THIS WEEK I’ve been covering some interesting instances of new gene evolution. The one I’m covering today is hard to boil down into a short title. This is a case of a new gene in hominoids as the result of retrotransposition of an aberrant mRNA transcript. Transposons showed up in the last post as well, but here they play a different role. That example involved a class II transposon, a segment of DNA that can jump around the genome. This case involves a class I transposon, a retrotransposon that transcribes itself into RNA, then copies that transcript back into DNA, and inserts it elsewhere in the genome. Here the retrotransposon accidentally retrotransposed a gene transcript instead of a retrotransposon transcript. This is not an especially rare event, but this case is unusual because the transcript itself is unusual.

The new gene is called PIPSL, and is an intronless chimera of the genes PIP5K1A and S5a, also known as PSMD4. PIP5K1A and S5a are adjacent genes found on chromosome 1. PIP5K1A encodes the alpha isoform of phoshatidylinositol 4-phosphate 5-kinase type I (PIP5K). Kinases in this family localize to the cell membrane and there phosphorylate their substrate, phosphatidylinositol 4-phosphate, to produce phosphatidylinositol 4,5-bisphosphate. This is a key signalling molecule that helps control many different cellular events. The second gene, S5a, encodes part of the 26S proteasome, a complex that degrades proteins that have been tagged by polyubiquitination. The protein S5a recognizes polyubiquitin chains. Since the two genes are adjacent, sometimes when PIP5K1A is transcribed transcription continues through S5a, producing a chimeric transcript.

The human PIPSL gene is located on Chr 10 and is comprised of the first 13 exons of the 15-exon PIP5K1A gene joined to the last nine exons of the adjacent 10-exon S5a gene (Fig. 1). The two “parental” genes are located in tandem on Chr 1, separated by 5.2 kb, and normally form a low-abundance readthrough transcript spliced between PIP5K1A exon 13 and S5a exon 2 (Fig. 2B), both in the same phase, allowing for in-frame fusion of the greater part of both proteins (for intron analysis, see Supplemental Supplemental Table S1). The event that produced this PIP5K1A-S5a Transcription-Induced Chimeric mRNA (PIP5K1A-S5a TIC) is typical of intergenic splicing, going from an existing splice donor in PIP5K1A to the first splice acceptor of S5a, with <8.5 kb separating the two genes (Akiva et al. 2006; Parra et al. 2006).

To determine whether L1-mediated retrotransposition of PIP5K1A-S5a TIC was responsible for creation of the PIPSL gene, we examined the genomic site of PIPSL for signs of L1-mediated integration. The intronless 3.3-kb PIPSL gene is integrated at a canonical L1 insertion site 5-TTCT’GA-3, terminates in an 80-bp A-rich repeat (GAAA)n, and is flanked by 15-bp target site duplications, all typical of L1 retrotransposition (Ostertag and Kazazian 2001). Concordant divergence of both parts of the PIPSL gene (2.31% and 2.26%) underscores their integration in a single event. Moreover, older processed pseudogenes of PIP5K1A and S5a in the human genome confirm that both genes are expressed at a time and tissue site compatible with L1-mediated retrotransposition.

The diagram below shows the arrangement of the parent genes on chromosome 1 and the derived PIPSL gene on chromosome 10.


By a variety of methods, the authors determined the transcript was inserted 15-19 million years ago. A number of processed pseudogenes and retrotransposon remnants in the genome show that there was a burst of retrotransposon activity about this time. The gene is present in humans, chimpanzees, bonobos, and Sumatran orangutans, but not in gorillas and not in any monkeys. The gene originated after the apes diverged from the monkeys, but before the divergence of the gorillas. This indicates that either the gene was not fixed (present in 100% of the population) at the time gorillas diverged or gorillas later lost the gene. More research would be needed to determine which is the case.

By comparing PIPSL to its parent genes, they determined that PIPSL has mutated more rapidly, and showed especially high rates of nonsynonymous mutations. These change the sequence of the protein product and thus are more likely to cause change in protein function. In the parent genes such mutations faced negative selection when they occurred because the protein was long-adapted to its function and mutations were likely to be detrimental. However, the new gene was just becoming adapted to its new role, so mutations were more likely to produce beneficial structural changes. By examining the sequences of human and chimpanzee PIPSL, the authors determined that this strong selection has since abated. This is consistent with the pattern of initial rapid radiation that appears following genetic innovation.

Since strong selection in the past means the gene must have been serving some role, the next step was to see if PIPSL currently is active in chimpanzees and humans. What they found was interesting. The gene is transcribed at significant levels only in the testis, which has been described as a “transcriptionally permissive” environment, expressing high levels of RNA polymerase II. The high levels of this enzyme make possible transcription of genes without strong promoters, which such an odd new gene is unlikely to have. Humans expressed the gene at a low level, but chimpanzees significantly.

While the gene is transcribed, it does not appear to be translated at all, that is, protein PIPSL levels are undetectable. This is not so surprising in humans since a mutation deactivated the original Kozak consensus sequence that assisted translation initiation. A second weaker consensus sequence is present, and if translation initiated there a slightly shortened protein would be produced, but it appears translation does not occur. On the other hand, chimpanzees possess the intact gene and the protein can be expressed in vitro, but is undetectable in chimpanzee testicular tissue. This suggests strong post-transcriptional suppression of translation, although it is also possible that the protein is synthesized and then rapidly degraded. While PIPSL protein must have been useful in our ancestors, at some point it became unnecessary and perhaps even detrimental.

To determine what this past role might have been, they expressed the gene in vitro and examined the activity of the protein in phosphorylating phosphatidylinositol 4-phosphate and in binding ubiquitin chains. While a mutation has abolished kinase activity, the S5a-derived portion still binds ubiquitinated proteins. PIP5K1A is a membrane protein and phosphorylates its substrate there. Consistent with its lack of kinase activity, PIPSL also does not localize to the membrane but is spread throughout the cytoplasm. The original function of PIPSL was probably quite different from either of the parent protein’s activity, and the authors hypothesize it may have been involved in protein trafficking, endocytosis, or regulated protein degradation.

At some point this ancestral activity became detrimental, and the ancestors of humans and chimpanzees separately evolved mechanisms of silencing protein production. However, the gene is still transcribed at high levels in chimpanzee testis and lower but detectable levels in human testis, and the RNA transcript might act to help control the expression of the two parent genes. Since the gene is also present in several other ape species, it would be interesting to see whether they have likewise evolved mechanisms to suppress PIPSL production.

This is the last of three examples of unusual gene evolution that I will cover this week. Each of the three genes evolved by a novel mechanism. In the first case, an intragenic inversion converted one gene to two. In the second case a primate gene fused with a transposon gene. In this case, a retrotransposon converted an unusual exon-shuffled two-gene co-transcript into DNA and inserted it back into the genome. For all three new genes their sequences archive the story of how that gene was made.

Babushok, D. V.; Ohshima, K.; Ostertag, E. M.; Chen, X.; Wang, Y.; Mandal, P. K.; Okada, N.; Abrams, C. S.; Kazazian, H. H. “A novel testis ubiquitin-binding protein gene arose by exon shuffling in hominoids.” Genome Research 2007, 17, 1129-1138. DOI:10.1101/gr.6252107