Death and resurrection of the human IRGM gene

Abstract

Immunity-related GTPases (IRG) play an important role in defense against intracellular pathogens. One member of this gene family in humans, IRGM, has been recently implicated as a risk factor for Crohn's disease. We analyzed the detailed structure of this gene family among primates and showed that most of the IRG gene cluster was deleted early in primate evolution, after the divergence of the anthropoids from prosimians ( about 50 million years ago). Comparative sequence analysis of New World and Old World monkey species shows that the single-copy IRGM gene became pseudogenized as a result of an Alu retrotransposition event in the anthropoid common ancestor that disrupted the open reading frame (ORF). We find that the ORF was reestablished as a part of a polymorphic stop codon in the common ancestor of humans and great apes. Expression analysis suggests that this change occurred in conjunction with the insertion of an endogenous retrovirus, which altered the transcription initiation, splicing, and expression profile of IRGM. These data argue that the gene became pseudogenized and was then resurrected through a series of complex structural events and suggest remarkable functional plasticity where alleles experience diverse evolutionary pressures over time. Such dynamism in structure and evolution may be critical for a gene family locked in an arms race with an ever-changing repertoire of intracellular parasites.

Figure 1. Comparative structure of IRGM loci.

The structures of the IRGM loci are shown in the context of a generally-accepted primate phylogenetic tree. ORF, ERV9, intronic sequence, Alu sequence, and 5′ untranslated region (UTR) depicted in green, black, white, yellow and blue colors respectively. A red color denotes pseudogenes based on the accumulation of deleterious mutations in the ORF. Shaded orange color indicates an atypical GTPase because of mutations leading to the loss of a canonical GTPase binding motif (see Figure S1). The first ATG codon (green arrow) after the Alu repeat sequence is used as putative start codon for the open reading frame of IRGM. The transcription start site is marked with green flag. FS indicates frameshift mutation. TGA and TAA denote the position of stop codons (arrows). The shaded white, blue and green colors indicate predicted intron, UTR or exon, respectively. The genomic loci are not drawn to scale with the exception of the full-length sequence of IRGM ORF.

Figure 2. FISH analysis of IRGM.

The figure shows examples of FISH experiments on Hs (Homo sapiens), Rh (Macaca mulatta), Cja (Callithrix jacchus) and Lca (Lemur catta), with the use of human fosmid clone WIBR2-3607H18 (A, B, C) and lemur species-specific BAC clone LB2-77B23 (D).

Figure 3. Expression analysis of IRGM in human, macaque and marmoset.

A) RT-PCR results from cDNA prepared from total RNA extracted from various human (Hs) tissues are compared against . B) rhesus macaque (Rh) and marmoset (Cja) tissues. Total RNA was treated with DNAse I prior to cDNA synthesis. cDNA reactions established with (+) and without (−) reverse transcriptase as a control and RT-PCR compared against a positive UBE1 expression control. C) RT-PCR reveals splicing of the 5′ UTR region for human and chimp IRGM but not in rhesus macaque. Diagram is showing the promoter and 5′UTR region of IRGM. ORF, ERV9, intronic sequence, Alu sequence, and 5′ untranslated region (UTR) depicted in green, black, white, yellow and blue colors respectively. The first ATG codon after Alu sequence is used as putative start codon for the open reading frame of IRGM. Transcription start site is marked with green flag. Arrows indicate the position of the primers.

Figure 4. Selection of the IRGM locus during primate evolution.

Branch estimates for ω = d_N/d_S are shown using the free branch model in PAML. Species names are indicated as Hs (Homo sapiens), Ptr (Chimp, Pan trogylodytes), Ggo (Gorilla gorilla), Ppy (Orangutan, Pongo pygmaeus), Rh (Rhesus macaque, Macaca mulatta), Pha (Papio hamadryas), Cja (Marmoset, Callithrix jacchus), Mmu (Microcebes murinus), and Dog (Canis familiaris). Omega values (ω) and the number of nonsynonymous and synonymous substitutions (N*d_N/S*d_S) for each respective branch are indicated in parentheses. Branch estimates for d_N/d_S are shown for the three groups (see Text S1). Species names highlighted in red carry pseudogenes based on multiple stop codons in the ORF.

Figure 5. Evolution of IRGM loci.

A model for the evolution of primate IRGM genes is depicted. The mammalian IRGM tandem gene family contracts to a single-copy gene after the divergence of prosimians and anthropoids. The single-copy gene is pseudogenized in the anthropoid ancestor due to an AluS_c repeat integration into the second exon, disrupting the ORF of the sole remaining IRGM gene. Multiple stop codons and frameshift mutations accrue in all Old World and New World monkey lineages. Three mutation events restore the IRGM gene in the common ancestor of apes and humans: integration of the ERV9 element to serve as a new promoter, a single-nucleotide mutation that introduces a new ATG codon (green arrow) after the Alu repeat and the loss of a stop codon that is shared with Old World monkey species. The latter event is polymorphic in orangutans rendering both functional and nonfunctional copies in this species. (*) of the five gibbon species analyzed, H. gabriellae shows a heterozygote stop codon. In the human and African great ape, the functional copy becomes fixed. Frameshift mutation (Fs) and stop codons are indicated. The genomic loci are not drawn to scale with the exception of the full-length sequence of IRGM ORF.