U1 small nuclear ribonucleoparticle (U1 snRNP) plays a central role during RNA processing. Previous structures of U1 snRNP revealed how the ribonucleoparticle is organized and recognizes the pre-mRNA substrate at the exon-intron junction. As with many other ribonucleoparticles involved in RNA metabolism, U1 snRNP contains extensions made of low complexity sequences. Here, we developed a protocol to reconstitute U1 snRNP in vitro using mostly full-length components in order to perform liquid-state NMR spectroscopy. The accuracy of the reconstitution was validated by probing the shape and structure of the particle by SANS and cryo-EM. Using an NMR spectroscopy-based approach, we probed, for the first time, the U1 snRNP tails at atomic detail and our results confirm their high degree of flexibility. We also monitored the labile interaction between the splicing factor PTBP1 and U1 snRNP and validated the U1 snRNA stem loop 4 as a binding site for the splicing regulator on the ribonucleoparticle. Altogether, we developed a method to probe the intrinsically disordered regions of U1 snRNP and map the interactions controlling splicing regulation. This approach could be used to get insights into the molecular mechanisms of alternative splicing and screen for potential RNA therapeutics.

The allosteric regulation during the binding interactions between small nuclear RNAs (snRNAs) and the associated protein factors is critical to the function of spliceosomes in alternative RNA splicing. Although network models combined with molecular dynamics simulations have shown to be powerful tools for the analysis of protein allostery, the atomic-level simulations are, however, too expensive and with limited accuracy for the large-size systems. In this work, we use a residual network model combined with a coarse-grained Gaussian network model (GNM) to investigate the binding interactions between the snRNA and the human U1A protein which is a major component of the spliceosomal U1 small nuclear ribonucleoprotein particle, and to identify the residues that play an important role in the allosteric communication in U1A during this process. We also utilize the Girvan-Newman method to detect the structural organization in U1A-snRNA recognition and interactions. Our results reveal that: (Ι) not only the residues at the binding sites that are traditionally considered to play a major role in U1A-snRNA association, but those residues that are far away from the RNA binding interface participate in the U1A\'s allosteric signal transmission induced by the RNA binding; (Π) the structure of U1A protein is well organized with different communities acting different roles for its RNA binding and allosteric regulation. The study demonstrates that the combination of the residual network and elastic network models is an effective and efficient method which can be readily extended to the investigation of the allosteric communication for other macromolecular interaction systems.

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In this study we aimed to investigate foetal and maternal pregnancy outcomes from a large multicentre cohort of women diagnosed with MCTD and anti-U1RNP antibodies.
This multicentre retrospective cohort study describes the outcomes of 203 pregnancies in 94 consecutive women ever pregnant who fulfilled the established criteria for MCTD with confirmed U1RNP positivity.
The foetal outcomes in 203 pregnancies were as follows: 146 (71.9%) live births, 38 (18.7%) miscarriages (first trimester pregnancy loss of <12 weeks gestation), 18 (8.9%) stillbirths (pregnancy loss after 20 weeks gestation) and 11 (5.4%) cases with intrauterine growth restriction. Maternal pregnancy outcomes were as follows: 8 (3.9%) developed pre-eclampsia, 2 (0.9%) developed eclampsia, 31 (15.3%) developed gestational hypertension and 3 (1.5%) developed gestational diabetes. Women with MCTD and aPL and pulmonary or muscular involvement had worse foetal outcomes compared with those without. Moreover, we report a case of complete congenital heart block (0.45%) and a case of cutaneous neonatal lupus, both born to a mother with positive isolated anti-U1RNP and negative anti-Ro/SSA antibodies.
In our multicentre cohort, women with MCTD had a live birth rate of 72%. While the true frequency of heart block associated with anti-U1RNP remains to be determined, this study might raise the consideration of echocardiographic surveillance in this setting. Pregnancy counselling should be considered in women with MCTD.

Pulmonary arterial hypertension (PAH) is a rare and severe complication of primary Sjögren\'s syndrome (pSS). Cohort studies indicate that the underlying diseases of PAH associated with connective tissue disease (CTD-PAH) in Asian countries are different from that in the United States and in Europe. This study investigated the clinical characteristics, survival, and prognostic factors of pSS-PAH in Chinese patients. We enrolled 29 patients with pSS-PAH who visited our referral center during August 2007 and May 2015. PAH was confirmed by right heart catheterization (RHC). Baseline demographic data, clinical manifestations, laboratory tests, autoantibody results, hemodynamics data, and treatment regimens were analyzed. All patients were followed up at the Department of Rheumatology, Peking Union Medical College Hospital (PUMCH) every three to six months. All patients were female. Mean age of pSS onset was 34.4 ± 11.1 years. Mean age of PAH diagnosed by RHC was 40.6 ± 9.0 years. There was no significant difference in age, disease duration, and hemodynamic findings between the survivors and the non-survivors in this cohort. The overall one-, three-, and five-year survival rates were 80.2%, 74.8%, and 67.4%, respectively. Prognostic factors of mortality were time between pSS onset and PAH onset (HR 1.102, 95% CI 1.017-1.185, p = 0.018) and cardiac index < 2 l/min/m2 (HR 5.497, 95% CI 1.063-28.434, p = 0.042). In contrast, the use of immunosuppressants (HR 0.110, 95% CI 0.024-0.495, p = 0.004) was related to better survival. This study demonstrates that the predictors of mortality in patients with pSS-PAH include delayed onset of PAH in pSS and worse cardiac function. Immunosuppressants can improve the prognosis of pSS-PAH.

The literature reports that mixed connective tissue disease seems more frequent in the black population and among Asians. This study aims to determine the prevalence of mixed connective tissue disease (MCTD) among connective tissue disorders and all rheumatologic pathologies in a hospital population in Gabon as well as to describe the clinical features of this disease. We conducted a retrospective study by reviewing the medical records of patients treated for mixed connective tissue disease (Kasukawa criteria) and other entities of connective tissue disorders (ACR criteria) in the Division of Rheumatology at the University Hospital in Libreville between January 2010 and December 2015. For each case of MCTD the parameters studied were articular and extra-articular manifestations, anti-U1RNP antibodies levels, patient\'s evolution. Over a period of 6 years, data were collected by medical records of 7 patients out of 6050 patients and 67 cases of connective tissue disorders, reflecting a prevalence of 0.11% and 10.44% respectively. the 7 patients were women (100%), with an average age of 39.5 years. Articular manifestations included: polyarthritis, myalgias, chubby fingers and Raynaud\'s phenomenon in 87.5%, 87.5%, 28.6% and 14% respectively. The 7 patients had high anti-U1RNP antibodies levels, ranging between 5 and 35N (N≤ 7 IU). A case of death due to pulmonary arterial hypertension (PAH) was certified. This is the largest case series of MCTD reported in Black Africa. The disease seems to be rare among the black Africans; the reason could be genetic. The demographic and clinical aspects appear similar to those in Caucasians, Asians and Blacks except for a low frequency of Raynaud?s phenomenon among Blacks.

OBJECTIVE: Mixed connective tissue disease (MCTD) is characterized by the presence of anti-U1-snRNP autoantibodies and a variable set of associated clinical features. Some MCTD patients test positive over time to autoantibodies against Sm, proteins spatially related with U1-snRNP. This situation has been attributed to expanding of the autoimmune response by a phenomenon known as epitope spreading. Our aim was to study the frequency of this phenomenon in MCTD patients and the specific clinical features of those with epitope spreading.
METHODS: All anti-U1-RNP-positive patients (2010-2015) were retrospectively reviewed, and those meeting the MCTD criteria were included in the study. Patients showing epitope spreading were compared with the remainder of the MCTD cohort. In addition, the clinical features of patients with epitope spreading were compared before and after the phenomenon occurred.
RESULTS: Among 72 anti-U1-RNP-positive patients, 40 (37 women) were diagnosed with MCTD. Thirteen MCTD patients (43%) presented epitope spreading, mainly during the first 2 years after the diagnosis of the disease (median, 1.4 years). Patients with epitope spreading had a significantly lower prevalence of skin sclerosis (0% vs. 44%, P = 0.004) and a greater prevalence of interstitial lung disease (46% vs. 15%, P = 0.05) than those without. Arthritis (92% vs. 25%, P = 0.02) and muscle involvement (67% vs. 17%, P = 0.02) were less frequent after epitope spreading had occurred.
CONCLUSIONS: Epitope spreading is common in MCTD, occurring early after the diagnosis. The clinical manifestations in patients with this phenomenon differ from those without, and their clinical features change after the immunological phenomenon has occurred.

We sought to identify the clinical factors associated with calcinosis in an international multicenter collaborative effort with the Scleroderma Clinical Trials Consortium (SCTC).
This is a retrospective cohort study of 5218 patients with systemic sclerosis (SSc). Logistic regression was used to obtain odds ratios (OR) relating calcinosis to various clinical features in multivariate analyses.
A total of 1290 patients (24.7%) had calcinosis. In univariate analyses, patients with calcinosis were older than patients without calcinosis, more likely to be female, and had longer disease duration from the first non-Raynaud phenomenon symptom. Patients with calcinosis were more likely to have digital ulcers, telangiectasias, acro-osteolysis, cardiac disease, pulmonary hypertension, gastrointestinal involvement, arthritis, and osteoporosis, but less likely to have muscle disease. Anti-Scl-70, RNA-polymerase-III, and U1-RNP autoantibodies were significantly less common in patients with calcinosis, while anticentromere (ACA), anti-PM/Scl, and anticardiolipin antibodies were more frequent. In multivariate analysis, the strongest associations with calcinosis were digital ulcers (OR = 3.9; 95% CI: 2.7-5.5; p < 0.0001) and osteoporosis (OR = 4.2; 95% CI: 2.3-7.9; p < 0.0001).
One quarter of patients with SSc have calcinosis at some time during their illness. Our data confirm a strong association of calcinosis with digital ulcers, and support a novel association with osteoporosis.

The U1A/U2B″/SNF family of small nuclear ribonucleoproteins uses a phylogenetically conserved RNA recognition motif (RRM1) to bind RNA stemloops in U1 and/or U2 small nuclear RNA (snRNA). RRMs are characterized by their α/β sandwich topology, and these RRMs use their β-sheet as the RNA binding surface. Unique to this RRM family is the tyrosine-glutamine-phenylalanine (YQF) triad of solvent-exposed residues that are displayed on the β-sheet surface; the aromatic residues form a platform for RNA nucleobases to stack. U1A, U2B″, and SNF have very different patterns of RNA binding affinity and specificity, however, so here we ask how YQF in Drosophila SNF RRM1 contributes to RNA binding, as well as to domain stability and dynamics. Thermodynamic double-mutant cycles using tyrosine and phenylalanine substitutions probe the communication between those two residues in the free and bound states of the RRM. NMR experiments follow corresponding changes in the glutamine side-chain amide in both U1A and SNF, providing a physical picture of the RRM1 β-sheet surface. NMR relaxation and dispersion experiments compare fast (picosecond to nanosecond) and intermediate (microsecond-to-millisecond) dynamics of U1A and SNF RRM1. We conclude that there is a network of amino acid interactions involving Tyr-Gln-Phe in both SNF and U1A RRM1, but whereas mutations of the Tyr-Gln-Phe triad result in small local responses in U1A, they produce extensive microsecond-to-millisecond global motions throughout SNF that alter the conformational states of the RRM.

OBJECTIVE: We assessed the association between serum autoantibodies against the 70-kDa polypeptide of the U1-ribonucleoprotein (RNP) complex (U1-70k) and the central nervous system (CNS) syndromes in systemic lupus erythematosus (SLE) patients.
METHODS: We studied 106 hospitalized patients with active SLE, comparing those with (n = 32) and without (n = 74) CNS syndromes. CNS syndromes were further classified into neurologic (n = 21) and psychiatric (n = 15) disorders. Immunoglobulin G (IgG) anti-U1-70k antibodies were measured by enzyme-linked immunosorbent assay (ELISA) using recombinant antigens. IgG antibodies against whole U1-RNP were measured using commercial ELISA kits.
RESULTS: Although there was no significant difference in the levels of serum anti-U1-70k antibodies in SLE patients with or without CNS syndromes (p = 0.83), the levels were significantly elevated in SLE patients compared with patients without psychiatric syndromes (p = 0.030). In contrast, no significant difference was observed in the levels of serum anti-U1-RNP antibodies in SLE patients with or without psychiatric syndromes (p = 0.555).
CONCLUSIONS: These results indicate that serum anti-U1-70k antibodies are associated with psychiatric syndromes in SLE but that they are not associated with CNS syndromes as a whole or with neurologic syndromes. The anti-U1-70k antibodies might be involved in the pathological mechanisms of psychiatric syndromes in SLE.