Background: Antibodies directed to proteins containing the non-standard amino acid citrulline, are extremely specific for rheumatoid arthritis (RA). Peptidylcitrulline can be generated by post-translational conversion of arginine residues. This process, citrullination, is catalysed by a group of calcium dependent peptidylarginine deiminase (PAD) enzymes. Objective: To investigate the expression and activity of four isotypes of PAD in peripheral blood and synovial fluid cells of patients with RA. Results: The data presented here show that citrullination of proteins by PAD enzymes is a process regulated at three levels: transcription-in peripheral blood PAD2 and PAD4 mRNAs are expressed predominantly in monocytes; PAD4 mRNA is not detectable in macrophages, translation-translation of PAD2 mRNA is subject to differentiation stage-specific regulation by its 39 UTR, and activation-the PAD proteins are only activated when sufficient Ca 2+ is available. Such high Ca 2+ concentrations are normally not present in living cells. In macrophages, which are abundant in the inflamed RA synovium, vimentin is specifically citrullinated after Ca 2+ influx. Conclusion: PAD2 and PAD4 are the most likely candidate PAD isotypes for the citrullination of synovial proteins in RA. Our results indicate that citrullinated vimentin is a candidate autoantigen in RA.

SUMMARYWe analysed 112 idiopathic inflammatory myopathy (IIM) sera for the presence of anti-Ro, anti-La and anti-histidyl-tRNA synthetase (Jo-1) autoantibodies, and subsequently mapped B cell epitopes on the Ro52 protein recognized by anti-Ro52 þ IIM sera. Sera were characterized by immunoblotting, ELISA and RNA precipitation. Both anti-Ro60 and anti-La activity was found in 4% of IIM sera. Anti-Ro52 antibodies were present in 20% of IIM sera. However, in anti-Jo-1 þ IIM sera (21%), the frequency of the anti-Ro52 antibodies was found to be much higher (58%). No cross-reactivity between anti-Ro52 and anti-Jo-1 antibodies could be detected in these sera. To learn more about the nature of anti-Ro52 antibodies occurring in IIM sera, we analysed the major epitopes of the Ro52 protein targeted by antiRo52 þ IIM sera by immunoprecipitation of in vitro translated Ro52 deletion mutants. The major epitope was mapped in the region bordered by amino acids 126 and 252. This part of the protein includes a long a-helical region which contains two potential coiled-coil domains as well as a leucine zipper motif. Although no difference in Ro52 epitope recognition between anti-Jo-1 þ and anti-Jo-1 ¹ IIM sera could be observed, our results suggest that the autoimmune response against Ro52 and Jo-1 in IIM patients is coupled.

In this study, DNA-depleted nuclear protein matrices are isolated from HeLa S3 cells. These nuclear matrices consist of peripheral laminae, residual nucleoli, and internal fibrillar structures . High molecular weight, heterogeneous nuclear RNA (hnRNA) is quantitatively associated with these structures and can be released intact only by affecting the integrity of the matrices . It is, therefore, concluded that hnRNA is part of a highly organized nuclear structure.By irradiation of intact cells or isolated nuclear matrices with ultraviolet light, proteins tightly associated with hnRNA can be induced to cross-link with the RNA . Performing the crosslinking in vivo is an extra guarantee that only hnRNA-protein (hnRNP) complexes existing in the intact cell are covalently linked . Such hnRNP complexes were isolated and purified under conditions that completely dissociate nonspecific RNA-protein complexes .By comparison of the hnRNP found in nuclear matrices and the published data on the composition of hnRNP particles, it was found that the so-called hnRNP "packaging" proteins (32,000-38,000 mol wt) were not efficiently cross-linked to hnRNA by UV irradiation . They were, however, present in the matrix preparations, bound to hnRNA, because they were released from nuclear matrices after ribonuclease treatment of these structures . On the other hand, two major hnRNPs (41,500 and 43,000 mol wt) were efficiently cross-linked to hnRNA . These proteins were not released by ribonuclease treatment, which suggests that they are involved in the binding of hnRNA to the nuclear matrix .Althoughin the last decademuch knowledge has been acquired about hnRNA at the molecular level, little is known about the role ofvarious cellular structures and components in transcription, RNA processing, and RNA transport to the cytoplasm. To investigate such a role, studies have been undertaken to identify protein and RNA molecules that interact with heterogeneous nuclear RNA (hnRNA) . hnRNA can be extracted from nuclei in the form of hnRNA-protein (hnRNP) particles (reviewed by Heinrich et al. [1] and Van Venrooij and Janssen [2]) that have a repeating subunit structure composed of 200-300 A spherical particles connected by a ribonuclease-sensitive strand (3, 4). Small nuclear RNA seem to be integral parts of hnRNP particles (5-8). The protein composition of hnRNP particles is very complex and seems to be related to the isolation procedure used. Although nonspecific binding of proteins to the RNA during the isolation of the particles has never rigorously been excluded,most workers (9-11) agree on the presence of some predominant proteins in the 30,000-44,000 mol wt range.The isolation of hnRNP particles mostly involves nuclear breakage (for example, by sonication) or extraction of the nuclei with buffer solutions for prolonged periods at a relatively high temperature . In general, the amount of hnRNP released from the nuclei seems to depend very much on the degree of nuclear disintegration achieved (2) .Detergent-treated nuclei from euka...

The U2 snRNP contains two specific proteins, U2B″ and U2A'. Neither of these proteins, on its own, is capable of specific interactions with U2 RNA. Here, a complex between U2B″ and U2A' that forms in the absence of RNA is identified. Analysis of mutant forms of U2B″ shows that the smallest fragment able to bind specifically U2 RNA (amino acids 1‐88) is also the minimal region required for complex formation with U2A', and implies that this region must be largely structurally intact for U2A' interaction. Although this truncated U2B″ fragment is capable of making specific protein–RNA and protein‐protein interactions its structure, as measured by the ability to bind to U2A″, appears to depend on the rest of the protein. Hybrids between U2B″ and the closely related U1A protein are used to localize U2B″ specific amino acids involved in protein‐protein interaction. These can be divided into two functional groups. U2A' interaction with U2B″ amino acids 37‐46 permits binding to U2 RNA whereas interaction with U2B″ specific amino acids between positions 14 and 25 reduces non‐specific binding to U1 RNA. These two proteins may serve as a general example of how RNA binding may be modulated by protein‐protein interaction in the assembly of RNPs, particularly since the region of U2″ involved in interaction with U2A' consists mainly of a conserved RNP motif.

Objectives-Systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) can both present with an erosive arthritis with the small joints of the hands aVected. Therefore a serological marker would be useful to distinguish between these two diseases at onset. In this study anti-RA33 antibodies, which are found in patients with SLE and RA, and anti-citrullinated peptide antibodies (anti-CCP), which have recently been described as highly specific for RA, were assessed. Methods-Two hundred and thirty one patients receiving long term follow up for SLE were evaluated for arthritis and classified as erosive and non-erosive disease. Sixty six patients were tested for anti-RA33 and anti-CCP antibodies. All the patients were tested for rheumatoid factor (RF) and HLA-DR4 status. Results-Ten patients had erosive disease, six of whom were RF positive (60%), and six anti-RA33 positive (60%), whereas only two were anti-CCP positive (20%). Two hundred and twenty one patients had nonerosive disease, 40 of whom were RF positive (18%), 14 were anti-RA33 positive (6%), whereas only one patient was found to be anti-CCP positive (0.5%). Conclusion-The presence of anti-CCP antibodies may be useful in distinguishing RA from erosive SLE. Anti-RA33 antibodies and RF are unhelpful.