i>otu</i> 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).

<P>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).

<P>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 <i>nos-Gal4</i> &gt; UASp-<i>otu</i>::GFP were markedly reduced in <i>sakura</i> 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 <i>sakura</i> and <i>mycbp</i> 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 <i>otu</i> promoter-clearly demonstrated that Otu protein levels were not reduced in <i>mycbp<sup>null</sup></i> or <i>sakura<sup>null</sup></i> 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).

<P>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).
