Hechos y controversias en la enfermedad mixta del tejido conectivo / Facts and controversies in mixed connective tissue disease

La enfermedad mixta del tejido conectivo (EMTC) es una enfermedad reumática autoinmunitaria sistémica (ERAS) caracterizada por la asociación de manifestaciones clínicas de lupus eritematoso sistémico (LES), esclerosis sistémica cutánea (ESC) y polimiositis-dermatomiositis en presencia de títulos elevados de anticuerpos anti-U1-RNP en el suero de los pacientes. Sus principales síntomas son la poliartritis, el edema de manos, el fenómeno de Raynaud, la esclerodactilia, la miositis y la hipomotilidad esofágica. Actualmente, la mayoría de los autores acepta que la EMTC es una entidad independiente, pero algunos mantienen que estos pacientes podrían presentar una ERAS, definida en su fase precoz como LES o ESC, o ser, en realidad, un síndrome de solapamiento de la ERAS (AU)

Mixed connective tissue disease (MCTD) is a systemic autoimmune rheumatic disease (SARD) characterised by the combination of clinical manifestations of systemic lupus erythematosus (SLE), cutaneous systemic sclerosis (SSc) and polymyositis-dermatomyositis, in the presence of elevated titers of anti-U1-RNP antibodies. Main symptoms of the disease are polyarthritis, hand oedema, Raynaud's phenomenon, sclerodactyly, myositis and oesophageal hypomobility. Although widely discussed, most authors today accept MCTD as an independent entity. Others, however, suggest that these patients may belong to subgroups or early stages of certain definite connective diseases, such as SLE or SSc, or are, in fact, SARD overlap syndromes (AU)

Genome-wide RNA-binding analysis of the trypanosome U1 snRNP proteins U1C and U1-70K reveals cis/trans-spliceosomal network.

Trans-splicing in trypanosomes adds a 39-nucleotide mini-exon from the spliced leader (SL) RNA to the 5' end of each protein-coding sequence. On the other hand, cis-splicing of the few intron-containing genes requires the U1 small nuclear ribonucleoprotein (snRNP) particle. To search for potential new functions of the U1 snRNP in Trypanosoma brucei, we applied genome-wide individual-nucleotide resolution crosslinking-immunoprecipitation (iCLIP), focusing on the U1 snRNP-specific proteins U1C and U1-70K. Surprisingly, U1C and U1-70K interact not only with the U1, but also with U6 and SL RNAs. In addition, mapping of crosslinks to the cis-spliced PAP [poly(A) polymerase] pre-mRNA indicate an active role of these proteins in 5' splice site recognition. In sum, our results demonstrate that the iCLIP approach provides insight into stable and transient RNA-protein contacts within the spliceosomal network. We propose that the U1 snRNP may represent an evolutionary link between the cis- and trans-splicing machineries, playing a dual role in 5' splice site recognition on the trans-spliceosomal SL RNP as well as on pre-mRNA cis-introns.

Analytical performance of RNA isolated from BD SurePath™ cervical cytology specimens by the PreTect™ HPV-Proofer assay.

Several commercial HPV ancillary tests are available for detection of E6/E7 RNA. It is not clear how storage of a cervical Pap affects the analytical and clinical performance of the PreTect™ HPV-Proofer assay. To investigate the qualitative performance of RNA extracted from BD SurePath™ liquid-based cytology (LBC) specimens for the detection of human papillomavirus (HPV) E6/E7 mRNA using the PreTect™ HPV-Proofer assay, studies including stability, reproducibility, residual specimen analysis, and storage medium comparison assays were performed. Cervical cytology specimens were collected and stored in BD SurePath™ LBC preservative fluid and/or PreTect™ Transport Media. RNA was isolated using the RecoverAll™ Total Nucleic Acid Isolation kit and RNA integrity was evaluated in the PreTect™ HPV-Proofer assay. The performance of RNA isolated from cervical cells collected and stored in BD SurePath™ LBC preservative fluid or PreTect™ Transport Media was also evaluated through a storage medium comparison study. The RNA was found to be stable for a minimum of 21 days when stored at ambient temperature and displayed high reproducibility with the mean percentage reproducibility ranging from 90.5% to 100% for the HPV types detected by the PreTect™ HPV-Proofer assay. The prevalence rate of HPV types in this study cohort was consistent with published reports. A 93.7% first pass acceptance rate was demonstrated across all cytology grades. The positive human U1 snRNP specific A protein (U1A) and HPV rate for BD SurePath™ LBC and PreTect™ Transport Media specimens was statistically equivalent for both normal and abnormal specimens. This data support the use of RNA isolated from BD SurePath™ LBC for ancillary HPV testing and demonstrates the feasibility of using BD SurePath™ preservative fluid as a specimen type with the PreTect™ HPV-Proofer assay.

Strategies for H-score normalization of preanalytical technical variables with potential utility to immunohistochemical-based biomarker quantitation in therapeutic response diagnostics.

Digital quantitative immunohistochemical analysis of protein biomarker expression offers a broad dynamic range against which clinical outcomes may be measured. Semi-quantitative expression data represented as an H-score is produced by computer generated average intensity of positive staining given weight by the percentage of cells showing positive staining. While patient H-scores vary for biological reasons, variation may also arise from preanalytic technical issues, such as differences in fixation protocols. In this study, we present data on two candidate calibrator nuclear-localized proteins, SNRPA and SnRNP70, with robust and consistent expression levels across breast cancers. Quantitative expression measurement of these two candidate biomarkers may potentially be used to eliminate the effect of differences in preanalytic processing of specimens by normalizing H-scores derived from test biomarkers of interest. To examine the effects of preanalytical fixation variation on biomarker quantitation and potential utility of candidate calibrators to address such issues, 6 surgically-resected human breast cancer patient specimens were divided into 6 portions and fixed under distinct conditions (fixation following resection in formalin for 2 hr, 8 hr or 48 hr, or held overnight at 4°C in buffered saline prior to formalin fixation for 2 hr, 8 hr, or 48 hr). We find H-score variation between fixation conditions within individual patient's tumors that were stained for XPF, ATM, BRCA1, pMK2 and PARP1. Most interestingly, detectable expression of SNRPA and SnRNP70 is covariant to test biomarkers under distinct fixation conditions and so these hold the potential for serving as calibration standards for general antigen preservation and reactivity.

Interpreting a low resolution map of human U1 snRNP using anomalous scatterers.

We recently determined the crystal structure of the functional core of human U1 snRNP, consisting of nine proteins and one RNA, based on a 5.5 A resolution electron density map. At 5-7 A resolution, alpha helices and beta sheets appear as rods and slabs, respectively, hence it is not possible to determine protein fold de novo. Using inverse beam geometry, accurate anomalous signals were obtained from weakly diffracting and radiation sensitive P1 crystals. We were able to locate anomalous scatterers with positional errors below 2 A. This enabled us not only to place protein domains of known structure accurately into the map but also to trace an extended polypeptide chain, of previously undetermined structure, using selenomethionine derivatives of single methionine mutants spaced along the sequence. This method of Se-Met scanning, in combination with structure prediction, is a powerful tool for building a protein of unknown fold into a low resolution electron density map.

[The spliceosome and its interest for lupus therapy]. / Le splicéosome et son intérêt dans la recherche thérapeutique sur le lupus.

INTRODUCTION: The spliceosome, which is a particle containing a molecule of U-RNA and proteins that are specific to each U ribonuclear particle (U-snRNP) or common to every U-snRNPs, is one of the numerous nuclear targets recognized by the antibodies (Abs) and CD4+ T cells from patients with systemic lupus erythematosus and lupus mice. EXEGESIS: We recently characterized a peptide from the spliceosomal protein U1-70K (sequence 131-151), which is recognized by the Abs and CD4+ T cells from lupus mice and patients. This peptide contains a conserved RNP1 motif, which is also present in other spliceosomal proteins targeted by the Abs from individuals with lupus. We further showed that peptide 131-151 containing a phosphoserine at position 140 (peptide P140) possessed tolerogenic properties in lupus mice and was recognized by the Abs and CD4+ T cells from lupus patients. CONCLUSION: Thanks to its RNP1 motif, the peptide P140 might play an important role in the initiation and perpetuation steps of the humoral and cellular immune response diversification in lupus individuals. Therapeutic and particularly immunomodulating properties of P140 peptide are being evaluated in humans (a phase III clinical trial will be undertaken in the next weeks).

Regulated nucleo-cytoplasmic shuttling of human aci-reductone dioxygenase (hADI1) and its potential role in mRNA processing.

Bacterial aci-reductone dioxygenase (ARD), a member of the cupin superfamily, has evolutionarily primitive protein folding and functions in the methionine recycling pathway. Recently, a human ARD orthologue (human ADI1, hADI1) has been identified and exhibits functions other than ARD activity. The hADI1 localizes mainly to the cytoplasm, but a substantial fraction is nuclear, suggesting functions in both cellular compartments. In this study, we report that nucleo-cytoplasmic transport of hADI1 is regulated by a non-canonical nuclear export signal (NES) located in the N-terminal region of hADI1. The NES is composed of multiple basic amino-acid residues instead of the canonical leucine-rich sequence. Nuclear export of hADI1 was not mediated by CRM1, a major transporter that binds to leucine-rich NES. Substitution of the basic residues with alanines abolished NES activity. Mutant hADI1 accumulated in the nucleus and formed speckles frequently observed with splicing factors and some transcription factors. Indeed, hADI1 specifically co-localized with the splicing factor U1-70K to the nucleus but not with another splicing factor, SC35. U1-70K over-expression induced nuclear accumulation of hADI1. Nuclear hADI1 expression significantly altered the splicing pattern of the adenovirus E1A mini-gene, which generates multiple alternatively spliced transcripts. Thus, hADI1 may have acquired a novel role in nuclear mRNA processing possibly by modulating U1-70K-related functions, an activity negatively regulated by a non-classical NES sequence.

Visual screening for localized RNAs in yeast revealed novel RNAs at the bud-tip.

Several RNAs, including rRNAs, snRNAs, snoRNAs, and some mRNAs, are known to be localized at specific sites in a cell. Although methods have been established to visualize RNAs in a living cell, no large-scale visual screening of localized RNAs has been performed. In this study, we constructed a genomic library in which random genomic fragments were inserted downstream of U1A-tag sequences under a GAL1 promoter. In a living yeast cell, transcribed U1A-tagged RNAs were visualized by U1A-GFP that binds the RNA sequence of the U1A-tag. In this screening, many RNAs showed nuclear signals. Since the nuclear signals of some RNAs were not seen when the U1A-tag was connected to the 3' ends of the RNAs, it is suggested that their nuclear signals correspond to nascent transcripts on GAL1 promoter plasmids. Using this screening method, we successfully identified two novel localized mRNAs, CSR2 and DAL81, which showed bud-tip localization.

Intracellular trafficking of LET-756, a fibroblast growth factor of C. elegans, is controlled by a balance of export and nuclear signals.

The superfamily of fibroblast growth factors (FGF), which counts 22 members in humans, exerts many functions during animal development and adult life. LET-756 is one of the two FGFs of the nematode C. elegans. Re-introduction of LET-756 in a null mutant strain restores viability, allowing the study of structural requirements for LET-756 trafficking and function. LET-756 protein has several regions and motifs, including a non-classical internal motif required for secretion. We show here that a main difference in the wild-type LET-756 molecule and a truncated molecule that mimics a partial loss-of-function mutant lies on subnuclear expression. Using Cos-1 cells and rescue activity we show that: (i) nuclear localization is due to various redundant NLS, one of them acting as a nucleolar localization signal; (ii) nuclear LET-756 is addressed to the speckles by a stretch of glutamine residues; (iii) nuclear LET-756 is trafficking between speckles and nucleoli; (iv) in the nucleolus, LET-756 is associated with proteins of the rRNA splicing compartment; (v) changing LET-756 secretion signal prevents its nuclear localization. We propose that LET-756 exerts its functions through a balance between secreted and nuclear forms due to two opposite addressing signals, (i) synergy of several NLS and (ii) attenuated secretion signal.

Multiple properties of the splicing repressor SRp38 distinguish it from typical SR proteins.

The SR protein SRp38 is a general splicing repressor that is activated by dephosphorylation during mitosis and in response to heat shock. Here we describe experiments that provide insights into the mechanism by which SRp38 functions in splicing repression. We first show that SRp38 redistributes and colocalizes with snRNPs, but not with a typical SR protein, SC35, during mitosis and following heat shock. Supporting the functional significance of this association, a micrococcal nuclease-sensitive component, i.e., an snRNP(s), completely rescued heat shock-induced splicing repression in vitro, and purified U1 snRNP did so partially. SRp38 contains an N-terminal RNA binding domain (RBD) and a C-terminal RS domain composed of two subdomains (RS1 and RS2 domains). Unexpectedly, an RS1 deletion mutant derivative specifically inhibited the second step of splicing, while an RS2 deletion mutant retained significant dephosphorylation-dependent repression activity. Using chimeric SRp38/SC35 proteins, we show that SC35-RBD/SRp38-RS can function as a general splicing activator and that the dephosphorylated version can act as a strong splicing repressor. SRp38-RBD/SC35-RS, however, was essentially inactive in these assays. Together, our results help to define the unusual features of SRp38 that distinguish it from other SR proteins.

Autoantibodies that stabilize U1snRNP are a significant component of human autoantibodies to snRNP and delay proteolysis of sm antigens in vitro.

OBJECTIVE: Autoantibodies to U1-C have been considered a minor component of anti-snRNP (nRNP, Sm) response based on Western blotting. However, we have previously shown that virtually all human anti-nRNP sera contain antibodies to native U1-C, as well as novel autoantibodies that stabilize the molecular interaction of the U1-C-Sm core particle. The biological significance of stabilizing antibodies was investigated by titering anti-U1-C/U1-A compared to stabilizing antibodies, and by examining the effects of stabilizing antibodies on antigen processing. METHODS: Autoantibodies to individual native components of U1snRNP (immunoprecipitation of RNase-treated cell extract) and stabilizing antibodies (dissociation of snRNP on anti-Sm monoclonal antibodies by 1 M MgCl2) in human autoimmune sera were titered. Effects of stabilizing antibodies on proteolysis were assessed by incubating UsnRNP with anti-snRNP/Sm autoimmune sera prior to protease digestion. RESULTS: Autoantibodies to native U1-C and U1-C-Sm core particle stabilizing antibodies were universally present in human anti-nRNP or anti-nRNP + anti-Sm sera, but not in anti-Sm sera. Antibodies to U1-A were less common. The titers of stabilizing antibodies were higher than those of antibodies to U1-C (p < 0.01), indicating that the stabilizing antibodies were a significant component of the anti-snRNP response. The stabilizing anti-nRNP, but not anti-Sm antibodies, protects the Sm core particle from dissociation and proteolysis. CONCLUSION: Autoantibodies stabilizing the U1-C-Sm core particle were universally present in anti-nRNP sera and delay proteolysis of the Sm core particle. They may suppress spreading of the autoimmune response to Sm by delaying or altering processing of the Sm core proteins by antigen-presenting cells.

Use of fluorescent protein tags to study nuclear organization of the spliceosomal machinery in transiently transformed living plant cells.

Although early studies suggested that little compartmentalization exists within the nucleus, more recent studies on metazoan systems have identified a still increasing number of specific subnuclear compartments. Some of these compartments are dynamic structures; indeed, protein and RNA-protein components can cycle between different domains. This is particularly evident for RNA processing components. In plants, lack of tools has hampered studies on nuclear compartmentalization and dynamics of RNA processing components. Here, we show that transient expression of fluorescent protein fusions of U1 and U2 small nuclear ribonucleoprotein particle (snRNP)-specific proteins U1-70K, U2B", and U2A ', nucleolar proteins Nop10 and PRH75, and serine-arginine-rich proteins in plant protoplasts results in their correct localization. Furthermore, snRNP-specific proteins also were correctly assembled into mature snRNPs. This system allowed a systematic analysis of the cellular localization of Arabidopsis serine-arginine-rich proteins, which, like their animal counterparts, localize to speckles but not to nucleoli and Cajal bodies. Finally, markers for three different nuclear compartments, namely, nucleoli, Cajal bodies, and speckles, have been established and were shown to be applicable for colocalization studies in living plant protoplasts. Thus, transient expression of proteins tagged with four different fluorescent proteins is a suitable system for studying the nuclear organization of spliceosomal proteins in living plant cells and should therefore allow studies of their dynamics as well.

Identification of both shared and distinct proteins in the major and minor spliceosomes.

In metazoans, two distinct spliceosomes catalyzing pre-messenger RNA splicing have been identified. Here, the human U11/U12 small nuclear ribonucleoprotein (snRNP), a subunit of the minor (U12-dependent) spliceosome, was isolated. Twenty U11/U12 proteins were identified, including subsets unique to the minor spliceosome or common to both spliceosomes. Common proteins include four U2 snRNP polypeptides that constitute the essential splicing factor SF3b. A 35-kilodalton U11-associated protein homologous to the U1 snRNP 70K protein was also identified. These data provide fundamental information about proteins of the minor spliceosome and shed light on its evolutionary relationship to the major spliceosome.

A critical evaluation of enzyme immunoassays for detection of antinuclear autoantibodies of defined specificities. I. Precision, sensitivity, and specificity.

OBJECTIVE: To determine the performance characteristics of enzyme-based immunoassay (EIA) kits for the detection of antinuclear and other autoantibodies of defined specificities. METHODS: Nine manufacturers of EIA kits to detect antibodies of defined specificities participated in a study in which they received coded sera from the Centers for Disease Control and Prevention. These coded sera contained different dilutions of antibody of one specificity mixed with sera containing antibodies of other specificities. The manufacturers were asked to use their standard technology to determine antibody content and send the data to a committee of the International Union of Immunological Societies for analysis. The data were analyzed for sensitivity and specificity in the detection of anti-double-stranded DNA (anti-dsDNA), anti-single-stranded DNA, antihistone, anti-Sm, anti-U1 RNP, anti-SSA/Ro, anti-SSB/La, anti-Scl-70 (DNA topoisomerase I), anticentromere, and anti-Jo-1 antibodies. In addition, replicate samples were included in the coded sera to evaluate the precision of each EIA method. RESULTS: Lack of sensitivity and specificity was most evident in the anti-dsDNA and anti-Sm kits, although 2 kits for anti-dsDNA achieved acceptable sensitivity and specificity. Generally, anti-SSA/Ro, anti-SSB/La, anti-Scl-70, anticentromere, and anti-Jo-1 kits performed well. Many false-positive results were obtained with a multiple myeloma serum containing cryoprecipitates, but multiple myeloma sera without cryoprecipitates presented no problem in the EIA system. Precision, based on evaluation of replicate samples, varied from very good to poor. CONCLUSION: No single manufacturer was clearly superior to others in terms of their products' overall sensitivity, specificity, and precision. Areas that needed improvement were in kits for the detection of antibodies to dsDNA and to Sm antigen. Some EIA kits achieved good sensitivity and specificity. Individual manufacturers were informed of the performance of their respective kits so they could take measures to correct perceived deficiencies and thus improve the reliability of a group of important diagnostic assays used in the evaluation of systemic rheumatic diseases.

Identification of a novel, non-snRNP protein complex containing U1A protein.

Mouse monoclonal antibodies (MAbs) were generated against Escherichia coli-produced U1snRNP-A (U1A) protein. U1A-specific MAbs as well as MAbs that reacted with both U1A and U2snRNP-B" (U2B") were isolated. MAb 12E12 was unique among the characterized MAbs because it failed to immunoprecipitate U1A protein produced by in vitro transcription and translation using rabbit reticulocyte lysates. However, when U1A protein was made using a wheat germ extract, MAb 12E12 could immunoprecipitate U1A quite readily, as did the other MAbs. These data suggest that the MAb 12E12 epitope is masked when U1A is prepared in reticulocyte lysate. Further studies showed that MAb 12E12 recognizes an epitope that is masked when U1A protein is bound to U1 RNA. The unique nature of MAb 12E12 was used to demonstrate that U1A could be immunoprecipitated from whole-cell extracts in a form that was free of U1 RNA and other snRNP components. However, this snRNP-free U1A (SF-A) was found to co-immunoprecipitate with a unique set of non-snRNP proteins. In order to confirm that U1A exists in at least two distinct complexes (snRNP bound and snRNP free), [35S]-labeled nucleoplasmic extracts were analyzed by sucrose density gradient fractionation and immunoprecipitation. MAb 12E12 specifically immunoprecipitated SF-A, which migrated in a novel non-snRNP complex. Specifically, proteins of approximately 58, 59, 63, 65, and 105 kDa co-sedimented and co-immunoprecipitated with SF-A. Our data show that a significant portion of the cellular U1A (at least 3% or approximately 30,000 molecules) exists in the nucleoplasm in one or more novel complexes. Our previous studies have demonstrated an effect of purified U1A on polyadenylation of pre-mRNAs and, consistent with this finding, purified antibodies to SF-A significantly diminish polyadenylation in vitro.

Comparison of two different methods using overlapping synthetic peptides for localizing linear B cell epitopes in the U1 snRNP-C autoantigen.

We have compared the performances of two different approaches using overlapping synthetic peptides to identify the location of linear epitopes of the U1 snRNP-C autoantigen. The first method was based on the use of 15 overlapping peptides (16-30 residue-long) synthesized using conventional Fmoc chemistry, removed from the resin by a standard cleavage procedure, and tested by ELISA after direct coating to polyvinyl microtiter plates. The second approach used a commercial kit (SPOT) to synthesize 75 overlapping decapeptides on cellulose membrane which were assayed by a direct immunoenzymatic test. Both standard and SPOTscan methods were evaluated with antibodies raised in rabbits against synthetic peptides of U1C and sera from patients with autoimmune diseases. In addition to inherent problems linked to the SPOT synthesis (in particular the impossibility of checking the quality of peptides), a number of limitations in the SPOTscan method were identified (e.g. a certain lack of sensitivity and, in one case, the complete lack of peptide reactivity due to the removal of charged end groups at both extremities). However, we found no background with sera from autoimmune patients in the SPOTscan and the antigenic maps obtained using the two approaches generally agreed. This study shows that the SPOTscan approach represents a simple, relatively non expensive and rapid method for initial screening to identify candidate sequences that may be dominant linear epitopes in a protein. Subsequent analysis and controls should include the preparation of conventionally synthesized peptides for formal immunochemical investigations.

Prevalence and clinical significance of antibodies to ribonucleoproteins in systemic lupus erythematosus in Malaysia.

One hundred and seventy patients with systemic lupus erythematosus (SLE) were studied for the prevalence of antibodies to the small RNA-associated proteins Ro/SSA, La/SSB, Sm, U1RNP and Sm. The relationship of these autoantibodies to different races, sexes and clinical manifestations of SLE was evaluated. Passive immunodiffusion was employed using human spleen extract as antigen source for Ro and rabbit thymus extract for La, Sm and U1RNP. We found the prevalence of antibodies to be as follows: anti-Ro/SSA, 36%; anti-La/SSB, 8%; anti-Sm, 15% ; anti-U1RNP, 21%. Except for a low prevalence of anti-La, the prevalence of these antibodies was similar to that in Western studies, The prevalence of anti-Ro/SSA is similar to that reported in the Western studies, but lower than that reported in the Oriental patients from Singapore and Hong Kong. Linkages of anti-Ro with anti-La antibodies were usual; however, although anti-Sm antibodies were usually associated with anti-U1RNP, they were more frequently associated with anti-Ro antibodies. The Malay patients had a high prevalence of anti U1RNP compared to other races. No gender difference was detected. Anti-Sm antibody was associated with serositis and anti-U1RNP antibodies with Raynaud's phenomenon. No association was found between the presence of skin renal or cerebral manifestations and any specific antibodies or combination of antibodies.

Members of a family of proteins (the RD family) detected by a U1 70K monoclonal antibody are present in spliceosomal complexes.

We have characterized a monoclonal antibody (mAb) to the U1 snRNP component U1 70K. We find that this antibody recognizes several proteins, in addition to U1 70K, in purified spliceosomal complexes and in total HeLa cell nuclear extract preparations. The novel mAb U1 70K antigens can also be specifically immunoprecipitated by the antibody. Similarly to U1 70K, many of the mAb U1 70K antigens can be phosphorylated by a co-purifying kinase activity. The epitope recognized by mAb U1 70K was previously shown to be a repeating arginine/aspartate (RD) dipeptide. Thus we have designated the novel mAb U1 70K antigens the RD family. Comparison of mAb U1 70K with a recently characterized antibody, mAb 16H3, whose epitope is a repeating R/D or R/E motif, showed that a large subset of the antigens are common. In contrast, most of the mAb U1 70K antigens are distinct from the proteins detected by mAb 104, an antibody to the SR family of splicing factors.