Frequent and recent retrotransposition of orthologous genes plays a role in the evolution of sperm glycolytic enzymes

Abstract

Background: The central metabolic pathway of glycolysis converts glucose to pyruvate, with the net production of 2 ATP and 2 NADH per glucose molecule. Each of the ten reactions in this pathway is typically catalyzed by multiple isozymes encoded by a multigene family. Several isozymes in this pathway are expressed only during spermatogenesis, and gene targeting studies indicate that they are essential for sperm function and male fertility in mouse. At least three of the novel glycolytic isozymes are encoded by retrogenes (Pgk2, Aldoart1, and Aldoart2). Their restricted expression profile suggests that retrotransposition may play a significant role in the evolution of sperm glycolytic enzymes.

Results: We conducted a comprehensive genomic analysis of glycolytic enzymes in the human and mouse genomes and identified several intronless copies for all enzymes in the pathway, except Pfk. Within each gene family, a single orthologous gene was typically retrotransposed frequently and independently in both species. Several retroposed sequences maintained open reading frames (ORFs) and/or provided evidence of alternatively spliced exons. We analyzed expression of sequences with ORFs and <99% sequence identity in the coding region and obtained evidence for the expression of an alternative Gpi1 transcript in mouse spermatogenic cells.

Conclusions: Our analysis detected frequent, recent, and lineage-specific retrotransposition of orthologous glycolytic enzymes in the human and mouse genomes. Retrotransposition events are associated with LINE/LTR and genomic integration is random. We found evidence for the alternative splicing of parent genes. Many retroposed sequences have maintained ORFs, suggesting a functional role for these genes.

Figure 1

Gene and species-specific divergence of human and mouse retroposed sequences. Percent substitution at the nucleotide level in the entire retroposed sequences (ORFs and UTRs) compared to each parent gene. Retroposed sequences matching each enzyme are represented by a different color, as shown in the inset. Retroposed sequences for each parent gene are shown in the same order as the inset. Each retroposed sequence in represented once at the appropriate substitution level and each bar represents the number of sequences (y-axis) with the percent substitution compared to the parent gene (x-axis). Human sequences are shown on the top and mouse sequences are shown on the bottom.

Figure 2

Retroposed sequences support the expression of novel transcripts. The structure of each parent gene is diagrammed with the coding sequence denoted by alternating yellow and red exons. Retroposed sequences with ORFs have red and yellow segments corresponding to exons in the parent gene. Upstream start codons (black exons), and/or alternatively spliced exons (diagonal lined boxes) are also shown. Sequences containing LINE elements are denoted by horizontal lines. Coding regions for retroposed sequences with ORFs were compared to their parent gene, and the percent identity at the nucleotide level is shown next to the corresponding gene structure.

Figure 3

Human ORFs with divergent sequences are not expressed in testis. (A) Diagram of the RT-PCR approach used to distinguish expression of transcripts. Black arrows denote primer sets used to amplify both parent gene and retroposed sequences. The fraction next to each retroposed sequence shows the number of unique nucleotide residues in the amplified product. (B) TPI1-rs1, PGAM1- rs7, and ENO1- rs1 transcripts were not detected in pooled human testis RNA samples with RT-PCR using primers that amplify both the retroposed sequence and the parent glycolytic enzyme, followed by single-strand conformation polymorphism (SSCP) gel electrophoresis. PCR products amplified from human genomic DNA (G1 and G2; two individuals) show the expected position of transcripts from retroposed sequences. PCR products amplified from pooled testis total RNA are shown in lanes T1, T2, and T3 (triplicate cDNA preparations).

Figure 4

Expression of an alternative Gpi1 transcript in mouse spermatogenic cells. (A) Diagram of RT-PCR approach used to distinguish expression of Gpi1-related transcripts. Gray arrows denote the primer set used to differentiate transcripts containing alternatively spliced exons 5 and 6 (boxes with diagonal lines). Black arrows denote the Gpi1-rs1-specific primer set. (B) Transcripts from Gpi1 were detected in all mouse tissues and isolated testicular cells. Gpi1-rs1 was amplified from genomic DNA to identify the expected size of PCR products from Gpi1 transcripts not containing exons 5 and 6. A product of the same size was detected in isolated pachytene spermatocytes (PS) and round spermatids (RS), but not condensing spermatids (CS). This PCR fragment appears to be derived from Gpi1_v2, since Gpi1_rs1-specific primers did not amplify a product. (C) A smaller GPI1_V2 protein was not detected by western analysis using a polyclonal antibody raised against human GPI1. A larger protein product was seen in isolated testicular cell, but not in mouse or human (Hs) sperm. S/N fraction contains proteins solubilized from sperm tail following brief sonication and centrifugation. Tail fraction contains proteins left insoluble following sonication and centrifugation.

Figure 5

Abundance of repetitive elements flanking retroposed sequences and (G + C) content. (A) Diagram comparing the frequency of LINE and LTR elements in regions flanking retroposed sequences (grey) or genes encoding all glycolytic enzymes (black). (B) (G + C) content (%) of combined upstream and downstream 10 kb sequence flanking human (grey) and mouse (black) retroposed sequences.