Supplementary Material for: Functional divergence between the Z and W alleles of the UBAP2 gene revealed by differences in their expression patterns in Japanese quail

Introduction: In birds, males have the homogametic sex chromosomes ZZ, whereas females have the heterogametic ZW. Similar to mammalians, avian Z and W chromosomes are believed to have originated from a pair of autosomes. Over evolutionary time, the W chromosome degenerated, losing many genes. The remaining W-linked genes retain homologs on the Z chromosome. One such gene is UBAP2, which participates in cellular metabolism and signaling through the ubiquitin–proteasome pathway in mammals. However, the functions of its avian homologs, UBAP2Z (Z-linked) and UBAP2W (W-linked), remain poorly understood. To investigate the functional divergence between them in birds, we analyzed their mRNA and protein expression in embryonic gonads of Japanese quail. Methods: Using RNA-seq data of embryonic gonads of Japanese quail, contigs were generated by de novo assembly. The nucleotide sequences were predicted from the contigs by blastn analysis. Expression levels were calculated by bowtie2 and RSEM. Immunohistochemistry and western blotting employing monoclonal antibodies specific to UBAP2Z and UBAP2W were generated to investigate protein expression and localization. Additionally, far-western blotting was performed to examine protein–protein interactions. Results: UBAP2Z and UBAP2W nucleotide and amino acids sequences showed high similarity and shared a conserved N-terminal UBA domain. However, UBAP2W expression was consistently lower than that of UBAP2Z and showed a distinct localization pattern from UBAP2Z in embryonic gonads. In males, UBAP2Z was expressed in seminiferous tubules, while in females, it localized to the medulla. By contrast, UBAP2W was exclusively observed in the cortex of female gonads, particularly at developmental stage HH38. Furthermore, UBAP2Z and UBAP2W interacted with different binding partners, indicating divergence in their molecular function. Conclusion: These findings indicate that UBAP2W has a distinct female-specific functional role in avian gonadal differentiation. We propose that UBAP2W contributes to ovarian development and oogenesis through the ubiquitin–proteasome pathway, while UBAP2Z is involved in general cellular regulation across sexes. This study highlights functional divergence between Z- and W-linked paralogs in birds and provides new insights into sex chromosome evolution and gonadal development.

引言：在鸟类中，雄性拥有同型性染色体ZZ，而雌性拥有异型性染色体ZW。与哺乳动物类似，鸟类的Z、W染色体被认为起源于一对常染色体。在进化过程中，W染色体发生退化，丢失了大量基因，剩余的W连锁基因在Z染色体上保留有同源基因。其中一个基因为UBAP2，在哺乳动物中该基因通过泛素-蛋白酶体通路参与细胞代谢与信号转导。然而，其鸟类同源基因——Z连锁的UBAP2Z与W连锁的UBAP2W的功能仍不甚明确。为探究二者在鸟类中的功能分化，我们对日本鹌鹑胚胎性腺中二者的信使RNA（mRNA）与蛋白质表达水平进行了分析。

方法：利用日本鹌鹑胚胎性腺的RNA测序（RNA-seq）数据，通过从头组装（de novo assembly）生成重叠群（contigs）。通过blastn分析从重叠群中预测核苷酸序列。使用bowtie2与RSEM计算基因表达水平。制备针对UBAP2Z和UBAP2W的特异性单克隆抗体，采用免疫组织化学与蛋白质印迹（Western blotting）技术探究蛋白质的表达与定位情况。此外，通过远缘蛋白质印迹（far-western blotting）检测蛋白质-蛋白质相互作用。

结果：UBAP2Z与UBAP2W的核苷酸及氨基酸序列具有高度相似性，且共享保守的N端UBA结构域。但UBAP2W的表达水平始终低于UBAP2Z，且在胚胎性腺中的定位模式与UBAP2Z存在显著差异。在雄性个体中，UBAP2Z表达于生精小管；在雌性个体中，其定位于髓质。与之相反，UBAP2W仅在雌性性腺的皮质中被检测到，尤其在HH38发育阶段表达显著。此外，UBAP2Z与UBAP2W分别结合不同的结合伴侣，提示二者的分子功能存在分化。

结论：上述研究结果表明，UBAP2W在鸟类性腺分化中具有独特的雌性特异性功能。我们推测，UBAP2W通过泛素-蛋白酶体通路参与卵巢发育与卵子发生过程，而UBAP2Z则在两性中参与普遍的细胞调控。本研究阐明了鸟类Z连锁与W连锁旁系同源基因的功能分化，为性染色体进化与性腺发育研究提供了新的见解。