Patterns and predictors of skin score change in early diffuse systemic sclerosis from the European Scleroderma Observational Study.

OBJECTIVES: Our aim was to use the opportunity provided by the European Scleroderma Observational Study to (1) identify and describe those patients with early diffuse cutaneous systemic sclerosis (dcSSc) with progressive skin thickness, and (2) derive prediction models for progression over 12 months, to inform future randomised controlled trials (RCTs). METHODS: The modified Rodnan skin score (mRSS) was recorded every 3 months in 326 patients. 'Progressors' were defined as those experiencing a 5-unit and 25% increase in mRSS score over 12 months (±3 months). Logistic models were fitted to predict progression and, using receiver operating characteristic (ROC) curves, were compared on the basis of the area under curve (AUC), accuracy and positive predictive value (PPV). RESULTS: 66 patients (22.5%) progressed, 227 (77.5%) did not (33 could not have their status assessed due to insufficient data). Progressors had shorter disease duration (median 8.1 vs 12.6 months, P=0.001) and lower mRSS (median 19 vs 21 units, P=0.030) than non-progressors. Skin score was highest, and peaked earliest, in the anti-RNA polymerase III (Pol3+) subgroup (n=50). A first predictive model (including mRSS, duration of skin thickening and their interaction) had an accuracy of 60.9%, AUC of 0.666 and PPV of 33.8%. By adding a variable for Pol3 positivity, the model reached an accuracy of 71%, AUC of 0.711 and PPV of 41%. CONCLUSIONS: Two prediction models for progressive skin thickening were derived, for use both in clinical practice and for cohort enrichment in RCTs. These models will inform recruitment into the many clinical trials of dcSSc projected for the coming years. TRIAL REGISTRATION NUMBER: NCT02339441.

Imaging RNA polymerase III transcription using a photostable RNA-fluorophore complex.

Quantitative measurement of transcription rates in live cells is important for revealing mechanisms of transcriptional regulation. This is particularly challenging when measuring the activity of RNA polymerase III (Pol III), which transcribes growth-promoting small RNAs. To address this issue, we developed Corn, a genetically encoded fluorescent RNA reporter suitable for quantifying RNA transcription in cells. Corn binds and induces fluorescence of 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime, which resembles the fluorophore found in red fluorescent protein (RFP). Notably, Corn shows high photostability, enabling quantitative fluorescence imaging of mTOR-dependent Pol III transcription. We found that, unlike actinomycin D, mTOR inhibitors resulted in heterogeneous transcription suppression in individual cells. Quantitative imaging of Corn-tagged Pol III transcript levels revealed distinct Pol III transcription 'trajectories' elicited by mTOR inhibition. Together, these studies provide an approach for quantitative measurement of Pol III transcription by direct imaging of Pol III transcripts containing a photostable RNA-fluorophore complex.

RNA polymerase III autoantibodies may indicate renal and more severe skin involvement in systemic sclerosis.

BACKGROUND: Systemic sclerosis (SSc) is a multiorgan autoimmune disorder characterized by sclerosis of the skin and organs as well as the presence of antinuclear autoantibodies. Several types of antinuclear autoantibodies have been described in SSc, associated with distinct disease entities and differences in prognosis. METHODS: The aim of this study was to screen for the presence of antibodies reacting with RNA polymerase III (anti-RNAP3) in a large cohort of patients with SSc treated at a tertiary referral center and to evaluate correlations with disease severity. RESULTS: Anti-RNAP3 antibodies were detected in 11 of 158 patients (7.0%). Eight of the 11 (72.7%) anti-RNAP3-positive patients had diffuse cutaneous SSc (P < 0.01). A higher modified Rodnan skin score, associated with diffuse SSc, correlated with the presence of anti-RNAP3 (P < 0.0001). The detection of anti-RNAP3 antibodies strongly correlated with the presence of renal involvement (P < 0.0001). The odds ratio of RNAP3-positive patients to develop renal involvement was 80.1 (95% CI 9.3-690.1). CONCLUSIONS: This study demonstrates that the detection of anti-RNAP3 antibodies in patients with SSc correlates with renal crisis and severe cutaneous involvement. The possibility to detect specific antibodies with a prognostic value can lead to a better risk management of patients with SSc.

Rbs1, a new protein implicated in RNA polymerase III biogenesis in yeast Saccharomyces cerevisiae.

Little is known about the RNA polymerase III (Pol III) complex assembly and its transport to the nucleus. We demonstrate that a missense cold-sensitive mutation, rpc128-1007, in the sequence encoding the C-terminal part of the second largest Pol III subunit, C128, affects the assembly and stability of the enzyme. The cellular levels and nuclear concentration of selected Pol III subunits were decreased in rpc128-1007 cells, and the association between Pol III subunits as evaluated by coimmunoprecipitation was also reduced. To identify the proteins involved in Pol III assembly, we performed a genetic screen for suppressors of the rpc128-1007 mutation and selected the Rbs1 gene, whose overexpression enhanced de novo tRNA transcription in rpc128-1007 cells, which correlated with increased stability, nuclear concentration, and interaction of Pol III subunits. The rpc128-1007 rbs1Δ double mutant shows a synthetic growth defect, indicating that rpc128-1007 and rbs1Δ function in parallel ways to negatively regulate Pol III assembly. Rbs1 physically interacts with a subset of Pol III subunits, AC19, AC40, and ABC27/Rpb5. Additionally, Rbs1 interacts with the Crm1 exportin and shuttles between the cytoplasm and nucleus. We postulate that Rbs1 binds to the Pol III complex or subcomplex and facilitates its translocation to the nucleus.

Human RNA polymerase III transcriptomes and relationships to Pol II promoter chromatin and enhancer-binding factors.

RNA polymerase (Pol) III transcribes many noncoding RNAs (for example, transfer RNAs) important for translational capacity and other functions. We localized Pol III, alternative TFIIIB complexes (BRF1 or BRF2) and TFIIIC in HeLa cells to determine the Pol III transcriptome, define gene classes and reveal 'TFIIIC-only' sites. Pol III localization in other transformed and primary cell lines reveals previously uncharacterized and cell type-specific Pol III loci as well as one microRNA. Notably, only a fraction of the in silico-predicted Pol III loci are occupied. Many occupied Pol III genes reside within an annotated Pol II promoter. Outside of Pol II promoters, occupied Pol III genes overlap with enhancer-like chromatin and enhancer-binding proteins such as ETS1 and STAT1. Moreover, Pol III occupancy scales with the levels of nearby Pol II, active chromatin and CpG content. These results suggest that active chromatin gates Pol III accessibility to the genome.

Pol II and its associated epigenetic marks are present at Pol III-transcribed noncoding RNA genes.

Epigenetic control is an important aspect of gene regulation. Despite detailed understanding of protein-coding gene expression, the transcription of noncoding RNA genes by RNA polymerase III (Pol III) is less well characterized. Here we profile the epigenetic features of Pol III target genes throughout the human genome. This reveals that the chromatin landscape of Pol III-transcribed genes resembles that of Pol II templates in many ways, although there are also clear differences. Our analysis also uncovered an entirely unexpected phenomenon: namely, that Pol II is present at the majority of genomic loci that are bound by Pol III.

Active site labeling of the RNA polymerases A, B, and C from yeast.

RNA polymerases A, B, and C from yeast were modified by reaction with 4-formylphenyl-gamma-ester of ATP as priming nucleotide followed by reduction with NaBH4. Upon phosphodiester bond formation with [alpha-32P]UTP, only the second largest subunit, A135, B150, or C128, was labeled in a template-dependent reaction. This indicates that these polypeptide chains are functionally homologous. The product covalently bound to B150 subunit was found to consist of a mixture of ApU and a trinucleotide. Enzyme labeling exhibited the characteristic alpha-amanitin sensitivity reported for A and B RNA polymerases. Labeling of both large subunits of enzyme A and B but not of any of the smaller subunits was observed when the reduction step stabilizing the binding of the priming nucleotide was carried out after limited chain elongation. These results illustrate the conservative evolution of the active site of eukaryotic RNA polymerases.

Yeast RNA polymerase C and its subunits. Specific antibodies as structural and functional probes.

Yeast RNA polymerase C purified by a simple large scale method was resolved into multiple components by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Specific antibodies directed against each polypeptide chain were prepared in rabbits and used as structural and functional probes. With minor exceptions, each antibody recognized specifically the corresponding polypeptide by blot-immunodetection. Cross-reactions with purified RNA polymerases A and B confirmed our previous description of the subunits shared by the three nuclear RNA polymerases. Immunoadsorption of RNA polymerase C at different stages of purification using antibodies to subunits C160 and C128 yielded the same collection of polypeptides as found in the purified enzyme: C160, C128, C82, C53, C40, C37, C34, C31, C27, C25, C23, C19, C14.5, C12.5, and C10. Subunit-specific antibodies were used to probe the activity of RNA polymerase C in a specific, reconstituted transcription system as well as on a nonspecific template. Transcription of the tRNAGlu3 gene in vitro was inhibited when RNA polymerase C was preincubated with antibodies directed to C128, C82, C53, C34, C23, or C19. Antibodies to C82, C53, and C34 were much less inhibitory in the nonspecific assay. Inhibition by anti-C128 or anti-C23 was relieved by preincubation of enzyme C with plasmid DNA prior to antibody addition. These results are discussed in terms of the participation of these polypeptides to the active enzyme molecule, and of their possible role in DNA binding or transcription factor recognition.

Nucleotide sequence of the 5S ribosomal RNA gene repeat of Trypanosoma brucei.

The 750 base pair tandem repeat specifying the 5S ribosomal RNA of Trypanosoma brucei brucei has been cloned and four independent clones sequenced to completion. The repeat specifies only a single 5S rRNA and is unusually long when compared with other 5S gene repeats. The sequenced copies contain a number of sequence polymorphisms similar to those seen in the ribosomal genes of other organisms. By comparison with the consensus sequence for RNA polymerase III promoters ('internal control regions') a possible 'internal control region' for RNA polymerase III can be located in the predicted position within the 5S ribosomal RNA sequence.