Functional persistence of exonized mammalian-wide interspersed repeat elements (MIRs)

Abstract

Exonization of retroposed mobile elements, a process whereby new exons are generated following changes in non-protein-coding regions of a gene, is thought to have great potential for generating proteins with novel domains. Our previous analysis of primate-specific Alu-short interspersed elements (SINEs) showed, however, that during their 60 million years of evolution, SINE exonizations occurred in some primates, only to be lost again in some of the descendent lineages. This dynamic gain and loss makes it difficult to ascertain the contribution of exonization to genomic novelty. It was speculated that Alu-SINEs are too young to reveal persistent protein exaptation. In the present study we examined older mobile elements, mammalian-wide interspersed repeats (MIRs) that underwent active retroposition prior to the placental mammalian radiation approximately 130 million years ago, to determine their contribution to protein-coding sequences. Of 107 potential cases of MIR exonizations in human, an analysis of splice sites substantiates a mechanism that benefits from 3' splice site selection in MIR sequences. We retraced in detail the evolution of five MIR elements that exonized at different times during mammalian evolution. Four of these are expressed as alternatively spliced transcripts; three in species throughout the mammalian phylogenetic tree and one solely in primates. The fifth is the first experimentally verified, constitutively expressed retroposed SINE element in mammals. This pattern of highly conserved, alternatively and constitutively spliced MIR sequences evinces the potential of exonized transposed elements to evolve beyond the transient state found in Alu-SINEs and persist as important parts of functional proteins.

Figure 1.

Distribution and orientation of exonized MIR sequences located in protein-coding sequences (CDS). The 107 human exonized MIR sequence regions are aligned against a schematic of a MIR consensus element. The tRNA-related part, including the internal promoter A and B boxes (black), is shown without shading (white). The region shown in dark gray comprises the 70 nt conserved central domain including a highly conserved 15-bp core sequence. The location of the MIR internal, antisense cryptic splice site AG is indicated on the sense strands (CT) by a vertical line. The 9-nt natural 3′ MIR splice site (5′-ATTTTACAG-3′) is shown as the inverse consensus sequence (Supplemental Fig. S3). The exonized MIR regions are represented as black lines; intronic or untranslated region (UTR) portions of the MIRs are not shown. The five experimentally analyzed examples are indicated (CHRNA1, Zfp384, ZNF639, LAS1L, and NTRK3).

Figure 2.

Structures of five selected genes harboring internal exonized MIR elements. Numbered, thick gray boxes represent protein-coding sequences (CDS). The 5′ and 3′ untranslated regions (UTRs) are shown as medium thick gray bars. Introns are indicated as black lines; double slashes denote gaps in larger introns. Orientations and recognizable lengths of the MIR elements are indicated by arrows. The short white boxes represent the intronic parts of the MIR elements, and the adjacent tall white boxes the exonized regions. In three cases the exonization exceeds the MIR boundaries and includes anonymous intronic sequences (black bars adjacent to the exonized regions). (A) Human neurotrophic tyrosine kinase receptor type 3 gene (NTRK3; BT007291). (B) Human zinc finger protein 639 gene (ZNF639; NM_016331). (C) Human LAS1-like gene (LAS1L; NM_031206). (D) Rat zinc finger protein 384 gene (Zfp384; AF216807). (E) Human cholinergic receptor nicotinic alpha1 gene (CHRNA1; NM_001039523). The substructures in exon 7 of ZNF639 and in exons 6 and 7 of Zfp384 represent sequences encoding zinc finger domains.

Figure 3.

Phylogenetic tree of investigated mammals showing the species distributions of the five internal MIR exonizations. In DNA, “+” indicates the presence of the intronic MIR element leaving the splice sites and reading frame intact, “(+)” its presence, without leaving the reading frame intact, and “−” its absence. In RNA, “+” indicates the presence of the exonized MIR sequence in cDNA derived by RT-PCR, and “−” the presence of a cDNA that does not include the exonized MIR sequence. Both symbols indicate that both splice forms are expressed. EST, expressed sequence tag, “+” including or “−” without the exonized MIR region, available at GenBank (http://www.ncbi.nlm.nih.gov/BLAST/Blast.cgi). Empty boxes indicate that the corresponding sequence information is not available or not PCR amplifiable; n/a, not analyzed. ¹A third splice variant exists, derived from the human MegaMan Human Transcriptome Library (Stratagene). ²Five splice variants exist, three of which include the exonized MIR element. ³RT-PCR performed in the nine-banded armadillo instead of sloth. ⁴Not shown are “+” symbols under the DNA for chimpanzee and orangutan. ⁵Unrelated exonized intronic sequence in chicken and a corresponding exonization in the ostrich.

Figure 4.

Evolutionary scenario after exonization of sequences, from the ancestral constitutive to alternative and, occasionally, to novel constitutive splicing. Intensities of selection are indicated at the left and right bars. For evolution of alternative splice sites, the intensities of selection vary over time. Initially, inclusion of the novel exon might be slightly deleterious, neutral, or slightly advantageous. Subsequently, selection changes, perhaps during a period of positive selection to increasing levels of negative selection (bar at the left with a gradient from white to red). The numeration refers to intronic insertion of a transposed element (red box) (1), followed by acquisition of necessary components for alternative splicing (2). Under relaxed selective pressure, the exonized condition might revert or become fixed in different lineages (vertical double arrow). In an extreme case, (3), as shown for ZNF639, the novel alternative splice form might, over time, completely replace the ancestral constitutive splice form, thus representing a different constitutive form (bar at the right side, with gradients from black via white to red). However, the constitutive inclusion of the exonized sequence in ZNF639 could also have been acquired directly after acquisition of the necessary splice sites. Examples corresponding to different stages of exonization taken from previous works (Singer et al. 2004; Krull et al. 2005; Mola et al. 2007) or from the present work (bold letters) are presented in ovals.