Tandem donor splice sites (5'ss) are unique regions with at least two GU dinucleotides serving as splicing cleavage sites. The Δ3 tandem 5'ss are a specific subclass of 5'ss separated by 3 nucleotides which can affect protein function by inserting/deleting a single amino acid. One 5'ss is typically preferred, yet factors governing particular 5'ss choice are not fully understood. A highly conserved exon 21 of the STAT3 gene was chosen as a model to study Δ3 tandem 5'ss splicing mechanisms. Based on multiple lines of experimental evidence, endogenous U1 snRNA most likely binds only to the upstream 5'ss. However, the downstream 5'ss is used preferentially, and the splice site choice is not dependent on the exact U1 snRNA binding position. Downstream 5'ss usage was sensitive to exact nucleotide composition and dependent on the presence of downstream regulatory region. The downstream 5'ss usage could be best explained by two novel interactions with endogenous U6 snRNA. U6 snRNA enables the downstream 5'ss usage in STAT3 exon 21 by two mechanisms: (i) binding in a novel non-canonical register and (ii) establishing extended Watson-Crick base pairing with the downstream regulatory region. This study suggests that U6:5'ss interaction is more flexible than previously thought.

• MeSH
• Exons * MeSH
• HeLa Cells MeSH
• Humans MeSH
• RNA Splice Sites * MeSH
• RNA, Small Nuclear * metabolism   genetics   MeSH
• Base Sequence MeSH
• RNA Splicing MeSH
• STAT3 Transcription Factor * metabolism   genetics   MeSH
• Protein Binding MeSH
• Binding Sites genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

• MeSH
• Periodicals as Topic MeSH
• General Practice MeSH
• Societies, Medical MeSH
• Publication type
• Personal Narrative MeSH

• About
• Hotová, Marta, 1947- Authority

Kniha představuje průřez životního díla francouzské historičky a bohemistky Marie-Élizabeth Ducreux. Výbor textů si klade za cíl umožnit českému čtenáři plně pochopit rozsah badatelských zájmů Marie-Élizabeth Ducreux i způsob a metodu jejího přemýšlení o české historii důsledně zasazené ve středoevropském kontextu, neboť je sice historičkou, která zná příslušné prameny a dobovou literaturu středoevropské provenience hlouběji než mnozí "domácí" autoři, ale současně jako Francouzka dokáže nahlížet zdejší dějiny "zvenčí", bez zátěže našich traumat a stereotypů.; Kniha představuje průřez životního díla francouzské historičky a bohemistky Marie-Élizabeth Ducreux.Výbor textů si klade za cíl umožnit českému čtenáři plně pochopit rozsah badatelských zájmů Marie-Élizabeth Ducreux i způsob a metodu jejího přemýšlení o české historii důsledně zasazené ve středoevropském kontextu, neboť je sice historičkou, která zná příslušné prameny a dobovou literaturu středoevropské provenience hlouběji než mnozí „domácí“ autoři, ale současně jako Francouzka dokáže nahlížet zdejší dějiny „zvenčí“, bez zátěže našich traumat a stereotypů.

Hereditary angioedema (HAE) is a rare genetic disorder with variable expressivity even in carriers of the same underlying genetic defect, suggesting other genetic and epigenetic factors participate in modifying HAE severity. Recent knowledge indicates the role of immune cells in several aspects of HAE pathogenesis, which makes monocytes and macrophages candidates to mediate these effects. Here we combined a search for HAE phenotype modifying gene variants with the characterization of selected genes' mRNA levels in monocyte and macrophages in a symptom-free period. While no such gene variant was found to be associated with a more severe or milder disease, patients revealed a higher number of dysregulated genes and their expression profile was significantly altered, which was typically manifested by changes in individual gene expression or by strengthened or weakened relations in mutually co-expressed gene groups, depending on HAE severity. SERPING1 showed decreased expression in HAE-C1INH patients, but this effect was significant only in patients carrying mutations supposedly activating nonsense-mediated decay. Pro-inflammatory CXC chemokine superfamily members CXCL8, 10 and 11 were downregulated, while other genes such as FCGR1A, or long non-coding RNA NEAT1 were upregulated in patients. Co-expression within some gene groups (such as an NF-kappaB function related group) was strengthened in patients with a severe and/or mild course compared to controls. All these findings show that transcript levels in myeloid cells achieve different activation or depression levels in HAE-C1INH patients than in healthy controls and/or based on disease severity and could participate in determining the HAE phenotype.

• Publication type
• Journal Article MeSH

BACKGROUND: The PLAUR gene encodes the urokinase-like plasminogen activator receptor (uPAR) and may undergo alternative splicing. Excluding cassette exons 3, 5 and 6 from the transcript results in truncated protein variants whose precise functions have not been elucidated yet. The PLAUR gene is one of several expressed in myeloid cells, where uPAR participates in different cellular processes, including the contact activation system and kallikrein-kinin system, which play an important role in hereditary angioedema (HAE) pathogenesis. A hypothesis about the PLAUR splicing pattern impact on HAE severity was tested. METHODS AND RESULTS: The RT-PCR quantified by capillary electrophoresis was used. Although no significant difference in alternative transcript frequency was observed between healthy volunteers and HAE patients, a significant increase in all cassette exon inclusion variants was revealed during monocyte-to-macrophage differentiation. CONCLUSIONS: PLAUR alternative splicing in monocytes and macrophages neither was different between HAE patients and healthy controls, nor reflected disease severity. However, the results showed an PLAUR splicing pattern was changing during monocyte-to-macrophage differentiation, but the significance of these changes is unknown and awaits future clarification.

• MeSH
• Alternative Splicing genetics   MeSH
• Angioedemas, Hereditary * genetics   pathology   MeSH
• Leukocytes MeSH
• Humans MeSH
• Macrophages pathology   MeSH
• Monocytes * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Among alternative splicing events in the human transcriptome, tandem NAGNAG acceptor splice sites represent an appreciable proportion. Both proximal and distal NAG can be used to produce two splicing isoforms differing by three nucleotides. In some cases, the upstream exon can be alternatively spliced as well, which further increases the number of possible transcripts. In this study, we showed that NAG choice in tandem splice site depends considerably not only on the concerned acceptor, but also on the upstream donor splice site sequence. Using an extensive set of experiments with systematically modified two-exonic minigene systems of AFAP1L2 or CSTD gene, we recognized the third and fifth intronic upstream donor splice site position and the tandem acceptor splice site region spanning from -10 to +2, including NAGNAG itself, as the main drivers. In addition, competition between different branch points and their composition were also shown to play a significant role in NAG choice. All these nucleotide effects appeared almost additive, which explained the high variability in proximal versus distal NAG usage.

• MeSH
• Alternative Splicing genetics   MeSH
• Exons genetics   MeSH
• HeLa Cells MeSH
• Introns genetics   MeSH
• Humans MeSH
• RNA Splice Sites genetics   MeSH
• Cell Line, Tumor MeSH
• Nucleotides genetics   MeSH
• Tandem Repeat Sequences genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Acceptor splice site recognition (3' splice site: 3'ss) is a fundamental step in precursor messenger RNA (pre-mRNA) splicing. Generally, the U2 small nuclear ribonucleoprotein (snRNP) auxiliary factor (U2AF) heterodimer recognizes the 3'ss, of which U2AF35 has a dual function: (i) It binds to the intron-exon border of some 3'ss and (ii) mediates enhancer-binding splicing activators' interactions with the spliceosome. Alternative mechanisms for 3'ss recognition have been suggested, yet they are still not thoroughly understood. Here, we analyzed 3'ss recognition where the intron-exon border is bound by a ubiquitous splicing regulator SRSF1. Using the minigene analysis of two model exons and their mutants, BRCA2 exon 12 and VARS2 exon 17, we showed that the exon inclusion correlated much better with the predicted SRSF1 affinity than 3'ss quality, which were assessed using the Catalog of Inferred Sequence Binding Preferences of RNA binding proteins (CISBP-RNA) database and maximum entropy algorithm (MaxEnt) predictor and the U2AF35 consensus matrix, respectively. RNA affinity purification proved SRSF1 binding to the model 3'ss. On the other hand, knockdown experiments revealed that U2AF35 also plays a role in these exons' inclusion. Most probably, both factors stochastically bind the 3'ss, supporting exon recognition, more apparently in VARS2 exon 17. Identifying splicing activators as 3'ss recognition factors is crucial for both a basic understanding of splicing regulation and human genetic diagnostics when assessing variants' effects on splicing.

• MeSH
• Alternative Splicing genetics   MeSH
• Exons genetics   MeSH
• HeLa Cells MeSH
• Introns genetics   MeSH
• Humans MeSH
• RNA Splice Sites genetics   physiology   MeSH
• RNA-Binding Proteins metabolism   MeSH
• Regulatory Sequences, Nucleic Acid genetics   MeSH
• Base Sequence genetics   MeSH
• Serine-Arginine Splicing Factors metabolism   MeSH
• RNA Splicing physiology   MeSH
• RNA Splicing Factors metabolism   physiology   MeSH
• Splicing Factor U2AF metabolism   MeSH
• Spliceosomes metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

PURPOSE: Hereditary angioedema (HAE) is a rare autosomal dominant life-threatening disease characterized by low levels of C1 inhibitor (type I HAE) or normal levels of ineffective C1 inhibitor (type II HAE), typically occurring as a consequence of a SERPING1 mutation. In some cases, a causal mutation remains undetected after using a standard molecular genetic analysis. RESULTS: Here we show a long methodological way to the final discovery of c.1029 + 384A > G, a novel deep intronic mutation in intron 6 which is responsible for HAE type I in a large family and has not been identified by a conventional diagnostic approach. This mutation results in de novo donor splice site creation and subsequent pseudoexon inclusion, the mechanism firstly described to occur in SERPING1 in this study. We additionally discovered that the proximal part of intron 6 is a region potentially prone to pseudoexon-activating mutations, since natural alternative exons and additional cryptic sites occur therein. Indeed, we confirmed the existence of at least two different alternative exons in this region not described previously. CONCLUSIONS: In conclusion, our results suggest that detecting aberrant transcripts, which are often low abundant because of nonsense-mediated decay, requires a modified methodological approach. We suggest SERPING1 intron 6 sequencing and/or tailored mRNA analysis to be routinely used in HAE patients with no mutation identified in the coding sequence.

• MeSH
• Child MeSH
• Adult MeSH
• Exons genetics   MeSH
• Hereditary Angioedema Types I and II genetics   MeSH
• Complement C1 Inhibitor Protein genetics   MeSH
• Introns genetics   MeSH
• Middle Aged MeSH
• Humans MeSH
• RNA Splice Sites genetics   MeSH
• Adolescent MeSH
• Young Adult MeSH
• Mutation genetics   MeSH
• DNA Mutational Analysis methods   MeSH
• Pedigree MeSH
• Aged MeSH
• Protein Splicing genetics   MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Common variable immunodeficiency disorders (CVID) represent a group of primary immunodeficiency diseases characterized by hypogammaglobulinemia and impaired specific Ab response, resulting in recurrent infections due to dysfunctional immune response. The specific mechanisms mediating immune deficiency in CVID remain to be determined. Previous studies indicated that immune dysregulation in CVID patients is associated with chronic microbial translocation, systemic immune activation, and altered homeostasis of lymphocytic and myeloid lineages. A detailed phenotypic, functional characterization of plasma markers and immune cell populations was performed in 46 CVID patients and 44 healthy donors. CVID patients displayed significantly elevated plasma levels of a marker of neutrophil activation neutrophil gelatinase-associated lipocalin. Neutrophils from CVID patients exhibited elevated surface levels of CD11b and PD-L1 and decreased levels of CD62L, CD16, and CD80, consistent with a phenotype of activated neutrophils with suppressive properties. Neutrophils from CVID patients actively suppressed T cell activation and release of IFN-γ via the production of reactive oxygen species. Furthermore, CVID was associated with an increased frequency of low-density neutrophils (LDNs)/granulocytic myeloid-derived suppressor cells. LDN/granulocytic myeloid-derived suppressor cell frequency in CVID patients correlated with reduced T cell responsiveness. Exogenous stimulation of whole blood with bacterial LPS emulated some but not all of the phenotypic changes observed on neutrophils from CVID patients and induced neutrophil population with LDN phenotype. The presented data demonstrate that neutrophils in the blood of CVID patients acquire an activated phenotype and exert potent T cell suppressive activity. Specific targeting of myeloid cell-derived suppressor activity represents a novel potential therapeutic strategy for CVID.

• MeSH
• Neutrophil Activation MeSH
• CD11b Antigen metabolism   MeSH
• B7-H1 Antigen metabolism   MeSH
• Common Variable Immunodeficiency immunology   MeSH
• Adult MeSH
• Granulocytes physiology   MeSH
• Immune Tolerance MeSH
• Cells, Cultured MeSH
• Middle Aged MeSH
• Humans MeSH
• Lipocalin-2 blood   MeSH
• Young Adult MeSH
• Myeloid-Derived Suppressor Cells physiology   MeSH
• Neutrophils physiology   MeSH
• Reactive Oxygen Species metabolism   MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• T-Lymphocytes immunology   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Male MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Background: Complex cardiovascular procedures may initiate a systemic inflammatory response syndrome (SIRS) with a massive cytokine release, which is involved in postoperative myocardial injury. Intraoperative cytokine hemoadsorption (HA) mitigates the inflammatory response. Micro ribonucleic acids (miRNAs) are emerging as a marker of myocardial injury. Methods: This study evaluated if intraoperative cytokine reduction by HA modulates SIRS and affects myocardial injury as measured by miRNA-126, 223 and miRNA-1, 133a, respectively. Twenty-eight patients were assigned into HA (n = 15) and control (C) (n = 13) groups. HA was performed by integrating CytoSorb™ into the extracorporeal circuit. Results: MiRNA-133a plasma levels were increased postoperatively in both groups but were much higher in the HA group than in the C group at 3 h (P = 0.037) and 18 h (P = 0.017) after reperfusion. MiRNA-1 and miRNA-223 plasma levels were significantly increased postoperatively, but did not differ between groups. The vascular miRNA-126 was not affected. Conclusion: Intraoperative cytokine HA in cardiovascular operations increased the plasma levels of miRNA-133a, suggesting higher myocardial injury.

• Publication type
• Journal Article MeSH