Functional Divergence and Origin of the Vertebrate Praja Family

The Praja family is an E3 ubiquitin ligase, promoting polyubiquitination and subsequent degradation of substrates. It comprises two paralogs, praja1 and praja2. Prior research suggests these paralogs have undergone functional divergence, with examples, such as their distinct roles in neurite outgrowth. However, the specific evolutionary trajectories of each paralog remain largely unexplored preventing mechanistic understanding of functional differences between paralogs. Here, we investigated the phylogeny and divergence of the vertebrate Praja family through molecular evolutionary analysis. Phylogenetic examination of the vertebrate praja revealed that praja1 and praja2 originated from the common ancestor of placentals via gene duplication, with praja1 evolving at twice the rate of praja2 shortly after the duplication. Moreover, a unique evolutionary trajectory for praja1 relative to other vertebrate Praja was indicated, as evidenced by principal component analysis on GC content, codon usage frequency, and amino acid composition. Subsequent motif/domain comparison revealed conserved N terminus and C terminus in praja1 and praja2, together with praja1-specific motifs, including nuclear localization signal and Ala-Gly-Ser repeats. The nuclear localization signal was demonstrated to be functional in human neuroblastoma SH-SY5Y cells using deletion mutant, while praja2 was exclusively expressed in the nucleus. These discoveries contribute to a more comprehensive understanding of the Praja family's phylogeny and suggest a functional divergence between praja1 and praja2. Specifically, the shift of praja1 into the nucleus implies the degradation of novel substrates located in the nucleus as an evolutionary consequence.