Transcriptional activation of a chimeric retrogene PIPSL in a hominoid ancestor

Highlights

• We verified the expression of the PIPSL retrogene in white-handed gibbon.

• The promoter is located upstream of the PIPSL TSS, similar to human PIPSL.

• Promoter development occurred early in hominoid evolution before gibbon divergence.

Abstract

Retrogenes are a class of functional genes derived from the mRNA of various intron-containing genes. PIPSL was created through a unique mechanism, whereby distinct genes were assembled at the RNA level, and the resulting chimera was then reverse transcribed and integrated into the genome by the L1 retrotransposon. Expression of PIPSL RNA via its transcription start sites (TSSs) has been confirmed in the testes of humans and chimpanzee. Here, we demonstrated that PIPSL RNA is expressed in the testis of the white-handed gibbon. The 5′-end positions of gibbon RNAs were confined to a narrow range upstream of the PIPSL start codon and overlapped with those of orangutan and human, suggesting that PIPSL TSSs are similar among hominoid species. Reporter assays using a luciferase gene and the flanking sequences of human PIPSL showed that an upstream sequence exhibits weak promoter activity in human cells. Our findings suggest that PIPSL might have acquired a promoter at an early stage of hominoid evolution before the divergence of gibbons and ultimately retained similar TSSs in all of the lineages. Moreover, the upstream sequence derived from the phosphatidylinositol-4-phosphate 5-kinase, type I, alpha 5′ untranslated region and/or neighboring repetitive sequences in the genome possibly exhibits promoter activity. Furthermore, we observed that a TATA-box-like sequence has emerged by nucleotide substitution in a lineage leading to humans, with this possibly responsible for a broader distribution of the human PIPSL TSSs.

Introduction

Retrogenes are a class of functional genes derived from the mRNA of various intron-containing genes (Brosius, 1999; Wang, 2004; Casola and Betrán, 2017; Kubiak and Makałowska, 2017). They are postulated to have emerged through a mechanism similar to processed pseudogene (PP) formation; however, the detailed mechanism and their biological functions have yet to be elucidated. PPs are reverse-transcribed, intron-less cDNA copies of mRNA that have been reinserted into the genome (Vanin, 1985; Weiner et al., 1986) and are especially abundant in mammalian genomes (Ohshima et al., 2003; Zhang et al., 2003; Abyzov et al., 2013; Ewing et al., 2013; Schrider et al., 2013; Zhang, 2013; Kabza et al., 2014; Navarro and Galante, 2015; Wang, 2017). PPs are not usually transcribed, because they lack an external promoter; therefore, they have long been viewed as evolutionary dead ends with little biological relevance. In addition to PPs, there are two types of pseudogenes: DNA-duplicated (or non-processed) pseudogenes, which arose by segmental duplication (Torrents et al., 2003); and unitary pseudogenes, which are non-processed pseudogenes with no functional counterparts in the genome. Both pseudogene variants are generated by disruptive mutations occurring in functional genes (Matsui et al., 2010; Zhang et al., 2010).

Because pseudogenes generally accumulate many disruptive mutations during the course of evolution, many are considered to have lost transcriptional activity and coding potential (Mighell et al., 2000; Douglas et al., 2016). However, an increasing number of studies offer evidence that PPs can be transcribed by acquiring regulatory elements (Harrison et al., 2005; Sakai et al., 2007; Sorourian et al., 2014), with some having retained coding potential leading to new proteins (Shashidharan et al., 1994; Betrán et al., 2002; Betrán and Long, 2003; Rosso et al., 2008; Parker et al., 2009; Young et al., 2010; Ciomborowska et al., 2013; Abdelsamad and Pecinka, 2014). Others have acquired the regulatory function associated with their parental gene expression as an RNA molecule (Poliseno et al., 2010; Cheetham et al., 2013; Ha et al., 2014; Hirano et al., 2014). A recent large-scale survey of retrogene promoters revealed several patterns associated with promoter acquisition (Carelli et al., 2016).

Previously, we and our collaborators (Babushok et al., 2007) characterized a novel gene, PIPSL, created through a unique mechanism, whereby new combinations of functional domains were assembled at the RNA level from distinct genes [phosphatidylinositol-4-phosphate 5-kinase, type I, alpha (PIP5K1A) and proteasome 26S subunit, non-ATPase 4 (PSMD4)], with the resulting chimera (Akiva et al., 2006) then reverse transcribed and integrated into the genome by the L1 retrotransposon (Babushok et al., 2007; Doucet-O'Hare et al., 2015). The PIPSL gene encodes a chimeric protein with distinct cellular localization and minimal lipid kinase activity while exhibiting significant affinity for cellular ubiquitinated proteins (Babushok et al., 2007). The PIPSL sequence emerged in the genome of the common ancestor of extant hominoid species (Hylobatidae and Hominidae) (Ohshima and Igarashi, 2010), and the PIPSL locus is conserved within human populations and retains a novel haplotype with prominent amino acid changes (Ohshima and Igarashi, 2010). During hominoid diversification, ubiquitin-interacting motif (UIM)1 in the PSMD4-derived domain underwent critical amino acid replacements at an early stage, which were conserved under subsequent high levels of nonsynonymous substitutions to UIM2 and other domains, suggesting that adaptive evolution diversified these functional compartments (Ohshima and Igarashi, 2010).

Relatively high expression of PIPSL RNA along with its transcription start sites (TSSs) was confirmed in the testes of humans and chimpanzees (Babushok et al., 2007; Zhang et al., 2009); however, a full-length PIPSL protein has not been detected (Babushok et al., 2007), although partial amino acid sequences of PIPSL are available in a proteomics database (Ohshima and Igarashi, 2010). Because PIPSL has been inserted in an intergenic region without contact with neighboring genes, it is likely that PIPSL acquired its own transcription-control mechanism at an early stage of its evolution. In this study, to elucidate such processes, we investigated PIPSL expression in species other than human and chimpanzee and searched for possible promoter sequences.