Identification of a truncated splice variant of IL-18 receptor alpha in the human and rat, with evidence of wider evolutionary conservation

Abstract

Interleukin-18 (IL-18) is a pro-inflammatory cytokine which stimulates activation of the nuclear factor kappa beta (NF-κB) pathway via interaction with the IL-18 receptor. The receptor itself is formed from a dimer of two subunits, with the ligand-binding IL-18Rα subunit being encoded by the IL18R1 gene. A splice variant of murine IL18r1, which has been previously described, is formed by transcription of an unspliced intron (forming a 'type II' IL18r1 transcript) and is predicted to encode a receptor with a truncated intracellular domain lacking the capacity to generate downstream signalling. In order to examine the relevance of this finding to human IL-18 function, we assessed the presence of a homologous transcript by reverse transcription-polymerase chain reaction (RT-PCR) in the human and rat as another common laboratory animal. We present evidence for type II IL18R1 transcripts in both species. While the mouse and rat transcripts are predicted to encode a truncated receptor with a novel 5 amino acid C-terminal domain, the human sequence is predicted to encode a truncated protein with a novel 22 amino acid sequence bearing resemblance to the 'Box 1' motif of the Toll/interleukin-1 receptor (TIR) domain, in a similar fashion to the inhibitory interleukin-1 receptor 2. Given that transcripts from these three species are all formed by inclusion of homologous unspliced intronic regions, an analysis of homologous introns across a wider array of 33 species with available IL18R1 gene records was performed, which suggests similar transcripts may encode truncated type II IL-18Rα subunits in other species. This splice variant may represent a conserved evolutionary mechanism for regulating IL-18 activity.

Keywords: Comparative genomics; Interleukin-18; Splice variant; Toll-like receptor.

Figure 1. Schematic diagram of IL18R1 reference sequence and aligned expressed sequence tags.

IL18R1 reference sequence (ENST00000233957/NM_003855.2) intron-exon structure aligned with three identified expressed sequence tags containing portions of intron 8-9. Shown above in boxed region is the expected protein coding sequence of a putative human IL18R1 splice variant incorporating intron 8-9 of ENST00000233957. Underlined nucleotides indicate those from the preceding exon 8. Codons are indicated by alternate grey shading of nucleotides. Incorporation of intron 8-9 would be expected to translate into a protein with a novel 22 amino acid C-terminal followed by a stop codon. First nucleotide shown in boxed region is nt 1132 from ENST00000233957/NM_003855.2, first amino acid is residue 370 from ENSP00000233957.1/NP_003846.1.

Figure 2. Schematic diagram of IL18R1 reference sequence and aligned expressed sequence tags.

IL18R1 cDNA from human blood was amplified by PCR using two polymerases, Platinum and Accuprime Taq as indicated, using the primer pairs shown in Table 1 to amplify the IL18R1 reference transcript (‘IL18R1’, expected product size 687 bp) or predicted human type II IL18R1 splice variant (‘type II’, expected product size 847 bp). ‘RT+’ and ‘RT−’ indicate PCR template generated with the addition of reverse transcriptase or without, respectively. Left hand lane shows 100 bp DNA ladder; brighter band towards the centre of the gel is 600 bp.

Figure 3. Comparison of human type I and type II IL18R1.

(A) C-terminal ends of human IL-18Rα and predicted type II IL-18Rα amino acid sequences. First amino acid, underlined, corresponds to residue 370 from ENSP00000233957.1/NP_003846.1. Shown in red is the amino acid at which the IL-18Rα and predicted type II IL-18Rα sequences diverge, which is encoded across an exon/exon and exon/intron boundary in the case of the reference sequence and predicted type II sequence, respectively. Between the two sequences, similar residues are shown with the region of IL-18Rα corresponding to ‘Box 1’ of the TIR domain (Dunne & O’Neill, 2003) indicated. (B) shows pairwise alignment by EMBOSS Water (European Bioinformatics Institute, Cambridgeshire, UK) of the nucleotides from exon 9 of the human IL18R1 reference sequence (ENST00000233957/NM_003855.2) with those of intron 8-9. Codons are indicated by alternating grey shading, with the initial G nucleotide shown in brackets being the final nucleotide from exon 8 (nt 1135 of ENST00000233957/NM_003855.2). Above and below the respective sequences are shown the corresponding amino acid sequences.

Figure 4. Multiple sequence alignment of human IL-1 receptor family members.

The predicted protein coding sequence of human type II IL-18Rα aligned with IL-1 receptor family members. Shown are the amino acid residues surrounding the beginning of the TIR domain, where the sequence of type II IL-18Rα diverges from the reference sequence. Boxed regions are the ‘Box 1’ and ‘Box 2’ motifs present in the TIR domain.

Figure 5. Multiple sequence alignment of putative type II IL-18Rα C-terminal ends across multiple species.

Sequences are colour-coded as per MView (European Bioinformatics Institute, Cambridgeshire, UK): the human sequence is coloured according to residue properties, as indicated under ‘Key’, with residues from other species identical to human coloured with the same schema. Orangutan, Armadillo and Tarsier sequences are truncated by the indicated number of amino acids. Percentages reflect percent identity to human sequence as a reference.

Figure 6. Alignment of homologous intron sequences from IL18R1 across a range of species.

(A) When the reading frame is continued into the intron sequences, stop codons (red shading) are encountered shortly into the intron sequence, including a conserved stop codon position which resides within a region of high conservation (grey shading) as identified by Lindblad-Toh et al. (2011). Further downstream (B) an apparent conserved polyadenylation sequence (red shading, PolyA) is apparent, within a region of high conservation identified by Lindblad-Toh et al. (2011) (grey shading). A proposed transcription termination site (TTS) is indicated by the dotted line.