The expansion of the PRAME gene family in Eutheria

Abstract

The PRAME gene family belongs to the group of cancer/testis genes whose expression is restricted primarily to the testis and a variety of cancers. The expansion of this gene family as a result of gene duplication has been observed in primates and rodents. We analyzed the PRAME gene family in Eutheria and discovered a novel Y-linked PRAME gene family in bovine, PRAMEY, which underwent amplification after a lineage-specific, autosome-to-Y transposition. Phylogenetic analyses revealed two major evolutionary clades. Clade I containing the amplified PRAMEYs and the unamplified autosomal homologs in cattle and other eutherians is under stronger functional constraints; whereas, Clade II containing the amplified autosomal PRAMEs is under positive selection. Deep-sequencing analysis indicated that eight of the identified 16 PRAMEY loci are active transcriptionally. Compared to the bovine autosomal PRAME that is expressed predominantly in testis, the PRAMEY gene family is expressed exclusively in testis and is up-regulated during testicular maturation. Furthermore, the sense RNA of PRAMEY is expressed specifically whereas the antisense RNA is expressed predominantly in spermatids. This study revealed that the expansion of the PRAME family occurred in both autosomes and sex chromosomes in a lineage-dependent manner. Differential selection forces have shaped the evolution and function of the PRAME family. The positive selection observed on the autosomal PRAMEs (Clade II) may result in their functional diversification in immunity and reproduction. Conversely, selective constraints have operated on the expanded PRAMEYs to preserve their essential function in spermatogenesis.

Figure 1. Expression patterns of PRAME/PRAMEY in cattle.

A. RT-PCR results (lanes 2-10). PRAMEY is expressed specifically in the testis, whereas the autosomal PRAME is expressed in the testis (predominantly), kidney, brain and the muscle tissues. Bovine male genomic DNA-specific PCR (lanes 11–12) confirmed that PRAMEY is Y-specific. Te, testis; Li, liver; Ki, kidney; Sp, spleen; Br, brain (cerebrum); Ad, adrenal gland; Mu, muscle; Ly, lymph node; Ov, ovary; ♂, bovine male genomic DNA control; ♀, bovine female genomic DNA control; -, negative control (water); M, 1 kb DNA ladder. B. The expression of the PRAMEY loci by deep-sequencing analysis. The alignment of reads derived from deep-sequencing of selected cDNAs against coding regions of the PRAMEY loci (Table S2) reveals that seven of the 10 active PRAMEY genes are expressed differentially, six of which have significant numbers of both read-pairs matching exactly to the specific loci.

Figure 2. Genome structures of the bovine PRAMEY genes.

Schematic representations of PRAMEY1 and PRAMEY2. Compared to the PRAMEY2 (GenBank acc. no. GU144302) that contains five exons, the first exon of the PRAMEY1 (GenBank acc. no. GU144301) reads through to the second exon and forms a larger exon. The introns are drawn to scale. The open boxes represent UTR regions and the filled black boxes are coding segments (CDS). The numbers denote the length of exons, introns and CDS in bp. The polyA [(A)_n] sites are indicated.

Figure 3. Spatial and temporal expression patterns of the sense and antisense RNA of the bovine PRAMEY2 in adult bovine testis.

A. The sense RNA of PRAMEY2 is expressed specifically in spermatids. B. The antisense RNA of PRAMEY2 is expressed broadly across seminiferous tubules with a predominant expression in spermatids. Sense and antisense RNAs of PRAMEY2 were detected by DIG-labeled cRNA probes. C. The bovine PRM1gene was used as positive control, and there is no antisense mRNA of PRM1 detected in the bovine testis . D. Haematoxylin and Eosin (H&E) staining is shown. Scale: bar  = 200 µm. E. Temporal expression pattern of PRAMEY2. The relative expression levels of the PRAMEY2 sense and antisense transcripts at different ages (X-axis), measured by the strand-specific qPCR, were normalized by the 18S rRNA (Y-axis). The PRAMEY2 sense RNA is expressed very low in earlier stage, but up-regulated in the 8 months and 2 years-old testis. Similarly, antisense RNA of PRAMEY2 is detected in the 8 months and 2 years-old testis. Values are means ± SD of the three biological replicates.

Figure 4. Phylogenetic tree of the PRAME gene family.

Two major PRAME/PRAMEY clades are shown in this tree. The PRAME locus on HSA22 and its syntenic orthologs in other species are clustered with the bovine PRAME and PRAMEY loci in Clade I (branches in red). The orthologs on the X-chrs of horse and mouse are also clustered with Clade I. The PRAME orthologs syntenic to HSA1 are clustered in Clade II (branches in light blue), which contains three sub-clusters, IIa (Artiodactyla), IIb (Primates) and IIc (Rodentia). The tree was built based on the ML method and bootstrap values (1000 replicates) are shown above the branches. The branches corresponding to partitions reproduced in less than 80% bootstrap replicates are collapsed.

Figure 5. Positive selection on the PRAME and PRAMEY orthologs.

Two branches in Clade I and 14 branches in Clade II are under positive selection (red) based on the branch-site model tests (Model A versus Model A null). The branches under positive selection are numbered and the selected sites along each foreground lineage are detailed in Table S5. The nodes underwent duplication are marked with a yellow circle and speciation with a blue circle.

Figure 6. Selective pressures on the PRAME family.

A. The dN/dS ratio distributions in different clades. Clade I has the lowest mean and median dN/dS ratios. The vertical axis represents the dN/dS ratio. The asterisk (*) represents the outliers of the data. B. Map of the positively selected sites detected in Clade IIa to the PRAME protein model. The selected sites derived from PAML analyses are mapped to the protein homology model. Eight of the 12 selected sites (red) are clustered in the inner concave region of the protein model. The model was built based on the PRAME gene (GenBank acc. no. XM_001256020.1) on BTA16. The predicted DNA binding site is highlighted in orange. The LXXLL motifs are highlighted in pink.