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.