Identification and Characterization of Multiple Splice Forms of the Human Interleukin-23 Receptor a Chain in Mitogen-Activated Leukocytes

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 receptor superfamily. It associates constitutively with Janus kinase 2 and also allows binding of Signal Interleukin-23 (IL-23) is a newly identified cytokine that Transducer and Activator of Transcription (STAT) 3 in a belongs to the IL-6 superfamily.1 It is structurally similar ligand-dependent manner, whose phosphorylation and to IL-12, sharing a common p40 subunit with an IL-23- consequent dimerization trigger downstream expression specific p19 subunit, instead of the p35 subunit found in of genes, such as IL17 and IL22, but not IFNg.1–4 The IL-12.1 Similar to the IL-12 receptor (IL-12R), the IL-23R IL-23R is mainly expressed on activated/memory T cells, is a heterodimeric construct that consists of the common T-cell clones and NK cells. Low levels of IL-23R expression IL-12Rb1 chain (as found in the IL-12R) and a unique have also been detected on monocytes, macrophages and IL-23Ra chain; IL-23Ra is analogous to the other IL-12R dendritic cells,1,2 which are consequently IL-23 responsive. chain, IL-12Rb2. Both the IL-23Ra chain and the IL-12Rb2 Although several of the structural and functional chain are the main signalling chains of their respective similarities between IL-23/IL-23R and IL-12/IL-12R sig- receptors.1 nalling suggest possible related regulatory functions in The human IL23R gene is located on chromosome T-cell development. Both cytokines actually have distinct 1p32.1–p31.2, 150 kb telomeric of the adjacent gene, roles in regulating the T cell-mediated immune responses IL12Rb2. The ‘native form’ of human IL23R mRNA is that bridge the innate and adaptive immunity.5 IL-12/IL- 2.8 kb long and comprises 11 exons (NM_144701). The 12R signalling plays a pivotal role in Th1 cell responses by transcribed mRNA is translated into a protein of 629 promoting IFN-g production during infection. However, amino acids, resulting in a type-I transmembrane protein IL-23/IL-23R signalling is the central molecule of Th17 cell which contains a signal peptide, an N-terminal fibronectin differentiation by upregulation of IL-17 production in III-like domain and a 252-residue cytoplasmic domain inflammatory (and autoimmune) responses. with three potential tyrosine phosphorylation sites.1 Interleukin-23 receptor and a series of downstream Interleukin-23 receptor is a member of hemopoietin 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 Correspondence: Dr G Gallagher, Genetic Immunology Laboratory, from mature dendritic cells, which acts together with Humigen LLC, The Institute for Genetic Immunology, 2439 Kuser activated TGF-b to induce expression of the retinoic Road, Hamilton, NJ 08690, USA. E-mail: [email protected] orphan receptor RORgt and upregulate IL-23R, thus Received 29 May 2008; revised 14 July 2008; accepted 15 July 2008; making them competent for IL-17 production through published online 28 August 2008 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 The detection and identification of IL23R splice variants immunity through Th17 cells may underscore why the In this study, both restriction enzyme digestion and IL23R gene also has been demonstrated to confer sequence analysis were applied to define the various susceptibility to several autoimmune diseases, including IL23R splice variants in the activated PBMCs. To psoriasis,8,9 ankylosing spondylitis,10 multiple sclerosis11–13 distinguish the differences in IL23R cDNA because of and inflammatory bowel diseases (both Crohn’s disease splicing, restriction endonuclease digestion was first and/or ulcerative colitis).14,15 In addition, a separate performed on the cloned PCR products and band sizes study by the Wellcome Trust Case Control Consortium were resolved by agarose gel electrophoresis. The digestion and the Australo-Anglo-American Spondylitis Consor- was designed to observe skipping of various complete tium found evidence to suggest that IL23R may be a IL23R exons as well as partial exon-skipping variants. Two common susceptibility factor for the major ‘seronegative’ endogenous EcoRIsiteswithinexon7andexon8ofthe diseases.16 native form of the IL23R mRNA gave three DNA A recent genome-wide association (GWA) study first fragments of clearly differing size, as depicted in Figure 1a. demonstrated that IL23R is one of the genetic factors The EcoRI restriction digestion of IL23R P5/P6 PCR contributing to Crohn’s disease. Among the single fragment clones was comprised of three DNA fragments: nucleotide polymorphisms within IL23R, one missense exon 4–7 (403 bp), exon 7–8 (155 bp) and exon 8–11 polymorphism (rs11209026, R381Q) is negatively asso- (343 bp), covering the area after the translated signal ciated with Crohn’s disease, which implies that the Q peptide, which is possibly the area containing most of allele may play a protective role in the pathogenesis of the alternative splicing events (Figure 1a). The diversity Crohn’s diseases.14 Subsequently, more studies have also of the EcoRI digestion pattern suggested that highly associated this R/Q single nucleotide polymorphism polymorphic and dynamic splicing activities were pre- with other diseases from different populations.9,10,15,17–19 sent in the mRNAs of the IL23R gene (Figure 1b). Clones Although investigating the IL23R for novel poly- that revealed different digestion patterns after EcoRI morphic elements, we discovered a range of novel splice digestion, described above, were then sequenced to variants of IL23R transcripts, greatly extending an earlier define the exact position and nature of these splice study by Zhang et al.20 from early 2006. We believe that events (Figure 1b). understanding the role of these variants and how their Sequencing of the variant IL23R fragment cDNA presence is controlled will give us novel insight to the clones identified 22 novel IL23R mRNA splice variants role of IL-23 in human Crohn’s disease. In addition, the as well as two (D5: AY937251 and D9: AY937253) that had IL-23/IL-17 pathway is known to be associated with previously been identified;20 these are depicted in other autoimmune diseases.8–13 The purpose of this Figure 2, which shows both the simple and compound study was therefore to understand possible patho- deletion events discovered in this study. All the genic mechanisms of IL-23R’s contribution to disease sequences identified in this study have been submitted susceptibility. to Genbank (summarized in Figure 2). The simple exon- skipping 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; Results 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 The IL-23Ra protein is mainly expressed on T cells, 67 nt; two different versions of pD5–7 and pD7–8. NK cells, monocytes and dendritic cells,1 thus the Isoforms v11–14 and v19–20). All of the exon-skipping expression IL23R in unstimulated peripheral blood events affected the exons encoding parts of the extra- mononuclear cells (PBMC) is ordinarily low. In this cellular region and/or transmembrane domain of study, no detectable IL23R expression was obtained from IL-23Ra, but not the intracellular domain. the freshly isolated PBMC by reverse-transcriptase In addition to the 14 simple exon-skipping events polymerase chain reaction (RT-PCR; data not shown). shown in Figure 2, 10 more splicing events were However, a significant upregulation of the IL23R cDNA identified that involved multiple exon-skipping events. was detected in PBMC after 72 h cultured with or In these compound exon-skipping transcripts, several without mitogen stimulation. Two forward primers, P3 splicing events were evident from the pre-mRNA or P5, combined with a reverse primer P6 were applied processing (Figure 2). These compound splicing events in this study to generate amplicons of IL23R whose include the skipping of combinations of complete, non- predicted sizes are 1234 bp and 901 bp, respectively. consecutive exons (D5,8; D5,9; D5,6,8; D5,6,9. Isoforms However, multiple smaller-sized transcripts compared v15–18) and combinations of the whole exon-skipping with the expected sizes of the IL23R amplicons were and partial exon deletion (pD5 5 nt plus D8; pD5 5 nt plus observed, suggesting the existence of multiple splice D6,7; pD5 71 nt plus D8,9; D8 plus pD11 67 nt; pD7,8 plus variant transcripts of the mRNA from these PBMC pD11 67 nt and pD5,6,7 plus D9 plus pD11 67 nt. Isoforms samples. The total RT-PCR products were cloned into v19–24). Among these 10 mRNA transcripts, the most TOPO TA vector PCR2.1 (Invitrogen, Carlsband, CA, complicated variant (v20) involved three individual USA) to identify the variants of each PCR product splicing events in one transcript. These multiple exon- individually. Colony PCR was first applied using M13 skipping events all involved the entire or partial deletion forward and M13 reverse primers located on either side of exon 5/6 and exon 8/9, which implies that the of the TA cloning sites on the vector to initially screen the skipping of these exons are common events in the pre- colonies generating amplicon size smaller than the wild- mRNA processing. In addition, the only skipping event type IL23R. Internal sequencing primers were designed involving an exon encoding the intracellular part of the as necessary. IL-23Ra protein, pD11 67 nt, repetitively occurred with

Genes and Immunity Human IL-23 receptor splice variants S-h Kan et al 633

1 243 5 679 8 10 1112131415 WT WT

403 403 343 343

155 155

Lane Deletion Fragments Lane Deletion Fragments WT - 403, 343, 155 8 343, 332, 155 1 343, 242, 155 9 343, 175 2 343, 257, 155 10 403, 276, 155 3 403, 343 11 408, 242 4 408, 403 12 408, 96 5 403, 240, 155 13 240, 155, 96 6 343, 155, 96 14 403, 267 7 403, 302 15 264, 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 identified in this study. Figure 2 depicts these six importance in pre-mRNA processing of the IL23R alternative splice events that involve partial exon deletions transcript. instead of the skipping of complete exons. The splice sites The results from the intensive screening of the IL23R used are shown in Table 1. Three of these variants (pD55nt, splice variants greatly expand the knowledge of varied pD571ntandpD11 67 nt) still keep their original splicing IL23R gene expression in activated human leukocytes. donor site (GT) in the boundary of the exon and intron but The EcoRI endonuclease digestion results from a utilize the next possible legitimate splice acceptor sites representative panel of 15 out of the 24 different splice (AG) within the exon in the immediate vicinity area, isoforms demonstrated in Figure 1b, offers an effective instead of the ‘native’ acceptor sites. These three partial and quick method to distinguish different splice variants. deletion events all cause out-of-frame translation and, In the IL23R transcript variants we obtained from furthermore, introduce stop codons to the transcripts after human activated leukocytes, most were discovered the alternative splicing, thereby introducing an early repeatedly in different conditions of mitogen stimula- termination of protein translation. tion; this implies that specific splice variants do not The splicing events of the other three variants are very result from different stimuli. However, some of the complicated. The partial deletion pD7,8 used a pair of variants were detected only once in the screening, legitimate splice sites (50-GT/AG-30) within exon 7 and implying that these splicing events are rare and the exon 8 to execute a proper splicing event causing a transcript is scarce within the cDNA pool. 164 bp deletion and an out-of-frame amino acid change. For the remaining two variants (isoforms v14 and v20), Unusual splice site usages in IL23R splice variants not only is none of the original splice donor/acceptor As mentioned previously, six partial exon deletion sites used, but those that are used are very atypical events, of which five were previously unknown were (Table 1). Although the predicted polypyrimidine tracts

Genes and Immunity Human IL-23 receptor splice variants S-h Kan et al 634 Target of Genbank Isoforms Description Schematic diagram of IL23R splice forms NMD Entry Number Pathway

WT 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

p 7,8; p 11 Isoform_v19 N (0) AM990331 67 nt p 5,6,7; 9; n/a Isoform_v20 AM990332 p 11 67nt ? (65)

Isoform_v21 p 5, 5nt; 6,7 Y (106) AM990333

Isoform_v22 p 5,71nt; 8,9 N (5) AM990334

n/a Isoform_v23 8; p 11 67 nt AM990335 n/a

Isoform_v24 p 5, 5nt; 8 Y (106) AM990336

Genes and Immunity Human IL-23 receptor splice variants S-h Kan et al 635 were present at the AG splice acceptor sites, no intact, but change the extracellular regions. These legitimate splice site pairs could be identified in these isoforms still retain the ability to initiate downstream two transcripts (involving partial deletion exons 5–7). signalling; however, the change of extracellular domain Although these two atypical splicing events both may again vary the ligand binding ability and specificity. occurred within exon 5 and exon 7, causing the entire Finally, two isoforms containing the partial deletion of exon 6 and part of exon 5 and exon 7 to be deleted, the exon 11 (pD11 67 nt; D8 and pD11 67 nt) may inhibit or splicing points used in these two transcripts are not the prevent receptor signalling because of the lack of two out same. The intron area between exon 5/6 and exon 6/7 of the three tyrosine activation motifs.1 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 Discussion sequences were observed within these two intron areas This is the most comprehensive splice variant study of an to offer a legitimate set of possible splice donor/acceptor 0 0 interleukin receptor to date. Here, we describe 24 site pairs to fit the (5 -GT/AG-3 ) rule, thus it is unlikely different mRNA splice variants of human IL23R from that cryptic exon(s) within the intron may be involved the activated human leukocytes (including two pre- with these splice events (data not shown). viously known variant: D5 and D9).20 In addition, a recent publication describing a compound splice variant Translation potential of the IL-23Ra splice variants involving a novel insertion between exon 9 and exon 10 The predicted protein sequences translated from these as well as a partial deletion in exon 5 (pD5 71 nt) is also alternative spliced transcripts are summarized in included in this research.21 These 22 novel splice variants Figure 3. As the introduction of early termination codon greatly expand our knowledge of the isoforms of IL23R caused by an earlier exon(s) skipping in complex at the level of mRNA. However, the rich numbers of skipping events, several different IL23R isoforms may splice variants of IL23R were not only observed in share the same predicted translated protein variants. A activated human blood cells. Similar results of the majority of these variants introduce early termination multiple IL23R splice variants were also observed in codons to the open reading frames immediately after the the human embryonic kidney cell line (HEK-293T/17, alternative splicing event(s), which may produce pre- ATCC, Manassas, VA, USA) culture and also in the mature proteins with only short peptides (fewer than 250 ‘MegaMan’ human transcriptome library (Stratagene, amino acids) of IL-23Ra. Four possible IL-23Ra expres- La Jolla, CA, USA; data not shown). Furthermore, no sion patterns predicted from the splice variants can be specific correlations were observed between the isoforms defined: of splice variants and the particular mitogen used for stimulation, nor were we able to identify variants that (1) a short premature IL-23Ra extracellular peptide; were preferentially observed among individuals. These (2) a soluble form of IL-23Ra without transmembrane or data suggest that the IL23R gene expression is very intracellular domains; dynamic. (3) a structurally complete IL-23Ra with a truncated Alternative splicing plays an important role in regulating extracellular region and gene expression by generating multiple mRNA transcripts (4) a non-responsive receptor isoform of IL-23Ra with- from a single gene in specific spatial/temporal patterns out its intracellular signalling components. that greatly expands the proteome information content and The different forms of the IL-23Ra variants, except the flexibility for gene expression.22,23 Nonetheless, the me- short premature IL-23Ra extracellular domain, may chanisms and functional significance of the process are still possess different biological functions if they can be unclear. The IL23R gene is a recently discovered interleukin translated. Five of these transcripts (D7; D9; D8,9; pD7,8 receptor, which plays a pivotal role in human autoimmune and pD11 67 nt; pD5,6,7 and D9 and pD11 67 nt) are responses through Th17 pathway. Translated variants of potentially able to encode soluble forms of IL-23Ra such an important mRNA species may have important without the transmembrane and intracellular domains, modulatory functions in the development or mainte- and with various truncations of the extracellular region. nance of autoimmunity in humans. The change of extracellular region may alter the ligand- As shown here, the predicted protein products from receptor binding specificity or affinity and the lack of these variant mRNAs can be categorized to four possible transmembrane domain may cause the protein to be different groups: secreted from the cells instead of anchoring itself in the membrane. (1) short premature IL-23R extracellular peptides; Two transcripts are in-frame deletions (D6 and 7; D8) (2) soluble forms of IL-23R without transmembrane/ that keep the transmembrane/intracellular domains 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 Human IL-23 receptor splice variants S-h Kan et al 636 Table 1 Splice code usages of the partial exon-skipping in IL23R mRNA

Transcripts Splice donor site Splice acceptor sites Deletion caused by alternative splicing

p∆5 5 nt TGTGAAGA Ggtagg tttgttttaag tttag AGACAGAA 5 bp p∆5 71nt TGTGAAGA Ggtagg acactttcattac aag GTGGCAAGAAGTAC 71 bp p∆11 67nt ATGCTACA Ggtaaccta gatcccatgatta cag AGATAAAAGAAA 67 bp p∆5,6,7 CTGATTCA Ttacaaggtgg cactgtgtttttt cat AAAACACCTGAAAC 383 bp p∆5,6,7 TGGCAAGA Agtacttggtttgggtc tggagccaaacat taa GTACGTATTTCA 288 bp p∆7,8 AAACATTA Agtacgtatttcaag gcttccatctcta cag GGCACCTTACTTC 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 In cytokine signalling, the soluble cytokine receptors can region and regulate immune events by acting as agonists or (4) a non-responsive receptor isoform of IL-23R without antagonists.27 These potential soluble receptors intracellular signalling components. generated by exon variants therefore may block IL-23/ IL-23R signalling activity by acting as a ‘decoy receptor’, However, it is unlikely that all, or perhaps even any of greatly expanding the complexity of immunological these mRNA last to be translated because of a cellular regulation. process that degrades malformed mRNA, known as The D6,7 and D8 transcripts are in-frame deletion ‘nonsense-mediated decay (NMD)’. The NMD pathway variants and if translated would have the transmem- is a post-transcriptional regulation, which is proficient in brane and intracellular signalling elements of the native recognition of the ‘aberrant’ mRNA and rapid down- IL-23R intact. However, parts of the extracellular domain regulation of the mRNA. The NMD pathway is triggered (encoded by exons 6–7 or exon 8, respectively) are by a premature early termination codon located at least absent. These variants have the potential to display 50–55 bp upstream of the 30-most exon–exon junction.24–26 altered ligand binding properties in terms of affinity More than half (14 out of 24) of the alternative splicing and/or specificity relative to the wild-type IL-23Ra and events described in this report introduce termination to signal, because of the intact intracellular domain. codons to the mRNA immediately after the exon(s) that These changes within the extracellular domain may also is/are alternatively spliced (Figure 2). Among these introduce variety to the ligand–receptor kinetic interac- fourteen transcripts, it is interesting to note that 12 tion and further signal downstream genes in a different involve the deletion of part of, or the entire, exon 5 and manner than the wild-type IL-23R in Th17 pathway lead to the generation of a potential short peptide development. containing fewer than 250 amino acids (the full length Two of the alternatively spliced transcripts involved IL-23Ra is 629 amino acids). However, these transcripts the skipping of only the first 67 nucleotides of exon 11 are not likely to be translated into protein because their (D11 67 nt; D8 plus D11 67 nt). Therefore, if translated, mRNAs are possibly to be the target for the NMD these would encode a non-signalling, truncated protein, pathway. The NMD phenomenon is apparently common containing the complete IL-23Ra extracellular and in these IL23R mRNA, in that some are not translated to transmembrane parts, but with a truncated cytoplasmic protein. Overall, it has been estimated that one-third of tail that lacks two out of the three signalling tyrosine mRNA alternative transcripts containing premature residues and their box motifs. A recently reported IL23R termination codon are highly likely to be targets of spliced isoform, caused by a novel insertion between nonsense-mediated mRNA decay.24 Interleukin-23 recep- exon 9 and exon 10, also predicted a similar non- tor a variants that may be candidates for NMD are signalling truncated protein isoform by removing the annotated in Figure 2. entire three tyrosine residues in the intracellular region.21 Similarly, notwithstanding the likely importance of These transcripts are very unlikely to be targets of the NMD to the splice variants, we have observed that NMD pathway and could therefore be translated and several remain as potentially functionally interesting. perhaps expressed. Similarly, the extracellular region will Five splice variants are of interest because of their interact with the IL-23 ligand, but the normal down- yielding potentially functional transcripts: D7; D9; D8,9; stream signalling activity will be halted because of the pD7,8 and pD11 67nt; pD5,6,7 and D9 and pD11 67 nt. The absence of the intracellular domain. Therefore, these two first three transcripts are very likely to be translated to variant forms may encode non-responsive receptor various IL-23Ra protein ‘early termination’ variants, variants of the IL-23R. It seems the expression of such resulting in forms that lack the transmembrane and truncated proteins may act as negative regulators of the intracellular regions. However, they have most of, or the IL-23/IL-23R pathway and Th17 development. However, entire, extracellular region of the native protein. As the the compound variant D8 and pD11 67 nt may combine transmembrane domain does not exist in these tran- these two properties by having both an altered extra- scripts, the proteins translated from these variants are no celluar region and a non-functional intracellular region. longer capable of being anchored to the membrane and How such a molecule would function rationally within thus are possibly secreted from the activated leukocytes. the context of the IL-23 system is unclear.

Genes and Immunity Human IL-23 receptor splice variants S-h Kan et al 637 Predicted Predicted Biological Predicted IL-23R protein expression amino acids Function

WT 629 Wild type IL-23R

Short peptide, NMD 4 123 candidate Short peptide, NMD 5, 5,8, 5,9 174 candidate Short peptide, NMD 6 218 candidate

7 283 Soluble form

8 599 Varied extracellular domain

9 356 Soluble form

5,6, 5,6,8, Short peptide, NMD 192 5,6,9 candidate

6,7 528 Varied extracellular domain

8,9 326 Soluble form

Short peptide, NMD 5,6,7 174 candidate p 5 5 nt, p 5, Short peptide, NMD 5nt; 6,7; 178 candidate p 5, 5nt; 8 p 5 71 nt; 191 Short peptide, but no NMD p 5,71nt; 8,9 Non-responsive receptor p 11 67 nt 414 isoform Short peptide, NMD p 5,6,7 184 candidate p 7,8; p 11 67 293 Soluble form nt p 5,6,7; 9; 260 Soluble form p 11 67nt Varied extracellular domain 8; p 11 67 nt 384 Non-responsive receptor isoform 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 novel translation products may represent regulatory mRNA that is transcribed from the human IL23R moieties that participate in control of Th17 cells and gene. Twenty-four variants were observed in human their pro-inflammatory function, perhaps therefore lymphocytes of which 22 have not previously been representing an intrinsic protective mechanism against observed. Variants were observed according to stimula- the development of autoimmune disease or other chronic tion by various mitogens or simple exposure to complete inflammatory disorders. cell culture medium over a 3-day period. Although many In conclusion, this study has revealed the molecular of the variants are likely to be destroyed within the heterogeneity of the expression of IL23R in human cytoplasm, almost half (10/24) may be translated. These leukocytes. Although more studies are needed to clarify

Genes and Immunity Human IL-23 receptor splice variants S-h Kan et al 638 better the significance of the biological function of these colonies were selected and colony PCR was performed IL-23Ra variants in activated human leukocytes, these using the M13 forward primer and M13 reverse primer. findings have revealed new regulatory features in the The PCR products (5 ml) were electrophoresed on a 1.5% expression and possibly function of IL-23Ra, which may agarose gel to verify the product size. provide new clues for clarifying the pathogenesis of some auto-inflammatory diseases. Identification and verification of IL23R splice variants by restriction endonuclease digestion and sequence analysis The remaining PCR products were digested overnight at Materials and methods 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 Culture and stimulation of human PBMCs products were electrophoresed for 45 min in 2% agarose Peripheral blood mononuclear cells were isolated from gel at 150 V in Tris-borate/EDTA buffer. Sequence heparinized whole venous blood of healthy donors analysis was further performed on those clones showing by density gradient centrifugation using Ficoll-Paque different digestion patterns compared with those of the (Sigma-Aldrich, St Louis, MO, USA) according to the wild-type IL23R P5/P6-fragment digestion patterns manufacturer’s instructions. Blood was purchased as (Figure 1b) with CEQ DTCS kit on the CEQ 8000 Genetic anonymous buffy coats from the New Jersey blood Analysis System (Beckman Coulter, Fullerton CA, USA). transfusion service with no donor identifying details. DNA sequences were analysed and aligned with Isolated PBMC were maintained in RPMI-1640 medium MacVector 9.5.2 (Macvector, Cary, NC, USA). (Invitrogen-Gibco, Carlsband, CA, USA) supplemented with 10% heat-inactivated foetal bovine serum (Invitro- gen-Gibco) and 1 mM glutamine (Invitrogen-Gibco) in the Acknowledgements presence of different mitogens (5 mg/ml concanavalin A, 0.2 mg/ml lipopolysaccharide, 5 mg/ml phytohaemagglu- Mr S Srinivas, Drs J Dai and N Megjugorac for PBMC tinin and 20 ng/ml phorbol myristate acetate plus 1 mM isolation. This study was supported intramurally by 1 Ionomycin) for 72 h at 37 C in a 5% CO2-humidified HUMIGEN LLC. All authors are employees of HUMIGEN. atmosphere. These activated PBMC were used for RNA isolation and RT-PCR. References Nucleic acid isolation and RT-PCR for cDNA production Total RNA was isolated from activated PBMC with the 1 Parham C, Chirica M, Timans J, Vaisberg E, Travis M, Cheung ‘Absolutely RNA’ miniprep kit (Stratagene) following the J et al. A receptor for the heterodimeric cytokine IL-23 is manufacturer’s instructions. Purified RNA (300 ng) was composed of IL-12Rbeta1 and a novel cytokine receptor reverse-transcribed with AffinityScript cDNA Synthesis subunit, IL-23R. J Immunol 2002; 168: 5699–5708. 2 Langrish CL, McKenzie BS, Wilson NJ, de Waal Malefyt R, Kit (Stratagene) primed with 300 ng of random hexamers Kastelein RA, Cua DJ. IL-12 and IL-23: master regulators in a volume of 20 ml of reverse transcription mixture. of innate and adaptive immunity. Immunol Rev 2004; 202: Polymerase chain reaction mixture (20 ml) included 1 U 96–105. ‘Expand Long Template Enzyme’ mix (Roche Applied 3 Zheng Y, Danilenko DM, Valdez P, Kasman I, Science, Indianapolis, IN, USA), 1x ‘buffer 2’ and 0.25 mM Eastham-Anderson J, Wu J et al. Interleukin-22, a T(H)17 of each specific primer pair, with the following thermal cytokine, mediates IL-23-induced dermal inflammation and cycling parameters: 94 1C for 4 min, 1 cycle; 94 1C for 45 s, acanthosis. Nature 2007; 445: 648–651. 56 1C for 45 s, 72 1C for 90 s, 40 cycles; 72 1C for 10 min, 1 4 Lee E, Trepicchio WL, Oestreicher JL, Pittman D, Wang F, cycle. The primers used to amplify IL23R gene fragments Chamian F et al. Increased expression of interleukin 23 p19 20 and p40 in lesional skin of patients with psoriasis vulgaris. were from Zhang et al.: J Exp Med 2004; 199: 125–130. 0 0 5 van de Vosse E, Lichtenauer-Kaligis EG, van Dissel JT, P3: forward primer 5 -GTAGAACCAGCCACAATTTT-3 ; Ottenhoff TH. Genetic variations in the interleukin-12/ 0 P5: forward primer 5 -AATGCTGGGAAGCTCACCT interleukin-23 receptor (beta1) chain, and implications for ACATA-30 and IL-12 and IL-23 receptor structure and function. Immuno- P6: reverse primer 50-GCTTGTGTTCTGGGATGAAGA genetics 2003; 54: 817–829. TTTC-30. 6 Weaver CT, Hatton RD, Mangan PR, Harrington LE. IL-17 family cytokines and the expanding diversity of effector T cell The primer pairs P3/P6 and P5/P6 generated amplicons lineages. Annu Rev Immunol 2007; 25: 821–852. sized at 1234 bp and 901 bp, respectively, from the 7 Yang XO, Panopoulos AD, Nurieva R, Chang SH, Wang D, Watowich SS et al. STAT3 regulates cytokine-mediated published sequence (NCBI reference sequence generation of inflammatory helper T cells. J Biol Chem 2007; NM_144701). The primer pairs P3 and P6 spanned exons 282: 9358–9363. 3–11 and primer pairs P5 and P6 spanned exons 4–11 8 Capon F, Di Meglio P, Szaub J, Prescott NJ, Dunster C, (Figure 1a), covering all the possible exon–exon junctions Baumber L et al. Sequence variants in the genes for the of IL23R mRNA encoding the mature region of IL-23Ra interleukin-23 receptor (IL-23R) and its ligand (IL-12B) wild-type protein. confer protection against psoriasis. Hum Genet 2007; 122: 201–206. 9 Nair RP, Ruether A, Stuart PE, Jenisch S, Tejasvi T, Cloning and screening of splice variants Hiremagalore R et al. Polymorphisms of the IL12B and IL23R The total PCR products from each of the mitogen genes are associated with psoriasis. J Invest Dermatol 2008; 128: stimulation, each from four donors were cloned to the 1653–1661. TOPO TA vector (PCR2.1; Invitrogen) and transformed to 10 Rueda B, Orozco G, Raya E, Fernandez-Sueiro JL, Mulero J, TOP10 competent cells (Invitrogen). Over 1500 bacterial Blanco FJ et al. The IL23R Arg381Gln non-synonymous

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