Skip Navigation


Human Molecular Genetics Advance Access originally published online on September 12, 2007
Human Molecular Genetics 2007 16(24):3027-3036; doi:10.1093/hmg/ddm261
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
16/24/3027    most recent
ddm261v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (2)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Greetham, D.
Right arrow Articles by Wilson, A. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Greetham, D.
Right arrow Articles by Wilson, A. G.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Functional characterization of NF-{kappa}B inhibitor-like protein 1 (NF{kappa}BIL1), a candidate susceptibility gene for rheumatoid arthritis

Darren Greetham1, Charles D. Ellis1, Devesh Mewar1, Ursula Fearon2,3, Sinead Nic an Ultaigh2,3, Douglas J. Veale2,3, François Guesdon1,{dagger} and Anthony G. Wilson1,*,{dagger}

1 School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield S10 2RX, UK, 2 St. Vincent’s University Hospital, Dublin, Ireland and 3 The Conway Institute of Molecular Medicine, Dublin, Ireland

* To whom correspondence should be addressed. Tel: +44 1142712566; Fax: +44 1142711711; Email a.g.wilson{at}shef.ac.uk

Received June 13, 2007; Revised September 3, 2007; Accepted September 9, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Several studies have implicated the NF-{kappa}B inhibitor-like protein 1 (NFkBIL1) gene located in the class III region of the major histocompatibility complex (MHC) as a possible susceptibility locus for rheumatoid arthritis (RA). Based on limited homology, it has been suggested to be a member of the inhibitor of NF-{kappa}B (I{kappa}B) family of proteins, but a role in mRNA processing has also been proposed. We have investigated the expression of NFkBIL1 in RA synovial tissue and characterized its function. Real-time PCR showed the two NFkBIL1 mRNA splice variants are expressed in a tissue-specific manner. Dual immunofluorescent staining of human RA synovium with polyclonal anti-NFkBIL1 antibodies and anti-CD68, anti-CD3 or anti-factor VIII showed that NFkBIL1 was expressed in the rheumatoid synovial lining and sub-lining layers and co-localized in CD68+ and CD3+, but not Factor VIII+ cells. Confocal microscopy of cultured synovial fibroblasts revealed expression in speckled nuclear and homogenous cytoplasmic distributions, suggesting shuttling between the cytoplasmic and nuclear compartments. Functional tests showed that NFkBIL1 isoforms were incapable of associating with NF-{kappa}B and did not inhibit it, thus disproving the hypothesis that NFkBIL1 functions as an I{kappa}B. Affinity purification of endogenous NFkBIL1 proteins and co-immunoprecipitation experiments showed that NFkBIL1 can associate with mRNA and with three protein partners, identified by mass spectrometry as leukophysin, translation elongation factor 1 {alpha} and CTP synthase I. These data support a potential role for NFkBL1 in the pathogenesis of RA and indicates that it may be involved in mRNA processing or the regulation of translation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
The genetic contribution to rheumatoid arthritis (RA) has been estimated at 30–50% of total risk (1,2). Approximately one-third of this is believed to arise from the major histocompatibility complex (MHC) at 6p21.3, a gene-dense region that encodes a large number of proteins involved in the innate and adaptive immune systems (3,4). The DRB1 gene within this region has been reproducibly implicated (5) in both RA susceptibility and severity (6,7).

Recent studies have however revealed that the MHC contribution to RA is polygenic with additional susceptibility arising telomeric of DRB1. A large US family-based study reported two non-DRB1 risk loci, one in a 497 kb interval in the central MHC and a second in a class I region in a subset of DRB1*0404 haplotypes (8). We also reported DRB1-independent association with several single-nucleotide polymorphisms (SNPs) in the telomeric class III region in British RA families (9) and another British study reported differences of tumour necrosis factor (TNF) haplotypes between DRB1*04-matched RA patients and healthy controls (10). A large Japanese case–control study typed 88 SNPs and the DRB1 locus and detected an independent association of markers located around the class III and I junction (11).

The telomeric class III region bordering the class I region is particularly gene-dense containing at least 10 genes in addition to TNF within an 82 kb interval; BAT1, ATP6V1G2, NFkBIL1, LTA, TNF, LTB, LST1, NCR3, AIF-1, BAT3 and BAT2 (12). The function of most of these molecules remains poorly characterized, although evidence for a role in immune and inflammatory responses does exist for several (13,14). Despite the undoubted importance of TNF in RA pathogenesis there is little convincing evidence linking genetic variation at this locus to RA susceptibility (15,16).

The NFkBIL1 gene is located 30 kb telomeric of TNF and owes its name to limited homology with members of the inhibitor of nuclear factor {kappa}B (I{kappa}B) family of proteins (17). These proteins are critical components of the NF-{kappa}B signalling pathway, which regulate the transcription of many important mediators of inflammation and tissue destruction in the rheumatoid joint including TNF, IL-1 and IL-6 (18). In unstimulated cells, NF-{kappa}B exists as homo- or heterodimers complexed with I{kappa}B proteins. Cellular activation leads to the rapid phosphorylation of N-terminal serine residues of I{kappa}B-{alpha} by the I{kappa}B kinases resulting in the ubiquitination of I{kappa}B-{alpha} and subsequent degradation by the proteasome. Heterodimers of NF-{kappa}B subsequently translocate to the nucleus and activate target genes. Although on the basis of the presence of ankyrin repeat sequences it was suggested that NFkBIL1 may be a novel member of the I{kappa}B family, there is no functional data to support this, and indeed it is now appreciated that ankyrin repeats represent a versatile linear scaffold and do not imply any specific function (19).

A number of studies have examined genetic variation in NFkBIL1 and RA susceptibility. One case–control study involving 116 Japanese RA patients and 97 controls reported association with a promoter SNP at position -62 (rs2071592) in a consensus motif for the transcriptional repressor {delta}EF1, however, this study did not report on linkage disequilibrium (LD) with DRB1 alleles (20). Another much larger Japanese study reported DRB1-independent association of another NFkBIL1 promoter SNP at -293 (rs3219185) (21). Interestingly, no association of rs2071592 was detected in either our RA family-based study (9) or a Spanish case–control study (22). Hence, genetic susceptibility linked to NFkBIL1 may vary in different ethnic groups.

In this study, we examined the both the function of NFkBIL1 and expression in RA synovium and synovial fibroblasts. We demonstrate that NFkBIL1 is expressed in T cells and macrophages in the rheumatoid synovium and is mainly distributed in a nuclear speckled pattern. We also show that it does not bind NF-{kappa}B proteins or down-regulate inflammatory signalling but binds mRNA, suggesting a role in mRNA processing.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Detection of endogenous NFKBIL1 mRNA variants in cell lines, differentiated tissues and biopsies from inflamed tissues
Two splice variants of the NFkBIL1 mRNA called NFkBIL1-{alpha} and NFkBIL1-ß have been identified previously (23). They encode proteins of 381 and 366 aminoacids, respectively, which differ by the presence in variant {alpha} of a 15-aminoacid sequence (GELEDEWQEVMGRFE) that is absent from NFkBIL1-ß. We measured the levels of expression of endogenous mRNAs for NFkBIL1-{alpha} and NFkBIL1-ß in commercially available cDNA preparations from several differentiated tissues by real-time PCR. We also measured the expression of NFkBIL1 mRNA variants in skin biopsies from two donors and in peripheral blood mononuclear cells (PBMCs) prepared from three healthy donors and three RA patients. The results showed that at least one NFkBIL1 mRNA variant was expressed in all tissue tested (Fig. 1). However, there were significant differences in the expression levels of individual isoforms. Only the ovary, prostate and PBMCs were found to express both NFkBIL1 splice variants at similar levels. All the other tissues tested expressed preferentially or exclusively a specific variant. NFkBIL1-{alpha} was the only variant expressed in brain and endothelial tissue, whereas NFkBIL1-ß was the preferred variant in muscle and the only detected variant detected in small intestine, placenta and skin samples. Finally, comparison of the expression levels of the NFkBIL1 variants in PBMCs from healthy donors and RA patients showed no significant difference between the two groups (Fig. 1).


Figure 1
View larger version (13K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 1. Quantitative determination of the expression of NFkBIL1-{alpha} and NFkBIL1-ß mRNA variants in differentiated tissues. The mRNA variants encoding the {alpha} and ß isoforms of NFkBIL1 were detected by real-time PCR. Individual results were expressed relative to the values measured for GAPDH in the same samples. Averages and standard deviations (SDs) of three to six determinations are shown.

 
Localization of NFKBIL1protein in rheumatoid synovium
We generated three polyclonal antibodies to synthetic NFkBIL1 peptides. The first antibody, Ab1, was designed to detect both NFkBIL1 variants, whereas the other two were raised against peptide sequences specific to each individual variant. Immunoblotting tests carried out with lysates of transfected cells expressing each individual variant showed that all three antibodies specifically detected their intended antigens (data not shown). The antibodies were then used to stain synovial membrane biopsy samples from five RA patients and three knee joint osteoarthritis (OA) synovial samples. Intense staining was found in both the synovial lining layer and the sub-lining synovium, in biopsy samples from all five patients (Fig. 2B and C). Antibodies to both NFkBIL1 variants gave positive results. However, those directed against NFkBIL1-{alpha}, Ab2, consistently stained more intensely and were used in all immunohistochemistry or immunofluorescence experiments shown. Staining was much less intense in the OA samples (Fig. 2E and F).


Figure 2
View larger version (80K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 2. Expression of NFkBIL1 protein in synovial tissue. Representative tissue sections from rheumatoid arthritis (A–C) and osteoarthritis (D–F) patients were stained with affinity-purified non-immune rabbit IgG (A and D, control, original magnification x20) or anti-I{kappa}BL-{alpha} (Ab2) antibodies in (B) and (E) (original magnification x10), (C) and (F) (original magnification x20).

 
To determine the cellular phenotype of cells expressing NFkBIL1, dual immunofluorescence staining was performed with the Ab2 antibody, anti-CD68 (macrophage), anti-CD3 (T cell) and anti-factor VIII (endothelial cell). Staining for NFkBIL1 co-localized with CD68+ cells in both the synovial lining and sub-lining layers (Fig. 3A–C). Expression was also observed on a proportion of T cells as shown in a perivascular CD3+ lymphoid aggregate (Fig. 3D–F), but minimal or no expression was detected in factor VIII-expressing synovial endothelial cells (Fig. 3G and H).


Figure 3
View larger version (100K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 3. Dual immunofluorescence analysis of NFkBIL1 expression in rheumatoid synovial tissue. Immunofluorescence staining of NFkBIL1 is shown in red (A, D and G) and immunofluorescence staining of CD68 (B), CD3 (E) or Factor VIII (H), in green (original magnification x20). Merging of red and green images (C and F) shows co-localization of I{kappa}BL with CD68-expressing cells (C) and sub-synovial CD3+ lymphoid aggregate (F). (I) shows IgG-matched control.

 
Intracellular localization of NFKBIL1 in synoviocytes
The intracellular distribution of NFkBIL1 was examined in rheumatoid fibroblast-like synoviocytes (FLS) between passages 5 and 8. Immunofluorescence confocal microscopy revealed discrete patterns of expression being either cytoplasmic (Fig. 4A and B), nuclear (Fig. 4C) or both (Fig. 4D).


Figure 4
View larger version (68K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 4. Intracellular distribution of endogenous NFkBIL1 in synoviocytes. Fibroblast-like synoviocytes from RA patients with were fixed and stained with Ab2 anti-NFkBIL1 antibody and fluorescent secondary antibody (Cy3). Four representative images acquired by confocal immunofluorescence microscopy are shown (A–D). The protein is found to adopt a uniform cytoplasmic distribution in some cells (A and B, long arrows), but also a speckled nuclear distribution (C, block arrowheads) or both (D) in others.

 
NFKBIL1L lacks I{kappa}B-like functions
Because NFkBIL1 owes its name to its structural similarity to I{kappa}B, we started our investigation of NFkBIL1 function by assessing whether or not it was involved in the regulation of NF-{kappa}B. We first investigated whether or not the NFkBIL1 proteins were capable of associating physically with the p50 and p65 subunits of NF-{kappa}B. Co-immunoprecipitations experiments showed that in marked contrast to I{kappa}B{alpha}, which was used as positive control, neither NFkBIL1-{alpha} nor NFkBIL1-ß were detected in p50 or p65 immunoprecipitates (Fig. 5A). We also determined if NFkBIL1 proteins were capable of regulating the expression of an NF-{kappa}B-driven promoter-reporter construct, pIL8-Luc. The results showed that unlike I{kappa}B{alpha}, whose expression in transfected cells strongly inhibited IL-1-induced activation of the reporter construct, the NFkBIL1 proteins had no significant effect (Fig. 5B). Similar results were obtained when the pIL8-Luc construct was activated by co-transfection of the signal transduction pathway components, IRAK-1 (data not shown) and TRAF6 (Fig. 5B). Altogether, these results show that NFkBIL1 is not involved in the regulation of NF-{kappa}B.


Figure 5
View larger version (20K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 5. NFkBIL1 proteins do not have I{kappa}B-like functions. (A) Lack of association of NF{kappa}B subunits with recombinant NFkBIL1. HEK 293 cells were co-transfected with constructs encoding NFkBIL1-{alpha}, NFkBIL1-ß or GST-I{kappa}B{alpha} (positive control) and either the p50 or p65/RelA subunits of NF-{kappa}B as indicated. Cell lysates were immunoprecipitated with pooled antibodies to p50 and p65. Immunoprecipitates (top) and samples of the lysates (bottom) were analysed by western blotting with the Ab1 anti-I{kappa}BL antibodies (right-hand and centre) or anti-GST antibodies (left-hand). The arrowheads indicate the positions of GST-I{kappa}B proteins. (B) Lack of Inhibition of IL-1- and TRAF6-induced IL-8-Luc promoter/reporter expression by NFkBIL1 proteins. COS-7 cells were co-transfected with the pIL8-Luc and pTK-Luc reporters and 50 ng of constructs encoding either GUS (negative control), I{kappa}B{alpha} (positive control), NFkBIL1-{alpha} or NFkBIL1-ß. The cells were stimulated either by co-transfection of 10 ng of TRAF6 expression plasmid (black bars) or by addition of IL-1-ß (3 ng/ml) to the medium 18 h after transfection (grey bars). The cells were lysed 24 h after transfection. Means and SDs of three independent normalized luciferase determinations are shown.

 
Association of NFKBIL1 with CTP-synthase I, elongation factor 1 {alpha} and leukophysin
To identify NFkBIL1 possible partners, the endogenous NFkBIL1 was immunoprecipitated from 35S labelled human embryonic kidney (HEK) 293 cells in non-denaturing conditions and the immunoprecipitated material was analysed by SDS–PAGE. The immunoprecipitates were found to contain a prominent 45-kD band, presumed to be NFkBIL1, and three weaker bands of ~60, 28 and 18 kD, designated, respectively, P1, P2 and P3. These bands were detected in immunoprecipitates obtained by all three anti-NFkBIL1 antibodies used either alone or in combination (Fig. 6A). When immunoprecipitations were carried out on a larger scale from unlabelled cells, the same co-precipitating bands were also detected by Coomassie blue staining (data not shown). The proteins present in these bands were identified by in-gel trypsin digestion followed by liquid chromatography and electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The results (Table 1) showed that the 60-kD P1 band contained CTP synthase 1 (CTPS) and two other proteins whose identifications were not secure. The 28-kD band, P2, corresponded to leukophysin (LKP). The 18-kDa band contained two components that were identified by mass spectrometry as translation elongation factor 1-{alpha} 1 (EF1{alpha}) and, less securely identified, the hsp90 co-chaperone, cdc37. Finally, mass spectrometry of the main 45-kD band confirmed its identity as NFkBIL1 and detected no other protein in that band (Table 1). The predicted molecular masses of CTPS and LKP, 66 and 25 kD respectively, are in agreement with the masses estimated from the migration positions of the P1 and P2 bands. However, both EF1{alpha} and cdc37 have predicted molecular masses that are much in excess of 18 kD (Table 1), which indicates that the 18-kD P3 band contained proteolytic fragments of the two proteins rather than their native functional forms.


Figure 6
View larger version (63K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 6. Identification of NFkBIL1 partners. (A) Immunoprecipitation of endogenous NFkBIL1 and associated proteins from 35S-labelled HEK293 cells. Immunoprecipitations were carried out using antibodies to both NFkBIL1 variants (Ab1), NFkBIL1-{alpha} alone (Ab2), NFkBIL1-ß alone (Ab3) or all three antibodies combined (lane 4). The immunoprecipitates were analysed by SDS–PAGE and the radiolabelled material was detected with a phosphorimager. The migration positions of NFkBIL1 and of the three bands containing candidate partners (P1–P3) are shown. (B) Co-immunoprecipitation of recombinant NFkBIL1 with CTP-synthase I, elongation factor-1{alpha} (EF1{alpha}) T7 and leukophysin (LKP). HeLa cells were co-transfected with expression constructs encoding NFkBIL1-{alpha} and T7-tagged versions of either CTP synthase I (CTPS), EF1{alpha}, cdc37 or LKP. The T7-tagged proteins were immunoprecipitated with anti-T7 antibody and the immunoprecipitates were subjected to western blotting with NFkBIL1-Ab1 (top) and with anti-T7 (loading control, middle). The bottom shows a western blot of samples of the whole cell lysates obtained with NFkBIL1-Ab1 (NFkBIL1 expression control). (C) Association of NFkBIL1 with mRNA. HeLa cell lysates were subjected to immunoprecipitations with antibodies specific for the poly-A-binding protein, NFkBIL1-{alpha}, NFkBIL1-ß, p50 and TAK1 or to a sham immunoprecipitation with protein G-sepharose beads only. The immunoprecipitates were then treated with proteinase K, after which material released in the supernatants were subjected to affinity purification on oligo-dT beads and analysed on a 1% agarose gel stained with ethidium bromide. A preparation of total cellular was used as molecular mass standards. The migration positions of 28S and 18S rRNA in this preparation are shown. (D) Degradation by RNAse of NFKBIL1-bound nucleic acids. The nucleic acids associated with PAPB, NFKBIL1-{alpha} and NFKBIL1-ß immunoprecipitates were prepared as in (C) and divided in two aliquots that were either incubated with RNAse A (+) or subjected to sham incubation without the enzyme (–). The samples were then analysed by electrophoresis on 8% polyacrylamide gels containing 8.3 M urea and staining with ethidium bromide. (E) Insensitivity of NFKBIL1-bound nucleic acids to DNAse I treatment. The nucleic acids associated with immunoprecipitates of PAPB or NFKBIL1 (both isoforms) were prepared as in (C) and divided in three aliquots that were incubated with RNAse-free DNAse I for the indicated times. Electrophoresis was carried out as in (D).

 


View this table:
[in this window]
[in a new window]

  		Table 1. Identification of proteins co-precipitating with NFkBIL1

 
To verify whether or not CTPS, EF1{alpha}, LKP and cdc37 were indeed capable of direct association with NFkBIL1, we expressed them as T7-tagged recombinant proteins and carried out co-immunoprecipitation experiments. The results showed that NFkBIL1-{alpha} can bind directly to EF1{alpha}, LKP and CTPS, but not to cdc37 (Fig. 6B). The same results were observed when NFkBIL1-ß was used instead of NFkBIL1-{alpha} (data not shown). This evidence thus confirms the ability of both NFkBIL1 variants to associate with CTPS, EF1{alpha} and LKP.

Association of NFKBIL1with mRNA
Because the co-localization of NFkBIL1 with the splicing factor, Sm, suggested a role in processing or nuclear export of mRNA (23), we also tested whether or not endogenous NFkBIL1 is associated with mRNA in HeLa cells. After immunoprecipitation of NFkBIL1, the immunoprecipitates were treated with proteinase K and the released soluble material was then subjected to an oligo-dT affinity-based mRNA purification procedure as described in the ‘Materials and Methods’ section. The results showed that both NFkBIL1-{alpha} and NFkBIL1-ß immunoprecipitates contained nucleic acids whose amount and size distribution was similar to that obtained in control precipitates obtained with an antibody to the poly-A-binding protein (PABP) (Fig. 6C), which is a well characterized mRNA-binding protein (24). In contrast, no such material was detected in control immunoprecipitations carried out with antibodies to the p50 NF-{kappa}B subunit or the TGF-ß-activated kinase 1-a (TAK1-a), used as negative controls, or in sham immunoprecipitations in which antibodies were omitted (Fig. 6C). The nucleic acids associated with the PABP and NFkBIL1 immunoprecipitates were totally degraded when incubated with RNAse (Fig. 6D) but resistant to the action of DNAse (Fig. 6E), thus confirming that they consisted of RNA. The size distribution of the mRNA found in the NFkBIL1 immunoprecipitates was similar to that of the mRNA associated with PABP (Fig. 6C) and to mRNA prepared directly from HeLa cells by standard methods (data not shown). These results suggest that like PABP, which binds mRNA with apparently no preference for specific sequence motifs or secondary structures (24), NFkBIL1 isoforms are associated with bulk cellular mRNAs.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Phylogenetic analysis indicates that the RA candidate gene NFkBIL1 has been strongly conserved in mammalian evolution, the rat and opossum orthologues displaying 92 and 61% identity to the human amino-acid sequence. However, only distantly related homologues were found in lower vertebrates (data not shown). This strong sequence conservation in mammals, together with the localization of the NFkBIL1 gene within the MHC and its proposed association with RA, suggest that NFkBIL1 may have a conserved function in mammalian immunity. However, the biological role of NFkBIL1 is at present poorly known.

NFkBIL1 is expressed as two isoforms denoted by {alpha} and ß, with the ß isoform being 15 amino acids shorter than the {alpha} isoform. Real-time PCR data from human tissues shows that some tissues or cell types, such as PBMCs, express both splice variants in similar amounts, while other tissues preferentially express NFkBIL1-{alpha} (e.g. brain and endothelial tissues) or NFkBIL1-ß (muscle, small-intestine, placenta and skin). These results indicate that expression of the two isoforms is regulated in a tissue-specific manner.

This study has shown that NFkBIL1 proteins are expressed in the rheumatoid, but not OA synovium suggestive of a role in the pathogenesis of RA. This is the first demonstration of NFkBIL1 protein expression in human cells or tissues. Protein expression was seen in both the lining and sub-lining layers, but remarkably was not uniform. The antigen was detected in CD68 positive macrophages, a proportion of T cells and cultured FLS from five patients but not at all in the endothelium. We also found that NFkBIL1 adopted predominantly a nuclear speckled distribution in cultured FLS, but was also observed in the cytoplasm of some cells. This result is in agreement with a previous report which showed that in transfected cells, fluorescent recombinant NFkBIL1 adopted the same localizations as reported here for the endogenous protein (23).

Although the partners of most MHC class III gene products have been identified by means of the yeast-two hybrid system, this approach was unsuccessful for NFkBIL1 (25). Based on limited sequence homology, it has been proposed that NFkBIL1 is a member of the I{kappa}B family (17). However our results, including the intracellular distribution of NFkBIL1 proteins, their inability to associate with two NF-{kappa}B subunits and their lack of activity in I{kappa}B functional tests clearly show that NFkBIL1 does not function as a inhibitor of NF-{kappa}B.

Our search for proteins associated with endogenous NFkBIL1 in HeLa cells revealed a diverse group of possible partners. Three of the candidates partners identified by mass spectrometry were then confirmed to be capable of directly binding to I{kappa}BL when expressed in transfected cells. They correspond to CTPS, EF1{alpha} and LKP. Although the functional significance of the interaction with CTPS, which catalyses the ATP-dependent amination of UTP to CTP, is unclear, the other two partners provide interesting insights into the molecular function of NFkBIL1. EF1{alpha}, which is also known as leukocyte receptor cluster member 7, is involved in the translation of mRNAs and is believed to be an autoantigen for Felty’s syndrome, which is characterized by systemic illness in a small subset of RA patients (26). LKP is a 235-residue protein of unknown function that owes its name to its initial detection in leukocyte secretory granules, but appears to be expressed in a variety of cell types. Our evidence clearly shows that it is produced in HeLa cells.

The sequence of LKP is identical to the C-terminal end of RNA helicase A (RHA), also known as DEAD box helicase 9 (DHX9). Indeed, LKP and RHA are produced by alternative mRNAs of the DHX9 gene. The association of NFkBIL1 with LKP thus suggests that it probably also associates with RHA. The physiological role of the helicase is much better known than that of LKP. It has an active role in transcription and has also been shown to stimulate the translation of specific messages by associating with regulatory secondary structures of their 5'-UTRs (27,28). Autoantibodies to helicaseA/DHX9 are considered an early marker for systemic lupus erythematosus (SLE) (28).

An earlier study has shown that recombinant NFkBIL1 proteins co-localized with the spliceosome component, Sm in transfected cells (23). Like RHA, Sm is an autoantigen associated with SLE (28). While this manuscript was in preparation, 13 other partners of NFkBIL1 were reported in the IntAct protein–protein interactions database (available on-line at http://www.ebi.ac.uk/intact/site/index.jsf). Similar to Sm, EF1{alpha} and RHA, many of these possible of partners of NFkBIL1 have functions associated with RNA processing or translation (29). Spliceosomal factors can redistribute into the cytoplasm during the cell cycle, and indeed can shuttle between nuclear speckles and the cytoplasm (30). Altogether, these findings and our observations that endogenous NFkBIL1 associates with mRNA in HeLa cells and can adopt either a nuclear speckled or a cytoplasmic distribution in synoviocytes thus strongly suggest that it is involved in mRNA processing.

In summary, we report the expression of NFkBIL1 in rheumatoid synovial tissue including T cells, macrophages and fibroblasts, but not endothelial cells supporting a potential role for this molecule in the pathogenesis of RA. While there is abundant evidence showing the presence a genetic susceptibility marker for RA telomeric of DRB1, its identification is complicated by the high gene density and large number of plausible candidate genes. The finding that NFkBIL1 is expressed in activated cells in the rheumatoid synovium lends support to a role in the pathogenesis of RA. Our evidence also indicates that NFkBIL1 proteins associate with mRNA and with proteins that are involved in transcription, processing and translation of mRNA and have been associated with SLE (RHA) or Felty’s syndrome (EFT1). These findings provide directions for future further biological and genetic studies of the mechanistic link between these proteins and chronic inflammation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
FUNDING
 REFERENCES
 
Reagents and established cell lines
Restriction enzymes and T4 ligase were from Promega (Madison, WI, USA). RNase A and RNase-free DNase I were from Qiagen (Crawley, UK). DNA amplifications were carried out with Pfu polymerase (Stratagene, La Jolla, CA, USA) unless otherwise stated. Preparations of cDNAs from differentiated cells and tissues were purchased from OriGene Technologies, Inc. (Rockville, MD, USA), except the cDNAs from skin biopsies and PBMCs, which were prepared as described previously (31). The IMAGE cDNA clones were obtained from the Rosalind Franklin Centre for Genomics Research (Hinxton, Cambridge, UK). The pCMV-IRAK and pI{kappa}B{alpha}EGFP expression constructs and the pIL8-LUc promoter/reporter construct have all been described before (32–34). Recombinant human IL-1ß was from R&D systems (Oxford, UK). Anti-p65 and anti-p50 antibodies were purchased from EMD Biosciences (Merck, Darmstadt, Germany), except for the RNA co-precipitation experiments, for which antibodies from Calbiochem (Merck) were used. The monoclonal anti-body to PABP and the anti-GST and anti-HA antibodies were from Sigma (St. Louis, MO, USA). The anti-EGFP and anti-T7 antibodies were from Roche (Mannheim, Germany) and Novagen (Merck), respectively.

COS-7 and HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% de-complemented foetal calf serum (FCS), penicillin G (100 ng/ml) and streptomycin (100 ng/ml) (all from Gibco, Paisley, UK). HEK 293-T cells were maintained in the same conditions except that high-glucose DMEM (Gibco, cat No 21969-035) was used.

NFKBIL1 antibodies
Three rabbit polyclonal antibodies to NFkBIL1 called Ab1, Ab2 and Ab3 were generated using KLH-conjugated synthetic peptides of sequences RWHPDRFLQRC, EDEWQEVMGR and RQKLQGDASH, respectively (Harlan Sera-Lab, Loughborough, UK). The peptide used to produce Ab1 is present in both NFkBIL1 variants, but the Ab2 and Ab3 antigens correspond to sequence motifs specific of NFkBIL1-{alpha} and NFkBIL1-ß, respectively. The specificity of each antiserum was confirmed by western blotting lysates of HEK 293 cells transiently transfected with plasmids encoding each splice variant of NFkBIL1 (data not shown). The antibodies were then purified by affinity chromatography using the immunizing peptide immobilized to a 4% beaded agarose support (AminoLink Plus Immobilization Kit, Pierce, Rockford, IL).

Synovial tissue samples
Samples for immunohistochemistry and confocal microscopy were obtained from knee joints of RA and OA patients recruited at St. Vincent’s University Hospital (Dublin, Ireland), and ethical permission was obtained from the Medical Research Ethics Committee in accordance with the Declaration of Helsinki principles. Briefly, arthroscopy and synovial biopsy was performed, under local anaesthesia and sterile conditions, using a Storz 2.7-mm needle arthroscope (Storz, Tuttlingen, Germany) and a 2-mm grasping forceps as previously described (35). Biopsy samples were obtained from all compartments of the knee joint, embedded in TissueTek OCT compound (Sakura Finetek, Zoeterwoude, The Netherlands), snap frozen and stored in liquid nitrogen until used.

Isolation of RA FLS
Fresh synovial biopsies were placed directly into RPMI (Invitrogen Ltd, Paisley, UK) and digested using 1 mg/ml collagenase type 1 (Worthington Biochemical, Freehold, NJ, USA) in RPMI for 4 h at 37°C in humidified air with 5% CO2. Dissociated cells were grown to confluence (~10 days) in RPMI, 10% FCS (Invitrogen Ltd), penicillin (100 units/ml), streptomycin (100 units/ml) and fungizone (0.25 µg/ml) before trypsinization and passage. Cells were used for experiments between passages 5–8. In the stimulation experiments, 200 000 FLS were plated per well and left for 48 h to recover from trypsinization. Cells were serum deprived overnight prior to stimulation with 0.1 ng/ml TNF (R&D Systems) for the times indicated. Stimulation was terminated by removal of the media and lysis of cells in Trizol (Invitrogen Ltd) prior to RNA extraction.

Quantitative determination of NFKBIL1 splice variants expression by real time-PCR
Intron-spanning primers were used in order to generate different sized PCR products from each transcript which were resolved by PAGE. All probes and primers were synthesized by MWG-Biotech (Edersberg, Germany). Oligonucleotide probes specific for NFkBIL1-{alpha} or NFkBIL-ß were designed using the manufacturer’s software. Variant-specific probes were labelled with FAM (5-Carboxyfluorescein) with TAMRA as a quencher; the nucleotide sequences specific for NFkBIL-{alpha} and NFkBIL-ß were 5'-AGTCATGGGGAGGTTTGAAG-3' and 5'-ATGGAGACAGAAGCTC CAGGGTGAT-3', respectively. RT–PCR data were normalized against GADPH as previously described (31).

Immunohistology
Cryostat sections (7 µM) were mounted on 3-aminopropyltriethoxysilane-coated glass slides, air dried overnight, wrapped in foil and stored at –80°C until immunohistochemical analysis was performed. Sections were fixed in acetone for 10 min. Prior to staining, endogenous peroxidase was quenched by treatment with 3% H2O2 for 5 min, followed by pre-treatment with 3% normal serum for 20 min. A routine three-stage immunoperoxidase labelling technique incorporating avidin-biotin-immunoperoxidase complex (Vector Laboratories, Burlingame, CA, USA) was used. Sections were incubated for 1 h with primary antibodies against rabbit polyclonal anti-NFKBIL1 or with an irrelevant isotype-matched monoclonal antibody as a negative control. Colour was developed in solution containing diaminobenzadine-tetrahydrochloride (Sigma), 0.5% H2O2 in PBS buffer (pH 7.6). Slides were counterstained with haemotoxylin and mounted.

Dual immunofluorescence staining
Synovial tissue sections were fixed in 4% paraformaldehyde and were incubated with mouse monoclonal antibodies against CD3, CD68 or Factor VIII alone or co-incubated with rabbit polyclonal anti-NFKBIL1 for 1 h. Sections were also incubated for 1 h with an irrelevant isotype-matched control. Following incubation, cells were washed three times in PBS and incubated with a goat anti-mouse secondary Cy2 antibody (1:200) (Jackson Laboratories, Cambridgeshire, UK) and a goat anti-rabbit Cy3 (1:500) (Jackson Laboratories). The sections were then washed and mounted with an anti-quenching agent, Citifluor (Citifluor Ltd, Leicester, UK). For immunofluorescence of cultured synoviocytes, the cells were plated on 8-well cell culture-treated glass chamber slides at a density of 5 000 per well and allowed to adhere overnight. After washing twice with PBS, the cells were fixed in 4% paraformaldehyde for 20 min at room temperature. They were then washed with PBS an incubated with primary antibody against rabbit polyclonal NFkBIL1 (1:50) or an irrelevant isotype-matched control. Following incubation, the cells were washed three times in PBS and incubated with a goat anti-rabbit Cy3 (1:500) (Jackson Laboratories). They were then washed and mounted with Citifluor.

NFKBIL1 cDNA expression constructs
The NFkBIL1, I{kappa}B{alpha} and ß-glucoronidase (Gus) expression constructs were generated by in vitro recombination using the Gateway system (Invitrogen Ltd). The I{kappa}B{alpha}, NFkBIL1-{alpha} and NFkBIL1-ß entry clones were generated by amplifying the open reading frames from IMAGE clones 3161686, 544778 and 6158780, respectively (GenBank accession numbers BC002601 [GenBank] , BM044744 and BQ425655). The forward and reverse primers used were 5'-CACCATGTTCCAGGCGGC-3' and 5'-CAGCGTCTGACGTTATGAGTGCA-3' for I{kappa}B{alpha}, 5'-CACCATGAGTAACCCCTCCCCCCAG-3' and 5'-CTAGGGTCACTTGAGGGCCTCTG-3' for NFkBIL1. The PCR products were ligated into the Gateway entry vector pENTR-D/TOPO (Invitrogen Ltd). The inserts of the entry constructs were fully sequenced to ensure that they did not encode mutant products. The Gus entry clone, pDNOR201-gus, was obtained commercially (Invitrogen Ltd). Mammalian expression constructs encoding GST-tagged and untagged products were then generated by in vitro recombination between the pENTR constructs and the pDEST27 or pcDNA3.2-DEST destination vectors, respectively.

Expression constructs encoding putative NFKBIL1 partners
The pCMV-relA and pCMV-p50 constructs, encoding untagged relA and p50 proteins, were generated by excising the CFP open-reading frame form pECFPrelA and pECFPp50 (33) with Bsr G1 and NheI. The recessed ends were then filled with Klenow polymerase and the plasmids were ligated. The T7-tagged constructs encoding other putative NFkBIL1 partners (CTPS, EF1{alpha}, LKP and cdc37) were prepared by amplifying their cDNAs with primers containing either EcoRI (forward) or SalI (reverse) sites and cloned into the pCMV-T7 vector. The primers used were 5'-CGCGAATTCATGAAGTACCCATCTCCC-3' and 5'-CGCGTCGACTTAATAGCCGCCACCTCCT-3' for LKP, 5'-CGCGAATTCATGGGAAAGGAAAAGACT-3' and 5'-CGCGTCGACTCATTTAGCCTTCTGAGC-3' for EF1{alpha}, 5'-CGCGAATTCATGGTGGACTACAGCGTG-3' and 5'-CGCGTCGACTCACACACTGACATCCTT-3' for CDC37 and 5'-CGCGAATTCATGAAGTAC ATTCTGTTAC-3' and 5'-CGCGTCGACAGTCAGTACTAAATAACTACCT-3' for CTPS. The inserts of the entry constructs were fully sequenced to ensure that they did not encode mutant products.

Immunoprecipitations and western blotting
For detection of associations between tagged constructs, HeLa cells were seeded at 1.6 x 106 cells per 10 cm dish and transfected the following day. At 24 h-post-transfection, cells were lysed in 400 µl lysis buffer (50 mM HEPES, pH7.9, 0.25% Nonidet P-40, 150 mM NaCl, 5 mM DTT, 1 mM EDTA, 20 mM ß-glycerophosphate, 10 mM sodium fluoride, 0.5 mM sodium orthovanadate) supplemented with protease inhibitors (Sigma). The cell lysates were then centrifuged (14 000g, 2 min) to remove cellular debris and pre-cleared by addition of Sepharose 4B beads (Sigma) to remove proteins binding non-specifically to the beads.

Immunoprecipitations were carried out by adding antibodies to the lysates at concentrations recommended by the manufacturers and the reactions were incubated for 2 h at 4°C. Protein A or G sepharose was then added and incubations were carried on for a further hour. The beads were then washed three times in lysis buffer. For western blotting, the samples (cell lysates, GST pull-downs or immunoprecipitates) were heated at 100°C for 5 min in 40 µl SDS–PAGE sample buffer, then separated on 10% SDS–PAGE gels and transferred to polyvinylidene difluoride membranes. The membranes were probed with appropriate antibodies, which were then visualized by incubation with horse-radish peroxidase-conjugated antibodies against rabbit or mouse immunoglobulin G (Cell Signalling Technology Inc., Danver, MA, USA) followed by chemoluminescent detection using the ECL detection reagents (GE Healthcare, Little Chalfont, UK).

Promoter-reporter assays
Cells were seeded into 96-well tissue culture plates at 1.0 x 104/well 24 h before transfection, which was performed using PolyFect reagent (Qiagen). Each well was transfected with 500 ng of pIL8-Luc reporter construct, 100 ng of pTK-rLuc control construct (Promega) and the cDNA expression constructs under test. Stimulations by IL1-ß (3 ng/ml) were done 18 h after transfection. Cells were lysed 24 h after transfection and luciferase reporters were assayed with the Dual luciferase kit (Promega). Assay results were normalized individually to Renilla luciferase activity measured from the same samples (32).

Immunoprecipitation of radiolabelled 35sNFKBIL1 and detection of putative binding partners
HEK 293 cells were seeded at 5 x 105 cells per well in 6-well plates. After 24 h, the cells were washed in methionine-free DMEM, then incubated for 18 h in DMEM containing 60 µCi 35S sulphonated amino acids (72% methionine and 27% cysteine) in methionine- and cysteine-free DMEM supplemented with 10% dialysed FCS. At the end of the labelling period, the cells were washed with PBS, then lysed as described before and the cleared lysates were incubated with anti-NFkBIL1 antibodies for 2 h at 4°C. Secondary antibodies were then added at concentrations recommended by the manufacturers and the reactions were incubated for a further 2 h at 4°C. Immunoprecipitates were collected with Protein G sepharose and washed three times in lysis buffer. The immunoprecipitates were analysed by SDS–PAGE gels followed by autoradiography.

Identification of proteins co-purifying with NFKBIL1
To identify the putative NFkBIL1 partners, NFkBIL1 complexes were immunoprecipitated from unlabelled cells grown in 175-cm2 flasks using anti-NFkBIL1 antibodies that had been covalently conjugated to sepharose beads. The purified proteins were then separated on SDS–PAGE gels and detected by Coomassie blue staining. The stained bands were excised from the gels and subjected to in-gel trypsin digestion. Tryptic peptides were recovered and subjected to LC-ESI-MS/MS as described by Sostaric et al. (36). Three MS runs were performed for each sample. The MS spectra were searched against the Mass Spectrometry Data Base in a sequence query search using MASCOT 2.0 software (www.matrixscience.com) (36). The taxonomy was limited to filter for only human matches. Trypsin was used as the cleavage enzyme, with one missed cleavage site allowed. The peptide tolerance was set to 0.5 Da and the MS/MS tolerance was set to 0.3 Da. Carbamidomethyl modification of cysteine and oxidized methionine were set as variable modifications.

Isolation of RNA associated with NF{kappa}BIL1
Endogenous NFkBIL1 and PABP were immunoprecipitated from HeLa cells seeded at 1.6 x 106 cells/10-cm dish and grown at 37°C for 16 h. Immunoprecipitations were carried out as described for transfected cells. The immunoprecipitates were then treated with proteinase K (30 min, 37°C). The soluble phase was then recovered and subjected to precipitation using phenol:chloroform:isoamyl alcohol (25:24:1) and centrifugation at 17 000g for 30 min. After centrifugation, the RNA pellet was washed in 70% ice-cold ethanol and resuspended in TE buffer (10 mM Tris pH 7.2 and 1 mM EDTA) and subjected to ethanol precipitation using 0.1 volume 3 M sodium acetate pH 5.2 and 2 volumes of ice-cold 100% ethanol. After a second centrifugation, the pellet was dissolved in TE buffer and the recovered mRNA was further purified by affinity to oligo-dT beads (GenElute kit, Sigma).


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
REFERENCES
 
Arthritis Research Campaign (14366); Sheffield Hospital Charitable Trust (7815).


   ACKNOWLEDGEMENTS
 
We would like to thank Dr Endre Kiss-Toth (University of Sheffield) for the pIL-8-Luc construct and Professor E. Qwarnström (University of Sheffield) for the pECFPrelA and pECFPp50 constructs.

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} These authors contributed equally to this work. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 

  1. Ollier W., Worthington J. Small fish in a big pond. Br. J. Rheumatol. (1997) 36:931–932.[Free Full Text]

  2. Vyse T.J., Todd J.A. Genetic analysis of autoimmune disease. Cell (1996) 85:311–318.[CrossRef][Web of Science][Medline]

  3. Deighton C.M., Walker D.J., Griffiths I.D., Roberts D.F. The contribution of HLA to rheumatoid arthritis. Clin. Genet. (1989) 36:178–182.[Web of Science][Medline]

  4. Consortium M.S. Complete sequence and gene map of a human major histocompatibility complex. Nature (1999) 401:921–923.[CrossRef][Medline]

  5. Gregersen P.K., Silver J., Winchester R.J. The shared epitope hypothesis. An approach to understanding the molecular genetics of susceptibility to rheumatoid arthritis. Arthritis Rheum. (1987) 30:1205–1213.[Web of Science][Medline]

  6. Weyand C.M., Xie C., Goronzy J.J. Homozygosity for the HLA-DRB1 allele selects for extraarticular manifestations in rheumatoid arthritis. J. Clin. Invest. (1992) 89:2033–2039.[Web of Science][Medline]

  7. de Vries N., van Elderen C., Tijssen H., van Riel P.L., van de Putte L.B. No support for HLA-DQ encoded susceptibility in rheumatoid arthritis. Arthritis Rheum. (1999) 42:1621–1627.[CrossRef][Web of Science][Medline]

  8. Jawaheer D., Li W., Graham R.R., Chen W., Damle A., Xiao X., Monteiro J., Khalili H., Lee A., Lundsten R., et al. Dissecting the genetic complexity of the association between human leukocyte antigens and rheumatoid arthritis. Am. J. Hum. Genet. (2002) 71:585–594.[CrossRef][Web of Science][Medline]

  9. Kilding R., Iles M.M., Timms J.M., Worthington J., Wilson A.G. Additional genetic susceptibility for rheumatoid arthritis telomeric of the DRB1 locus. Arthritis Rheum. (2004) 50:763–769.[CrossRef][Web of Science][Medline]

  10. Newton J., Brown M.A., Milicic A., Ackerman H., Darke C., Wilson J.N., Wordsworth B.P., Kwiatkowski D. The effect of HLA-DR on susceptibility to rheumatoid arthritis is influenced by the associated lymphotoxin {alpha}-tumor necrosis factor haplotype. Arthritis Rheum. (2003) 48:90–96.[CrossRef][Web of Science][Medline]

  11. Kochi Y., Yamada R., Kobayashi K., Takahashi A., Suzuki A., Sekine A., Mabuchi A., Akiyama F., Tsunoda T., Nakamura Y., et al. Analysis of single-nucleotide polymorphisms in Japanese rheumatoid arthritis patients shows additional susceptibility markers besides the classic shared epitope susceptibility sequences. Arthritis Rheum. (2004) 50:63–71.[CrossRef][Web of Science][Medline]

  12. Neville M.J., Campbell R.D. A new member of the Ig superfamily and a V-ATPase G subunit are among the predicted products of novel genes close to the TNF locus in the human MHC. J. Immunol. (1999) 162:4745–4754.[Abstract/Free Full Text]

  13. Pende D., Parolini S., Pessino A., Sivori S., Augugliaro R., Morelli L., Marcenaro E., Accame L., Malaspina A., Biassoni R., et al. Identification and molecular characterization of NKp30, a novel triggering receptor involved in natural cytotoxicity mediated by human natural killer cells. J. Exp. Med. (1999) 190:1505–1516.[Abstract/Free Full Text]

  14. Rollinger-Holzinger I., Eibl B., Pauly M., Griesser U., Hentges F., Auer B., Pall G., Schratzberger P., Niederwieser D., Weiss E.H., et al. LST1: a gene with extensive alternative splicing and immunomodulatory function. J. Immunol. (2000) 164:3169–3176.[Abstract/Free Full Text]

  15. Wilson A.G., de Vries N., van de Putte L.B.A., Duff G.W. A tumour necrosis factor {alpha} polymorphism is not associated with rheumatoid arthritis. Ann. Rheum. Dis. (1995) 54:601–603.[Abstract/Free Full Text]

  16. Hajeer A.H., Hutchinson I.V. TNF-{alpha} gene polymorphism: clinical and biological implications. Microsc. Res. Tech. (2000) 50:216–228.[CrossRef][Web of Science][Medline]

  17. Albertella M.R., Campbell R.D. Characterization of a novel gene in the human major histocompatibility complex that encodes a potential new member of the I{kappa}B family of proteins. Hum. Mol. Genet. (1994) 3:793–799.[Abstract/Free Full Text]

  18. Ghosh S., Karin M. Missing pieces in the NF-{kappa}B puzzle. Cell (2002) 109:S81–S96.[CrossRef][Web of Science][Medline]

  19. Sedgwick S.G., Smerdon S.J. The ankyrin repeat: a diversity of interactions on a common structural framework. Trends Biochem. Sci. (1999) 24:311–316.[CrossRef][Web of Science][Medline]

  20. Okamoto K., Makino S., Yoshikawa Y., Takaki A., Nagatsuka Y., Ota M., Tamiya G., Kimura A., Bahram S., Inoko H. Identification of I{kappa}BL as the second major histocompatibility complex-linked susceptibility locus for rheumatoid arthritis. Am. J. Hum. Genet. (2003) 72:303–312.[CrossRef][Web of Science][Medline]

  21. Tamiya G., Shinya M., Imanishi T., Ikuta T., Makino S., Okamoto K., Furugaki K., Matsumoto T., Mano S., Ando S., et al. Whole genome association study of rheumatoid arthritis using 27039 microsatellites. Hum. Mol. Genet. (2005) 14:2305–2321.[Abstract/Free Full Text]

  22. Collado L., Rueda B., Caliz R., Torres B., Garcia A., Nunez-Roldan A., Gonzalez-Escribano M.F., Martin J. Lack of association between the I{kappa}BL promoter polymorphism and rheumatoid arthritis. Arthritis Rheum. (2004) 50:2032–2033.[CrossRef][Web of Science][Medline]

  23. Semple J.I., Brown S.E., Sanderson C.M., Campbell R.D. A distinct bipartite motif is required for the localization of inhibitory {kappa}B-like (I{kappa}BL) protein to nuclear speckles. Biochem. J. (2002) 361:489–496.[CrossRef][Web of Science][Medline]

  24. Gorlach M., Burd C.G., Dreyfuss G. The mRNA poly(A)-binding protein: localization, abundance, and RNA-binding specificity. Exp. Cell Res. (1994) 211:400–407.[CrossRef][Web of Science][Medline]

  25. Lehner B., Semple J.I., Brown S.E., Counsell D., Campbell R.D., Sanderson C.M. Analysis of a high-throughput yeast two-hybrid system and its use to predict the function of intracellular proteins encoded within the human MHC class III region. Genomics (2004) 83:153–167.[CrossRef][Web of Science][Medline]

  26. Ditzel H.J., Masaki Y., Nielsen H., Farnaes L., Burton D.R. Cloning and expression of a novel human antibody-antigen pair associated with Felty’s syndrome. Proc. Natl. Acad. Sci. USA (2000) 97:9234–9239.[Abstract/Free Full Text]

  27. Fuller-Pace F.V. DExD/H box RNA helicases: multifunctional proteins with important roles in transcriptional regulation. Nucleic Acids Res. (2006) 34:4206–4215.[Abstract/Free Full Text]

  28. Hartman T.R., Qian S., Bolinger C., Fernandez S., Schoenberg D.R., Boris-Lawrie K. RNA helicase A is necessary for translation of selected messenger RNAs. Nat. Struct. Mol. Biol. (2006) 13:509–516.[CrossRef][Web of Science][Medline]

  29. Ewing R.M., Chu P., Elisma F., Li H., Taylor P., Climie S., McBroom-Cerajewski L., Robinson M.D., O’Connor L., Li M., et al. Large-scale mapping of human protein-protein interactions by mass spectrometry. Mol. Syst. Biol. (2007) 3:89–105.[Medline]

  30. Lamond A.I., Spector D.L. Nuclear speckles: a model for nuclear organelles. Nat. Rev. Mol. Cell Biol. (2003) 4:605–612.[CrossRef][Web of Science][Medline]

  31. Mewar D., Marinou I., Lee M.E., Timms J.M., Kilding R., Teare M.D., Read R.C., Wilson A.G. Haplotype-specific gene expression profiles in a telomeric major histocompatibility complex gene cluster and susceptibility to autoimmune diseases. Genes Immun. (2006) 7:625–631.[CrossRef][Web of Science][Medline]

  32. Ross K., Yang L., Dower S., Volpe F., Guesdon F. Identification of threonine 66 as a functionally critical residue of the interleukin-1 receptor-activated kinase. J. Biol. Chem. (2002) 277:37414–37421.[Abstract/Free Full Text]

  33. Yang L., Ross K., Qwarnstrom E.E. RelA control of I{kappa}B{alpha} phosphorylation: a positive feedback loop for high affinity NF-{kappa}B complexes. J. Biol. Chem. (2003) 278:30881–30888.[Abstract/Free Full Text]

  34. Kiss-Toth E., Wyllie D.H., Holland K., Marsden L., Jozsa V., Oxley K.M., Polgar T., Qwarnstrom E.E., Dower S.K. Functional mapping and identification of novel regulators for the toll/interleukin-1 signalling network by transcription expression cloning. Cell Signal. (2006) 18:202–214.[CrossRef][Web of Science][Medline]

  35. Youssef P.P., Smeets T.J., Bresnihan B., Cunnane G., Fitzgerald O., Breedveld F., Tak P.P. Microscopic measurement of cellular infiltration in the rheumatoid arthritis synovial membrane: a comparison of semiquantitative and quantitative analysis. Br. J. Rheumatol. (1998) 37:1003–1007.[Abstract/Free Full Text]

  36. Sostaric E., Georgiou A.S., Wong C.H., Watson P.F., Holt W.V., Fazeli A. Global profiling of surface plasma membrane proteome of oviductal epithelial cells. J. Proteome Res. (2006) 5:3029–3037.[CrossRef][Web of Science][Medline]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
16/24/3027    most recent
ddm261v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (2)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Greetham, D.
Right arrow Articles by Wilson, A. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Greetham, D.
Right arrow Articles by Wilson, A. G.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?