Genes and Immunity (2008) 9, 631–639 & 2008 Macmillan Publishers Limited All rights reserved 1466-4879/08 $32.00 www.nature.com/gene
ORIGINAL ARTICLE
Identification and characterization of multiple splice forms of the human interleukin-23 receptor a chain in mitogen-activated leukocytes S-h Kan, G Mancini and G Gallagher Genetic Immunology Laboratory, HUMIGEN LLC, The Institute for Genetic Immunology, Hamilton, NJ, USA
The signalling of interleukin-23 (IL-23) and its receptor (IL-23R) is a key element in the differentiation of T cells to the Th17 phenotype. Here, we present the identification and characterization of human IL23R splice variants resulting from alternative splicing of the IL23R mRNA, from activated human leukocytes, following the analysis of IL23R cDNA. Twenty-four different IL23R transcripts were observed in this study, which may potentially lead to an alteration in the protein coding region of IL-23Ra. Consequently, by analysing amino acid sequences deduced from alternatively spliced mRNA sequences, four different putative premature early termination forms of IL-23Ra: (1) a very short ‘IL-23Ra’, (2) an IL-23Ra containing only the extracellular region, (3) a IL-23Ra with truncated intracellular domain and (4) in-frame exon-skipping causing changes to the extracellular region of the IL-23Ra were revealed. These changes may affect the function of IL-23R by altering the ligand–binding interaction, producing a soluble form of the receptor to act as a decoy receptor and/or modify the IL-23/IL-23R signalling, respectively. Taken together, identification of potentially functional splice variants of IL23R underscores the biological diversity of the IL23R gene and will aid in the understanding of the gene’s function in normal and pathological conditions. Genes and Immunity (2008) 9, 631–639; doi:10.1038/gene.2008.64; published online 28 August 2008 Keywords: IL-23; IL-23R; Th17; splice variants; Crohn’s disease
Introduction Interleukin-23 (IL-23) is a newly identified cytokine that belongs to the IL-6 superfamily.1 It is structurally similar to IL-12, sharing a common p40 subunit with an IL-23specific p19 subunit, instead of the p35 subunit found in IL-12.1 Similar to the IL-12 receptor (IL-12R), the IL-23R is a heterodimeric construct that consists of the common IL-12Rb1 chain (as found in the IL-12R) and a unique IL-23Ra chain; IL-23Ra is analogous to the other IL-12R chain, IL-12Rb2. Both the IL-23Ra chain and the IL-12Rb2 chain are the main signalling chains of their respective receptors.1 The human IL23R gene is located on chromosome 1p32.1–p31.2, 150 kb telomeric of the adjacent gene, IL12Rb2. The ‘native form’ of human IL23R mRNA is 2.8 kb long and comprises 11 exons (NM_144701). The transcribed mRNA is translated into a protein of 629 amino acids, resulting in a type-I transmembrane protein which contains a signal peptide, an N-terminal fibronectin III-like domain and a 252-residue cytoplasmic domain with three potential tyrosine phosphorylation sites.1 Interleukin-23 receptor is a member of hemopoietin Correspondence: Dr G Gallagher, Genetic Immunology Laboratory, Humigen LLC, The Institute for Genetic Immunology, 2439 Kuser Road, Hamilton, NJ 08690, USA. E-mail:
[email protected] Received 29 May 2008; revised 14 July 2008; accepted 15 July 2008; published online 28 August 2008
receptor superfamily. It associates constitutively with Janus kinase 2 and also allows binding of Signal Transducer and Activator of Transcription (STAT) 3 in a ligand-dependent manner, whose phosphorylation and consequent dimerization trigger downstream expression of genes, such as IL17 and IL22, but not IFNg.1–4 The IL-23R is mainly expressed on activated/memory T cells, T-cell clones and NK cells. Low levels of IL-23R expression have also been detected on monocytes, macrophages and dendritic cells,1,2 which are consequently IL-23 responsive. Although several of the structural and functional similarities between IL-23/IL-23R and IL-12/IL-12R signalling suggest possible related regulatory functions in T-cell development. Both cytokines actually have distinct roles in regulating the T cell-mediated immune responses that bridge the innate and adaptive immunity.5 IL-12/IL12R signalling plays a pivotal role in Th1 cell responses by promoting IFN-g production during infection. However, IL-23/IL-23R signalling is the central molecule of Th17 cell differentiation by upregulation of IL-17 production in inflammatory (and autoimmune) responses. Interleukin-23 receptor and a series of downstream molecules are important components in triggering the Th17 cell responses in innate immunity. In Th17 cell development, native CD4 þ T cells are activated by IL-6 from mature dendritic cells, which acts together with activated TGF-b to induce expression of the retinoic orphan receptor RORgt and upregulate IL-23R, thus making them competent for IL-17 production through STAT 3 signalling.6,7
Human IL-23 receptor splice variants S-h Kan et al
632
The importance of IL-23R in controlling innate immunity through Th17 cells may underscore why the IL23R gene also has been demonstrated to confer susceptibility to several autoimmune diseases, including psoriasis,8,9 ankylosing spondylitis,10 multiple sclerosis11–13 and inflammatory bowel diseases (both Crohn’s disease and/or ulcerative colitis).14,15 In addition, a separate study by the Wellcome Trust Case Control Consortium and the Australo-Anglo-American Spondylitis Consortium found evidence to suggest that IL23R may be a common susceptibility factor for the major ‘seronegative’ diseases.16 A recent genome-wide association (GWA) study first demonstrated that IL23R is one of the genetic factors contributing to Crohn’s disease. Among the single nucleotide polymorphisms within IL23R, one missense polymorphism (rs11209026, R381Q) is negatively associated with Crohn’s disease, which implies that the Q allele may play a protective role in the pathogenesis of Crohn’s diseases.14 Subsequently, more studies have also associated this R/Q single nucleotide polymorphism with other diseases from different populations.9,10,15,17–19 Although investigating the IL23R for novel polymorphic elements, we discovered a range of novel splice variants of IL23R transcripts, greatly extending an earlier study by Zhang et al.20 from early 2006. We believe that understanding the role of these variants and how their presence is controlled will give us novel insight to the role of IL-23 in human Crohn’s disease. In addition, the IL-23/IL-17 pathway is known to be associated with other autoimmune diseases.8–13 The purpose of this study was therefore to understand possible pathogenic mechanisms of IL-23R’s contribution to disease susceptibility.
Results The IL-23Ra protein is mainly expressed on T cells, NK cells, monocytes and dendritic cells,1 thus the expression IL23R in unstimulated peripheral blood mononuclear cells (PBMC) is ordinarily low. In this study, no detectable IL23R expression was obtained from the freshly isolated PBMC by reverse-transcriptase polymerase chain reaction (RT-PCR; data not shown). However, a significant upregulation of the IL23R cDNA was detected in PBMC after 72 h cultured with or without mitogen stimulation. Two forward primers, P3 or P5, combined with a reverse primer P6 were applied in this study to generate amplicons of IL23R whose predicted sizes are 1234 bp and 901 bp, respectively. However, multiple smaller-sized transcripts compared with the expected sizes of the IL23R amplicons were observed, suggesting the existence of multiple splice variant transcripts of the mRNA from these PBMC samples. The total RT-PCR products were cloned into TOPO TA vector PCR2.1 (Invitrogen, Carlsband, CA, USA) to identify the variants of each PCR product individually. Colony PCR was first applied using M13 forward and M13 reverse primers located on either side of the TA cloning sites on the vector to initially screen the colonies generating amplicon size smaller than the wildtype IL23R. Internal sequencing primers were designed as necessary. Genes and Immunity
The detection and identification of IL23R splice variants In this study, both restriction enzyme digestion and sequence analysis were applied to define the various IL23R splice variants in the activated PBMCs. To distinguish the differences in IL23R cDNA because of splicing, restriction endonuclease digestion was first performed on the cloned PCR products and band sizes were resolved by agarose gel electrophoresis. The digestion was designed to observe skipping of various complete IL23R exons as well as partial exon-skipping variants. Two endogenous EcoRI sites within exon 7 and exon 8 of the native form of the IL23R mRNA gave three DNA fragments of clearly differing size, as depicted in Figure 1a. The EcoRI restriction digestion of IL23R P5/P6 PCR fragment clones was comprised of three DNA fragments: exon 4–7 (403 bp), exon 7–8 (155 bp) and exon 8–11 (343 bp), covering the area after the translated signal peptide, which is possibly the area containing most of the alternative splicing events (Figure 1a). The diversity of the EcoRI digestion pattern suggested that highly polymorphic and dynamic splicing activities were present in the mRNAs of the IL23R gene (Figure 1b). Clones that revealed different digestion patterns after EcoRI digestion, described above, were then sequenced to define the exact position and nature of these splice events (Figure 1b). Sequencing of the variant IL23R fragment cDNA clones identified 22 novel IL23R mRNA splice variants as well as two (D5: AY937251 and D9: AY937253) that had previously been identified;20 these are depicted in Figure 2, which shows both the simple and compound deletion events discovered in this study. All the sequences identified in this study have been submitted to Genbank (summarized in Figure 2). The simple exonskipping events can be categorized to: (a) simple deletions, that is skipping of complete single exons or consecutive exons (for example, D4; D5; D6; D7; D8; D9; D5,6; D6,7; D8,9; D5,6,7; isoforms v1–10) and (b) partial exon deletions (for example, pD5 5 nt; pD5 71 nt; pD11 67 nt; two different versions of pD5–7 and pD7–8. Isoforms v11–14 and v19–20). All of the exon-skipping events affected the exons encoding parts of the extracellular region and/or transmembrane domain of IL-23Ra, but not the intracellular domain. In addition to the 14 simple exon-skipping events shown in Figure 2, 10 more splicing events were identified that involved multiple exon-skipping events. In these compound exon-skipping transcripts, several splicing events were evident from the pre-mRNA processing (Figure 2). These compound splicing events include the skipping of combinations of complete, nonconsecutive exons (D5,8; D5,9; D5,6,8; D5,6,9. Isoforms v15–18) and combinations of the whole exon-skipping and partial exon deletion (pD5 5 nt plus D8; pD5 5 nt plus D6,7; pD5 71 nt plus D8,9; D8 plus pD11 67 nt; pD7,8 plus pD11 67 nt and pD5,6,7 plus D9 plus pD11 67 nt. Isoforms v19–24). Among these 10 mRNA transcripts, the most complicated variant (v20) involved three individual splicing events in one transcript. These multiple exonskipping events all involved the entire or partial deletion of exon 5/6 and exon 8/9, which implies that the skipping of these exons are common events in the premRNA processing. In addition, the only skipping event involving an exon encoding the intracellular part of the IL-23Ra protein, pD11 67 nt, repetitively occurred with
Human IL-23 receptor splice variants S-h Kan et al
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
WT
WT
633
403 343
403 343
155
155
Lane
Deletion
Fragments
Lane
WT 1 2 3 4 5 6 7
-
403, 343, 155 343, 242, 155 343, 257, 155 403, 343 408, 403 403, 240, 155 343, 155, 96 403, 302
8 9 10 11 12 13 14 15
Deletion
Fragments 343, 332, 155 343, 175 403, 276, 155 408, 242 408, 96 240, 155, 96 403, 267 264, 155
Figure 1 A representative panel of splice variants reverse-transcriptase polymerase chain reaction products digested with EcoRI endonuclease. (a) Schematic view of IL23R mRNA. The predicted size of the polymerase chain reaction fragment generated by IL23R primer P5 P6 is 901 bp. Two endogenous EcoRI recognition sites within exon 7 and exon 8 digest the IL23R P5/P6 PCR fragment to three DNA pieces: exon 4–7 (403 bp), exon 7–8 (155 bp) and exon 8–11 (343 bp). (b) Fifteen different splice isoforms digested with EcoRI and resolved in 2% agarose gel; lane 1–7: the splicing variants involved with simple exon(s)-skipping; lane 8–10: the splicing variants involved with partial exon(s)-skipping and lane 11–15: the splicing variants involved with compound exon(s)-skipping events. The information of the exon(s) deleted in each variant and the predicted digested fragment sizes is summarized below.
other splice events, also emphasizing its probable importance in pre-mRNA processing of the IL23R transcript. The results from the intensive screening of the IL23R splice variants greatly expand the knowledge of varied IL23R gene expression in activated human leukocytes. The EcoRI endonuclease digestion results from a representative panel of 15 out of the 24 different splice isoforms demonstrated in Figure 1b, offers an effective and quick method to distinguish different splice variants. In the IL23R transcript variants we obtained from human activated leukocytes, most were discovered repeatedly in different conditions of mitogen stimulation; this implies that specific splice variants do not result from different stimuli. However, some of the variants were detected only once in the screening, implying that these splicing events are rare and the transcript is scarce within the cDNA pool. Unusual splice site usages in IL23R splice variants As mentioned previously, six partial exon deletion events, of which five were previously unknown were
identified in this study. Figure 2 depicts these six alternative splice events that involve partial exon deletions instead of the skipping of complete exons. The splice sites used are shown in Table 1. Three of these variants (pD5 5 nt, pD5 71 nt and pD11 67 nt) still keep their original splicing donor site (GT) in the boundary of the exon and intron but utilize the next possible legitimate splice acceptor sites (AG) within the exon in the immediate vicinity area, instead of the ‘native’ acceptor sites. These three partial deletion events all cause out-of-frame translation and, furthermore, introduce stop codons to the transcripts after the alternative splicing, thereby introducing an early termination of protein translation. The splicing events of the other three variants are very complicated. The partial deletion pD7,8 used a pair of legitimate splice sites (50 -GT/AG-30 ) within exon 7 and exon 8 to execute a proper splicing event causing a 164 bp deletion and an out-of-frame amino acid change. For the remaining two variants (isoforms v14 and v20), not only is none of the original splice donor/acceptor sites used, but those that are used are very atypical (Table 1). Although the predicted polypyrimidine tracts Genes and Immunity
Human IL-23 receptor splice variants S-h Kan et al
634 Isoforms
Description
WT
Schematic diagram of IL23R splice forms
Target of NMD Pathway
Genbank Entry Number
n/a
NM_144701
Isoform_v1
4
Y (155)
AM990313
Isoform_v2
5
Y (112)
AM990314
Isoform_v3
6
Y (152)
AM990315
Isoform_v4
7
N (35)
AM990316
Isoform_v5
8
n/a
AM990317
Isoform_v6
9
? (65)
AM990318
Isoform_v7
5,6
Y (72)
AM990319
Isoform_v8
6,7
N
AM990320
Isoform_v9
8,9
? (65)
AM990321
Isoform_v10
5,6,7
? (56)
AM990322
Isoform_v11
p 5 5 nt
Y (106)
AM990323
Isoform_v12
p 5 71 nt
N (5)
AM990324
Isoform_v13
p 11 67 nt
n/a
AM990325
Isoform_v14
p 5,6,7
N (15)
AM990326
Isoform_v15
5,8
Y (112)
AM990327
Isoform_v16
5,9
Y (112)
AM990328
Isoform_v17
5,6,8
Y (72)
AM990329
Isoform_v18
5,6,9
Y (72)
AM990330
Isoform_v19
p 7,8; p 11 67 nt
N (0)
AM990331
Isoform_v20
p 5,6,7; 9; p 11 67nt
n/a ? (65)
AM990332
Isoform_v21
p 5, 5nt; 6,7
Y (106)
AM990333
Isoform_v22
p 5,71nt; 8,9
N (5)
AM990334
Isoform_v23
8; p 11 67 nt
n/a n/a
AM990335
Isoform_v24
p 5, 5nt; 8
Y (106)
AM990336
Genes and Immunity
Human IL-23 receptor splice variants S-h Kan et al
were present at the AG splice acceptor sites, no legitimate splice site pairs could be identified in these two transcripts (involving partial deletion exons 5–7). Although these two atypical splicing events both occurred within exon 5 and exon 7, causing the entire exon 6 and part of exon 5 and exon 7 to be deleted, the splicing points used in these two transcripts are not the same. The intron area between exon 5/6 and exon 6/7 was searched intensively for any possible cryptic exon events that may have caused these two very atypical alternative splicings, but none were found. No similar sequences were observed within these two intron areas to offer a legitimate set of possible splice donor/acceptor site pairs to fit the (50 -GT/AG-30 ) rule, thus it is unlikely that cryptic exon(s) within the intron may be involved with these splice events (data not shown). Translation potential of the IL-23Ra splice variants The predicted protein sequences translated from these alternative spliced transcripts are summarized in Figure 3. As the introduction of early termination codon caused by an earlier exon(s) skipping in complex skipping events, several different IL23R isoforms may share the same predicted translated protein variants. A majority of these variants introduce early termination codons to the open reading frames immediately after the alternative splicing event(s), which may produce premature proteins with only short peptides (fewer than 250 amino acids) of IL-23Ra. Four possible IL-23Ra expression patterns predicted from the splice variants can be defined: (1) a short premature IL-23Ra extracellular peptide; (2) a soluble form of IL-23Ra without transmembrane or intracellular domains; (3) a structurally complete IL-23Ra with a truncated extracellular region and (4) a non-responsive receptor isoform of IL-23Ra without its intracellular signalling components. The different forms of the IL-23Ra variants, except the short premature IL-23Ra extracellular domain, may possess different biological functions if they can be translated. Five of these transcripts (D7; D9; D8,9; pD7,8 and pD11 67 nt; pD5,6,7 and D9 and pD11 67 nt) are potentially able to encode soluble forms of IL-23Ra without the transmembrane and intracellular domains, and with various truncations of the extracellular region. The change of extracellular region may alter the ligandreceptor binding specificity or affinity and the lack of transmembrane domain may cause the protein to be secreted from the cells instead of anchoring itself in the membrane. Two transcripts are in-frame deletions (D6 and 7; D8) that keep the transmembrane/intracellular domains
intact, but change the extracellular regions. These isoforms still retain the ability to initiate downstream signalling; however, the change of extracellular domain may again vary the ligand binding ability and specificity. Finally, two isoforms containing the partial deletion of exon 11 (pD11 67 nt; D8 and pD11 67 nt) may inhibit or prevent receptor signalling because of the lack of two out of the three tyrosine activation motifs.1
635
Discussion This is the most comprehensive splice variant study of an interleukin receptor to date. Here, we describe 24 different mRNA splice variants of human IL23R from the activated human leukocytes (including two previously known variant: D5 and D9).20 In addition, a recent publication describing a compound splice variant involving a novel insertion between exon 9 and exon 10 as well as a partial deletion in exon 5 (pD5 71 nt) is also included in this research.21 These 22 novel splice variants greatly expand our knowledge of the isoforms of IL23R at the level of mRNA. However, the rich numbers of splice variants of IL23R were not only observed in activated human blood cells. Similar results of the multiple IL23R splice variants were also observed in the human embryonic kidney cell line (HEK-293T/17, ATCC, Manassas, VA, USA) culture and also in the ‘MegaMan’ human transcriptome library (Stratagene, La Jolla, CA, USA; data not shown). Furthermore, no specific correlations were observed between the isoforms of splice variants and the particular mitogen used for stimulation, nor were we able to identify variants that were preferentially observed among individuals. These data suggest that the IL23R gene expression is very dynamic. Alternative splicing plays an important role in regulating gene expression by generating multiple mRNA transcripts from a single gene in specific spatial/temporal patterns that greatly expands the proteome information content and flexibility for gene expression.22,23 Nonetheless, the mechanisms and functional significance of the process are still unclear. The IL23R gene is a recently discovered interleukin receptor, which plays a pivotal role in human autoimmune responses through Th17 pathway. Translated variants of such an important mRNA species may have important modulatory functions in the development or maintenance of autoimmunity in humans. As shown here, the predicted protein products from these variant mRNAs can be categorized to four possible different groups: (1) short premature IL-23R extracellular peptides; (2) soluble forms of IL-23R without transmembrane/ intracellular domains;
Figure 2 Summary of 24 different IL23R splice variants identified in this study. Schematic diagrams of IL23R splice isoforms showing the splicing patterns of wild type and 24 different variants detected in activated human leukocytes for IL23R. The shaded boxes are the skipped exon(s). An asterisk above exon 9 is labeled to indicate the position of the Crohn’s diseases susceptible single nucleotide polymorphism: R318Q (rs11209026). The target of nonsense-mediated decay (NMD) is determined by a premature early termination codon located at least 50–55 bp upstream of the 30 -most exon–exon junction.24–26 Transcripts which contain the stop codon located more than 70 bp upstream of the next splice junction are labelled ‘Y’; those lower than 50 bp are labelled ‘N’. The transcripts where the stop codon is located within the ambiguous distance between 55–70 nucleotides from the next splice junction are labelled with question marks. In-frame deletions or splicing involving the last exon are not subject to the nonsense-mediated decay pathway. The D4 isoform was revealed from a P3/P6 amplicon and all others were revealed from P5/P6 amplicons. All the splice variants’ sequences were deposited in Genbank with assigned accession numbers through The European Molecular Biology Laboratory (EMBL) Nucleotide Sequence Database. Genes and Immunity
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Table 1 Splice code usages of the partial exon-skipping in IL23R mRNA Transcripts
Splice donor site
Splice acceptor sites
p∆5 5 nt p∆5 71nt p∆11 67nt p∆5,6,7 p∆5,6,7 p∆7,8
TGTGAAGA Ggtagg TGTGAAGA Ggtagg ATGCTACA Ggtaaccta CTGATTCA Ttacaaggtg g TGGCAAGA Agtacttggtttgggtc AAACATTA Agtacgtatttcaag
tttgttttaag tttag AGACAGAA acactttcattac aag GTGGCAAGAAGTAC gatcccatgatta cag AGATAAAAGAAA cactgtgtttttt cat AAAACACCTGAAAC tggagccaaacat taa GTACGTATTTCA gcttccatctcta cag GGCACCTTACTTC
Deletion caused by alternative splicing 5 bp 71 bp 67 bp 383 bp 288 bp 164 bp
The possible splice acceptor/donor sites are boxed. The mRNA are in uppercases and the intron are in lowercases. The deleted exons compared with the wild type transcripts are in bold. The possible polypyrimidine tracts are underlined.
(3) a full length of IL-23R with truncated extracellular region and (4) a non-responsive receptor isoform of IL-23R without intracellular signalling components. However, it is unlikely that all, or perhaps even any of these mRNA last to be translated because of a cellular process that degrades malformed mRNA, known as ‘nonsense-mediated decay (NMD)’. The NMD pathway is a post-transcriptional regulation, which is proficient in recognition of the ‘aberrant’ mRNA and rapid downregulation of the mRNA. The NMD pathway is triggered by a premature early termination codon located at least 50–55 bp upstream of the 30 -most exon–exon junction.24–26 More than half (14 out of 24) of the alternative splicing events described in this report introduce termination codons to the mRNA immediately after the exon(s) that is/are alternatively spliced (Figure 2). Among these fourteen transcripts, it is interesting to note that 12 involve the deletion of part of, or the entire, exon 5 and lead to the generation of a potential short peptide containing fewer than 250 amino acids (the full length IL-23Ra is 629 amino acids). However, these transcripts are not likely to be translated into protein because their mRNAs are possibly to be the target for the NMD pathway. The NMD phenomenon is apparently common in these IL23R mRNA, in that some are not translated to protein. Overall, it has been estimated that one-third of mRNA alternative transcripts containing premature termination codon are highly likely to be targets of nonsense-mediated mRNA decay.24 Interleukin-23 receptor a variants that may be candidates for NMD are annotated in Figure 2. Similarly, notwithstanding the likely importance of NMD to the splice variants, we have observed that several remain as potentially functionally interesting. Five splice variants are of interest because of their yielding potentially functional transcripts: D7; D9; D8,9; pD7,8 and pD11 67nt; pD5,6,7 and D9 and pD11 67 nt. The first three transcripts are very likely to be translated to various IL-23Ra protein ‘early termination’ variants, resulting in forms that lack the transmembrane and intracellular regions. However, they have most of, or the entire, extracellular region of the native protein. As the transmembrane domain does not exist in these transcripts, the proteins translated from these variants are no longer capable of being anchored to the membrane and thus are possibly secreted from the activated leukocytes. Genes and Immunity
In cytokine signalling, the soluble cytokine receptors can regulate immune events by acting as agonists or antagonists.27 These potential soluble receptors generated by exon variants therefore may block IL-23/ IL-23R signalling activity by acting as a ‘decoy receptor’, greatly expanding the complexity of immunological regulation. The D6,7 and D8 transcripts are in-frame deletion variants and if translated would have the transmembrane and intracellular signalling elements of the native IL-23R intact. However, parts of the extracellular domain (encoded by exons 6–7 or exon 8, respectively) are absent. These variants have the potential to display altered ligand binding properties in terms of affinity and/or specificity relative to the wild-type IL-23Ra and to signal, because of the intact intracellular domain. These changes within the extracellular domain may also introduce variety to the ligand–receptor kinetic interaction and further signal downstream genes in a different manner than the wild-type IL-23R in Th17 pathway development. Two of the alternatively spliced transcripts involved the skipping of only the first 67 nucleotides of exon 11 (D11 67 nt; D8 plus D11 67 nt). Therefore, if translated, these would encode a non-signalling, truncated protein, containing the complete IL-23Ra extracellular and transmembrane parts, but with a truncated cytoplasmic tail that lacks two out of the three signalling tyrosine residues and their box motifs. A recently reported IL23R spliced isoform, caused by a novel insertion between exon 9 and exon 10, also predicted a similar nonsignalling truncated protein isoform by removing the entire three tyrosine residues in the intracellular region.21 These transcripts are very unlikely to be targets of the NMD pathway and could therefore be translated and perhaps expressed. Similarly, the extracellular region will interact with the IL-23 ligand, but the normal downstream signalling activity will be halted because of the absence of the intracellular domain. Therefore, these two variant forms may encode non-responsive receptor variants of the IL-23R. It seems the expression of such truncated proteins may act as negative regulators of the IL-23/IL-23R pathway and Th17 development. However, the compound variant D8 and pD11 67 nt may combine these two properties by having both an altered extracelluar region and a non-functional intracellular region. How such a molecule would function rationally within the context of the IL-23 system is unclear.
Human IL-23 receptor splice variants S-h Kan et al
637 Predicted amino acids
Predicted Biological Function
WT
629
Wild type IL-23R
4
123
Short peptide, NMD candidate
5, 5,8, 5,9
174
Short peptide, NMD candidate
6
218
Short peptide, NMD candidate
7
283
Soluble form
8
599
Varied extracellular domain
9
356
Soluble form
5,6, 5,6,8, 5,6,9
192
Short peptide, NMD candidate
6,7
528
Varied extracellular domain
8,9
326
Soluble form
5,6,7
174
Short peptide, NMD candidate
178
Short peptide, NMD candidate
191
Short peptide, but no NMD
p 11 67 nt
414
Non-responsive receptor isoform
p 5,6,7
184
Short peptide, NMD candidate
293
Soluble form
260
Soluble form
384
Varied extracellular domain Non-responsive receptor isoform
Predicted IL-23R protein expression
p 5 5 nt, p 5, 5nt; 6,7; p 5, 5nt; 8 p 5 71 nt; p 5,71nt; 8,9
p 7,8; p 11 67 nt p 5,6,7; 9; p 11 67nt 8; p 11 67 nt
Figure 3 Predicted IL-23Ra protein expression pattern from the splice variants discovered in this study. Each box represents the protein encoded by each exon. The grey boxes indicated the untranslated region (UTR) and the 50 -UTR is located in exon 1 and part of exon 2; the 30 -UTR is in the second half of exon 11. The signal peptide is encoded in exon 2 and indicated in red. The extracellular domain is encoded from the exons spanning from exon 3 to exon 8. The single spanning transmembrane region is encoded by exon 9 (in yellow; see the online version for colour figures). The intracellular domain is spanning from exon 10 to exon 11. The ‘ATG’ indicates the initial translation codon and the ‘STOP’ indicates the stop codon encoded by in each of the IL23R splice variant. An asterisk above exon 9 is labelled to indicate the position of the Crohn’s diseases susceptible single nucleotide polymorphism: R318Q (rs11209026). Splice variants causing the same predicted protein structures were grouped. Eighteen different deduced amino acid sequences, from the splice-variants and wild-type IL-23Ra protein are summarized in this figure. The predicted biological function from the variant mRNAs are categorized to four possible groups which are: short premature IL-23Ra extracellular peptide, which are similar to the nonsense-mediated decay (NMD) pathway candidates, soluble forms of IL-23Ra without transmembrane/intracellular domain and truncated IL-23Ra extracellular region with intact transmembrane/intracellular domain and a non-responsive receptor isoform form of IL-23Ra with a truncated intracellular domain. Some of the proteins may be characterized that belongs to more than one of these categories.
This study has revealed a great diversity in the mRNA that is transcribed from the human IL23R gene. Twenty-four variants were observed in human lymphocytes of which 22 have not previously been observed. Variants were observed according to stimulation by various mitogens or simple exposure to complete cell culture medium over a 3-day period. Although many of the variants are likely to be destroyed within the cytoplasm, almost half (10/24) may be translated. These
novel translation products may represent regulatory moieties that participate in control of Th17 cells and their pro-inflammatory function, perhaps therefore representing an intrinsic protective mechanism against the development of autoimmune disease or other chronic inflammatory disorders. In conclusion, this study has revealed the molecular heterogeneity of the expression of IL23R in human leukocytes. Although more studies are needed to clarify Genes and Immunity
Human IL-23 receptor splice variants S-h Kan et al
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better the significance of the biological function of these IL-23Ra variants in activated human leukocytes, these findings have revealed new regulatory features in the expression and possibly function of IL-23Ra, which may provide new clues for clarifying the pathogenesis of some auto-inflammatory diseases.
Materials and methods Culture and stimulation of human PBMCs Peripheral blood mononuclear cells were isolated from heparinized whole venous blood of healthy donors by density gradient centrifugation using Ficoll-Paque (Sigma-Aldrich, St Louis, MO, USA) according to the manufacturer’s instructions. Blood was purchased as anonymous buffy coats from the New Jersey blood transfusion service with no donor identifying details. Isolated PBMC were maintained in RPMI-1640 medium (Invitrogen-Gibco, Carlsband, CA, USA) supplemented with 10% heat-inactivated foetal bovine serum (Invitrogen-Gibco) and 1 mM glutamine (Invitrogen-Gibco) in the presence of different mitogens (5 mg/ml concanavalin A, 0.2 mg/ml lipopolysaccharide, 5 mg/ml phytohaemagglutinin and 20 ng/ml phorbol myristate acetate plus 1 mM Ionomycin) for 72 h at 37 1C in a 5% CO2-humidified atmosphere. These activated PBMC were used for RNA isolation and RT-PCR. Nucleic acid isolation and RT-PCR for cDNA production Total RNA was isolated from activated PBMC with the ‘Absolutely RNA’ miniprep kit (Stratagene) following the manufacturer’s instructions. Purified RNA (300 ng) was reverse-transcribed with AffinityScript cDNA Synthesis Kit (Stratagene) primed with 300 ng of random hexamers in a volume of 20 ml of reverse transcription mixture. Polymerase chain reaction mixture (20 ml) included 1 U ‘Expand Long Template Enzyme’ mix (Roche Applied Science, Indianapolis, IN, USA), 1x ‘buffer 2’ and 0.25 mM of each specific primer pair, with the following thermal cycling parameters: 94 1C for 4 min, 1 cycle; 94 1C for 45 s, 56 1C for 45 s, 72 1C for 90 s, 40 cycles; 72 1C for 10 min, 1 cycle. The primers used to amplify IL23R gene fragments were from Zhang et al.:20 P3: forward primer 50 -GTAGAACCAGCCACAATTTT-30 ; P5: forward primer 50 -AATGCTGGGAAGCTCACCT ACATA-30 and P6: reverse primer 50 -GCTTGTGTTCTGGGATGAAGA TTTC-30 . The primer pairs P3/P6 and P5/P6 generated amplicons sized at 1234 bp and 901 bp, respectively, from the published sequence (NCBI reference sequence NM_144701). The primer pairs P3 and P6 spanned exons 3–11 and primer pairs P5 and P6 spanned exons 4–11 (Figure 1a), covering all the possible exon–exon junctions of IL23R mRNA encoding the mature region of IL-23Ra wild-type protein. Cloning and screening of splice variants The total PCR products from each of the mitogen stimulation, each from four donors were cloned to the TOPO TA vector (PCR2.1; Invitrogen) and transformed to TOP10 competent cells (Invitrogen). Over 1500 bacterial Genes and Immunity
colonies were selected and colony PCR was performed using the M13 forward primer and M13 reverse primer. The PCR products (5 ml) were electrophoresed on a 1.5% agarose gel to verify the product size. Identification and verification of IL23R splice variants by restriction endonuclease digestion and sequence analysis The remaining PCR products were digested overnight at 37 1C with 1 U of EcoRI (Fermentas, Glen Burnie, MD, USA) in a final volume of 20 ml and 10 ml of the digested products were electrophoresed for 45 min in 2% agarose gel at 150 V in Tris-borate/EDTA buffer. Sequence analysis was further performed on those clones showing different digestion patterns compared with those of the wild-type IL23R P5/P6-fragment digestion patterns (Figure 1b) with CEQ DTCS kit on the CEQ 8000 Genetic Analysis System (Beckman Coulter, Fullerton CA, USA). DNA sequences were analysed and aligned with MacVector 9.5.2 (Macvector, Cary, NC, USA).
Acknowledgements Mr S Srinivas, Drs J Dai and N Megjugorac for PBMC isolation. This study was supported intramurally by HUMIGEN LLC. All authors are employees of HUMIGEN.
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