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. 2007 Aug;17(8):1129-38.
doi: 10.1101/gr.6252107. Epub 2007 Jul 10.

A novel testis ubiquitin-binding protein gene arose by exon shuffling in hominoids

Daria V Babushok  1 , Kazuhiko Ohshima, Eric M Ostertag, Xinsheng Chen, Yanfeng Wang, Prabhat K Mandal, Norihiro Okada, Charles S Abrams, Haig H Kazazian Jr
Affiliations

A novel testis ubiquitin-binding protein gene arose by exon shuffling in hominoids

Daria V Babushok et al. Genome Res. 2007 Aug.

Abstract

Most new genes arise by duplication of existing gene structures, after which relaxed selection on the new copy frequently leads to mutational inactivation of the duplicate; only rarely will a new gene with modified function emerge. Here we describe a unique mechanism of gene creation, whereby new combinations of functional domains are assembled at the RNA level from distinct genes, and the resulting chimera is then reverse transcribed and integrated into the genome by the L1 retrotransposon. We characterized a novel gene, which we termed PIP5K1A and PSMD4-like (PIPSL), created by this mechanism from an intergenic transcript between the phosphatidylinositol-4-phosphate 5-kinase (PIP5K1A) and the 26S proteasome subunit (PSMD4) genes in a hominoid ancestor. PIPSL is transcribed specifically in the testis both in humans and chimpanzees, and is post-transcriptionally repressed by independent mechanisms in these primate lineages. The PIPSL gene encodes a chimeric protein combining the lipid kinase domain of PIP5K1A and the ubiquitin-binding motifs of PSMD4. Strong positive selection on PIPSL led to its rapid divergence from the parental genes PIP5K1A and PSMD4, forming a chimeric protein with a distinct cellular localization and minimal lipid kinase activity, but significant affinity for cellular ubiquitinated proteins. PIPSL is a tightly regulated, testis-specific novel ubiquitin-binding protein formed by an unusual exon-shuffling mechanism in hominoid primates and represents a key example of rapid evolution of a testis-specific gene.

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Figures

Figure 1.
Figure 1.
(A) Neighboring 15-exon PIP5K1A and 10-exon S5a genes on Chr 1 are spliced to form PIP5K1A, S5a, and PIP5K1A-S5a TIC mRNAs. (Black rectangles) Exons; (curved lines) splicing. (B) PIP5K1A-S5a TIC was retrotransposed by L1 to create the PIPSL gene on Chr 10. (TSD) Target site duplication; (pA) A-rich repeat. (White rectangles) Regions corresponding to PIP5K1A exons; (gray rectangles) regions corresponding to S5a exons.
Figure 2.
Figure 2.
(A) Southern hybridization was performed on HindIII-digested primate DNA using a 32P-labeled probe to the S5a gene. The 3-kb band corresponds to PIPSL; it migrates at 4.8 kb in Gorilla because of an RFLP (arrowhead). The 5.9-kb and 5.6-kb doublet (**) corresponds to S5a and its Chr 21 pseudogene. Supplemental Figure S7 depicts probe locations. (B) Shown are duplicate RT-PCRs on total RNA from human testis or HeLa cells using the primers depicted in C. In the top gel of B, A* and D* primers specific for PIPSL amplified PIPSL from testis (lane 1), but not HeLa cDNA (lane 6). A and D primers specific for the PIP5K1A-S5a Transcription-Induced Chimera (TIC) amplified only trace amounts of TIC from testis (lane 2) and HeLa (lane 7) cDNAs. In the bottom gel, parental PIP5K1A (lanes 1,2) and S5a (lanes 6,7) transcripts were amplified from the same testis (T) and HeLa (H) cDNAs, serving as internal controls. (Lanes 3,4,8,9) RT-PCRs lacking reverse transcriptase; (lanes 5,10) no template PCRs; (M) 1-kb Plus DNA ladder. (C) Primers used in RT-PCRs are shown by arrowheads (A, B, C, D, A*, and D*). Probes used in Northern blots are shown by black (ex 15, ex 8–12, and PIPSL-specific) and white (ex 3–7) lines. (D) Northern blot of PIPSL in human testis (T) showing two PIPSL transcripts of ∼3.0–3.6 kb (bracket). HeLa RNA (H) is used as a negative control. PIPSL is visible with probe 2 in PIP5K1A exons 8–12, PIPSL-specific probe 3 spanning both parental genes, and probe 4 in S5a exons 3–7, but not with negative-control probe 1 to PIP5K1A exon 15. The 3.7-kb PIP5K1A and 1.3-kb S5a mRNAs serve as internal controls. (E) Northern blot on RNAs from multiple human tissues with PIPSL-specific probe 3 shows PIPSL transcripts only in the testis. PIP5K1A and S5a are ubiquitously present. To confirm comparable loading, the blot was stripped and reprobed for GAPDH and 7SL RNAs. (F) A Northern blot on RNAs from several chimpanzee tissues with PIPSL-specific probe 3 shows ∼3.0-kb PIPSL transcripts only in the testis. Internal control parental S5a is ubiquitously present. The human PIPSL-specific probe 3 does not hybridize to the chimpanzee PIP5K1A transcript.
Figure 3.
Figure 3.
PIPSL experienced a burst of positive selection shortly after its creation (A). The phylogenetic tree of the PIP5K1A region (A) and the S5a region of the PIPSL gene (B). Branches are drawn in proportion to the total number of substitutions incurred, with nonsynonymous-to-synonymous substitution rate ratios (Ka/Ks), and the nonsynonymous (n) and synonymous (s) substitutions in parentheses (n : s) shown above each branch. Nineteen nonsynonymous and 0 synonymous changes occurred in the S5a region of PIPSL along the branch leading to the human/chimp split (B, indicated by bold line). The n/s ratio is significantly greater than its neutral expectation (P = 0.0018, Fisher’s exact test; Table 1), indicating that the PIPSL gene was under strong positive selection shortly after its formation. (NA) Not applicable (Ks = 0).
Figure 4.
Figure 4.
(A) Diagram showing Renilla luciferase reporter constructs, in which the Renilla Kozak sequence is replaced by the 5′-UTR/Kozak regions from human PIPSL (1), chimp PIPSL (2), and PIP5K1A (3). (B) Average amounts of Renilla produced by in vitro transcription/translation reactions using the three constructs ±SD. For each construct, eight replicates were performed using six to eight independent DNA preparations. Human PIPSL translation was significantly reduced (P < 0.001).
Figure 5.
Figure 5.
Intracellular localization of GFP-fused PIP5K1A, S5a, and PIPSL proteins in 293T cells was observed by GFP fluorescence with confocal microscopy. (A) GFP control is seen throughout the cell, with nuclear predominance; (B) PIP5K1A is on the plasma membrane. (C) S5a is throughout the cell with cytoplasmic predominance and possibly nucleolar exclusion; (D) Human and (E) chimpanzee PIPSL proteins are cytoplasmic. A Western of IP’d GFP fusion proteins is shown in Supplemental Figure S6B.
Figure 6.
Figure 6.
PIPSL proteins lack PIP5 kinase activity, and bind cellular ubiquitinated proteins. (A) C-terminally HA-tagged human PIP5K1A (1), S5a (2) and PIPSL (3), and chimpanzee PIPSL (4) proteins were overexpressed in 293T cells, IP’d against HA epitope, and tested for phosphorylation of PI4P to produce PIP2. (B) The amounts of IP’d proteins were analyzed by an anti-HA Western blot. (C) Coimmunoprecipitation of HA-tagged human PIP5KA (1), S5a (2), S5AL (3), chimpanzee PIPSL (4), and untransfected 293T cells (5) with cellular ubiquitinated proteins (bracket). After anti-HA IP, proteins were immunoblotted for ubiquitin (C) or for HA-tag (D). (E) Anti-Ub Western blot of cellular lysates to demonstrate equal levels of endogenous ubiquitinated proteins. (F) Equal loading of lysates confirmed by Western against S10a. (G) Average band intensities of ubiquitinated protein from three independent co-IP experiments were normalized by HA-tagged protein amounts and scaled to the same overall blot intensity. Error bars denote ±1 SD. (*) Immunoglobulin chains; (**) a nonspecific band.
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