Figure 4. Three-dimensional structure of U2af1-rs proteins. (A) Disorder prediction. Positions of all the positively selected residues along the protein are shown as gray triangles. Position of U2af35- homologous domain shown as thick black line. Positively selected residue within this domain shown as white triangle. Thin black line denotes disorder probability 0.5. Values above this predict disorder. (B and C) Closeup view of the neighboring residues to the positively selected residue. (D) U2AF1-RS2 isoelucine. (E) U2AF1- RS1 valine. Residues within 6 Angstrom cut-off from the isoleucine residue or valine residues are colored by secondary structure: beta- sheets yellow, alpha helix purple, and coil white. RNA-binding domain showed evidence of positive selec- tion/adaptive evolution, changing from an isoleucine in the parent gene sequence to a valine in the retrogene. Our models show that this valine residue produces fewer perturbations within the core of the protein structure, compared to isoleucine. Although both residues are tolerated within the core of the modeled protein structure, the U2AF1-RS1 protein may therefore have enhanced stability compared with its parent protein. Most of the positively selected residue changes are focused in the disordered regions of the U2af1-rs1 retrogene. Here, con- straints to maintain a particular structure may be relaxed, and so the plasticity of these disordered regions might allow the protein to “experiment” with new residues. Investigations into the possi- ble structures of these disordered protein regions are required to ascertain the effects of these residue changes, but this is beyond the scope of this study. U2af1-rs1 has some parallels in the im- printed plant gene MEDEA (Spillane et al. 2007). MEDEA was formed through a whole-genome duplication in plants, and sub- sequently underwent neofunctionalization by means of positive selection. I M P R I N T E D R E T RO G E N E E VO L U T I O N Mcts2 IN SPERMATOGENESIS From previous studies, we know that all the parent genes in this study map to the X chromosome. It has been proposed that the significant excess of functional retrogenes produced from par- ent genes on the X chromosome is attributable to gene function during spermatogenesis (Emerson et al. 2004). X-linked genes are more likely to show a testis-specific expression pattern than would be expected by chance (Wang et al. 2001). However, dur- ing spermatogenesis, genes on the sex chromosomes are subject to epigenetic silencing during a process termed meiotic sex chro- mosome inactivation (MSCI). Many X-linked genes are down- regulated in their expression, particularly at the pachytene stages, whereas autosomal genes are not (Wang et al. 2005). Autoso- mal copies of X-linked genes, such as Mcts2, might compensate for their parent’s downregulation during the pachytene stages of spermatogenesis. Using microarray data (Namekawa et al. 2006), we tested the hypothesis that the imprinted genes in question can compensate for their parent genes. Figure 5 shows that expression of Mcts2 in mouse increases during the pachytene stage when MSCI takes place, with Mcts1 dropping dramatically. The strong purifying selection seen upon both genes might be acting to maintain the same protein function, with Mcts2 substituting for Mcts1 as it is inactivated during the later stages of spermatogenesis. The two other gene families for which microarray probes were present (Nap1l and U2af1-rs) do not exhibit expression pat- terns consistent with MSCI compensation (Fig. 5). The X-linked parent genes behave as expected; Nap1l2 remains at very low lev- els of expression as spermatogenesis progresses and U2af1-rs2 shows decreasing expression. However the corresponding retro- genes do not show increased expression levels as MSCI sets in, unlike Mcts2. This suggests that not all X-to-autosome retrogenes compensate for their parents during MSCI. EXPRESSION PROFILES OF RETROGENES ARE ATYPICAL It has been suggested that retrogenes tend to show an expression bias toward the testes (Shiao et al. 2007), reviewed in Wang et al. (2001), for both evolutionary and mechanistic reasons. Mecha- nistically, the testes provide a “permissive” environment for tran- scription (Schmidt 1996; Kleene 2001), and so retrogenes that have appeared de novo, and might not possess a strong promoter, still have a chance at expression. As discussed above, evolutionary pressures also act to confer male-specific functions upon many ret- rogenes, particularly those that originated on the X-chromosome. However, the retrogenes discussed here show a wide expression pattern. Indeed, the Nap1l genes and Inpp5f_v2 show very low or no expression in the testes. We compared our expression data with Potrzebowski et al. (2008), which contains expression data on the U2af1-rs1 retrogene. Potrzebowski et al. found U2af1-rs1 EVOLUTION MAY 2011 1 4 2 3