scholarly journals The Experimentally Obtained Functional Impact Assessments of 5' Splice Site GT>GC Variants Differ Markedly from Those Predicted

2020 ◽  
Vol 21 (1) ◽  
pp. 56-66 ◽  
Author(s):  
Jian-Min Chen ◽  
Jin-Huan Lin ◽  
Emmanuelle Masson ◽  
Zhuan Liao ◽  
Claude Férec ◽  
...  
Keyword(s):  
Splice Site ◽  
Disease Genes ◽  
Wild Type ◽  
Human Genetic Disease ◽  
Functional Impact ◽  
Prediction Tools ◽  
Human Disease Genes ◽  
The Impact

Introduction: 5' splice site GT>GC or +2T>C variants have been frequently reported to cause human genetic disease and are routinely scored as pathogenic splicing mutations. However, we have recently demonstrated that such variants in human disease genes may not invariably be pathogenic. Moreover, we found that no splicing prediction tools appear to be capable of reliably distinguishing those +2T>C variants that generate wild-type transcripts from those that do not. Methodology: Herein, we evaluated the performance of a novel deep learning-based tool, SpliceAI, in the context of three datasets of +2T>C variants, all of which had been characterized functionally in terms of their impact on pre-mRNA splicing. The first two datasets refer to our recently described “in vivo” dataset of 45 known disease-causing +2T>C variants and the “in vitro” dataset of 103 +2T>C substitutions subjected to full-length gene splicing assay. The third dataset comprised 12 BRCA1 +2T>C variants that were recently analyzed by saturation genome editing. Results: Comparison of the SpliceAI-predicted and experimentally obtained functional impact assessments of these variants (and smaller datasets of +2T>A and +2T>G variants) revealed that although SpliceAI performed rather better than other prediction tools, it was still far from perfect. A key issue was that the impact of those +2T>C (and +2T>A) variants that generated wild-type transcripts represents a quantitative change that can vary from barely detectable to an almost full expression of wild-type transcripts, with wild-type transcripts often co-existing with aberrantly spliced transcripts. Conclusion: Our findings highlight the challenges that we still face in attempting to accurately identify splice-altering variants.

scholarly journals The experimentally obtained functional impact assessments of GT>GC 5’ splice site variants differ markedly from those predicted

2019 ◽  
Author(s):  
Jian-Min Chen ◽  
Jin-Huan Lin ◽  
Emmanuelle Masson ◽  
Zhuan Liao ◽  
Claude Férec ◽  
...  
Keyword(s):  
Splice Site ◽  
Disease Genes ◽  
Wild Type ◽  
Human Genetic Disease ◽  
Functional Impact ◽  
Prediction Tools ◽  
Human Disease Genes ◽  
The Impact

ABSTRACTGT>GC 5’ splice site (or +2T>C) variants have been frequently reported to cause human genetic disease. However, although we have demonstrated that GT>GC variants in human disease genes may not invariably be pathogenic, none of the currently available splicing prediction tools appear to be capable of reliably distinguishing those GT>GC variants that generate wild-type transcripts from those that do not. Recently, SpliceAI, a novel deep residual neural network tool, has been developed for splicing prediction. Methodologically distinct from previous approaches that either rely on human-engineered features and/or which focus on short nucleotide windows adjoining exon-intron boundaries, SpliceAI assesses splicing determinants by evaluating 10,000 nucleotides of flanking contextual sequence to predict the functional role in splicing of each position in the pre-mRNA transcript. Herein, we evaluated the performance of SpliceAI in the context of three datasets of GT>GC variants, all of which had been characterized functionally in terms of their impact on mRNA splicing. The first two datasets refer to our recently described “in vivo” dataset of 45 disease-causing GT>GC variants and the “in vitro” dataset of 103 GT>GC substitutions. The third dataset comprised 12 BRCA1 GT>GC variants that were recently analyzed by saturation genome editing. We processed all GT>GC variants using the default settings of SpliceAI. Comparison of the SpliceAI-predicted and experimentally obtained functional impact assessments of the analyzed GT>GC variants revealed that although SpliceAI performed rather better than other prediction tools, it was still far from perfect. A key issue is that the impact of GT>GC (as well as GT>GA or +2T>A) variants that generated wild-type transcripts represents a quantitative change that can vary from barely detectable to almost full expression of wild-type transcripts, with wild-type transcripts often co-existing with aberrantly spliced transcripts. Our findings highlight the challenges that we still face in attempting to accurately identify splice-altering variants.

scholarly journals Production of Noncapped Genomic RNAs Is Critical to Sindbis Virus Disease and Pathogenicity

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
Author(s):  
Autumn T. LaPointe ◽  
V Douglas Landers ◽  
Claire E. Westcott ◽  
Kevin J. Sokoloski
Keyword(s):  
Sindbis Virus ◽  
Virus Disease ◽  
Severe Disease ◽  
Wild Type Virus ◽  
Wild Type ◽  
Genomic Rnas ◽  
Mortality And Morbidity ◽  
The Impact

ABSTRACT Alphaviruses are positive-sense RNA viruses that utilize a 5′ cap structure to facilitate translation of viral proteins and to protect the viral RNA genome. Nonetheless, significant quantities of viral genomic RNAs that lack a canonical 5′ cap structure are produced during alphaviral replication and packaged into viral particles. However, the role/impact of the noncapped genomic RNA (ncgRNA) during alphaviral infection in vivo has yet to be characterized. To determine the importance of the ncgRNA in vivo, the previously described D355A and N376A nsP1 mutations, which increase or decrease nsP1 capping activity, respectively, were incorporated into the neurovirulent AR86 strain of Sindbis virus to enable characterization of the impact of altered capping efficiency in a murine model of infection. Mice infected with the N376A nsP1 mutant exhibited slightly decreased rates of mortality and delayed weight loss and neurological symptoms, although levels of inflammation in the brain were similar to those of wild-type infection. Although the D355A mutation resulted in decreased antiviral gene expression and increased resistance to interferon in vitro, mice infected with the D355A mutant showed significantly reduced mortality and morbidity compared to mice infected with wild-type virus. Interestingly, expression of proinflammatory cytokines was found to be significantly decreased in mice infected with the D355A mutant, suggesting that capping efficiency and the production of ncgRNA are vital to eliciting pathogenic levels of inflammation. Collectively, these data indicate that the ncgRNA have important roles during alphaviral infection and suggest a novel mechanism by which noncapped viral RNAs aid in viral pathogenesis. IMPORTANCE Mosquito-transmitted alphaviruses have been the cause of widespread outbreaks of disease that can range from mild illness to lethal encephalitis or severe polyarthritis. There are currently no safe and effective vaccines or therapeutics with which to prevent or treat alphaviral disease, highlighting the need to better understand alphaviral pathogenesis to develop novel antiviral strategies. This report reveals production of noncapped genomic RNAs (ncgRNAs) to be a novel determinant of alphaviral virulence and offers insight into the importance of inflammation to pathogenesis. Taken together, the findings reported here suggest that the ncgRNAs contribute to alphaviral pathogenesis through the sensing of the ncgRNAs during alphaviral infection and are necessary for the development of severe disease.

scholarly journals Identification of a Bidirectional Splicing Enhancer: Differential Involvement of SR Proteins in 5′ or 3′ Splice Site Activation

1999 ◽  
Vol 19 (11) ◽  
pp. 7347-7356 ◽  
Author(s):  
Cyril F. Bourgeois ◽  
Michel Popielarz ◽  
Georges Hildwein ◽  
James Stevenin
Keyword(s):  
Splice Site ◽  
Sr Proteins ◽  
Dependent Manner ◽  
Wild Type ◽  
Adenovirus E1a ◽  
Differential Involvement ◽  
Splicing Enhancer

ABSTRACT The adenovirus E1A pre-mRNA undergoes alternative splicing whose modulation occurs during infection, through the use of three different 5′ splice sites and of one major or one minor 3′ splice site. Although this pre-mRNA has been extensively used as a model to compare the transactivation properties of SR proteins, no cis-acting element has been identified in the transcript sequence. Here we describe the identification and the characterization of a purine-rich splicing enhancer, located just upstream of the 12S 5′ splice site, which is formed from two contiguous 9-nucleotide (nt) purine motifs (Pu1 and Pu2). We demonstrate that this sequence is a bidirectional splicing enhancer (BSE) in vivo and in vitro, because it activates both the downstream 12S 5′ splice site through the Pu1 motif and the upstream 216-nt intervening sequence (IVS) 3′ splice site through both motifs. UV cross-linking and immunoprecipitation experiments indicate that the BSE interacts with several SR proteins specifically, among them 9G8 and ASF/SF2, which bind preferentially to the Pu1 and Pu2 motifs, respectively. Interestingly, we show by in vitro complementation assays that SR proteins have distinct transactivatory properties. In particular, 9G8, but not ASF/SF2 or SC35, is able to strongly activate the recognition of the 12S 5′ splice site in a BSE-dependent manner in wild-type E1A or in a heterologous context, whereas ASF/SF2 or SC35, but not 9G8, activates the upstream 216-nt IVS splicing. Thus, our results identify a novel exonic BSE and the SR proteins which are involved in its differential activity.

scholarly journals Different SUMO paralogues determine the fate of wild-type and mutant CFTRs: biogenesis versus degradation

2019 ◽  
Vol 30 (1) ◽  
pp. 4-16 ◽  
Author(s):  
Xiaoyan Gong ◽  
Yong Liao ◽  
Annette Ahner ◽  
Mads Breum Larsen ◽  
Xiaohui Wang ◽  
...  
Keyword(s):  
Physiological Role ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Specific Effects ◽  
Ubiquitin Conjugation ◽  
Protein Array Analysis ◽  
The Impact

A pathway for cystic fibrosis transmembrane conductance regulator (CFTR) degradation is initiated by Hsp27, which cooperates with Ubc9 and binds to the common F508del mutant to modify it with SUMO-2/3. These SUMO paralogues form polychains, which are recognized by the ubiquitin ligase, RNF4, for proteosomal degradation. Here, protein array analysis identified the SUMO E3, protein inhibitor of activated STAT 4 (PIAS4), which increased wild-type (WT) and F508del CFTR biogenesis in CFBE airway cells. PIAS4 increased immature CFTR threefold and doubled expression of mature CFTR, detected by biochemical and functional assays. In cycloheximide chase assays, PIAS4 slowed immature F508del degradation threefold and stabilized mature WT CFTR at the plasma membrance. PIAS4 knockdown reduced WT and F508del CFTR expression by 40–50%, suggesting a physiological role in CFTR biogenesis. PIAS4 modified F508del CFTR with SUMO-1 in vivo and reduced its conjugation to SUMO-2/3. These SUMO paralogue-specific effects of PIAS4 were reproduced in vitro using purified F508del nucleotide-binding domain 1 and SUMOylation reaction components. PIAS4 reduced endogenous ubiquitin conjugation to F508del CFTR by ∼50% and blocked the impact of RNF4 on mutant CFTR disposal. These findings indicate that different SUMO paralogues determine the fates of WT and mutant CFTRs, and they suggest that a paralogue switch during biogenesis can direct these proteins to different outcomes: biogenesis versus degradation.

scholarly journals FSMP-07. CYSTATHIONINE-Γ-LYASE DRIVES ANTIOXIDANT DEFENSE IN CYSTEINE-RESTRICTED IDH1 MUTANT ASTROCYTOMAS

2020 ◽  
Vol 3 (Supplement_1) ◽  
pp. i17-i17
Author(s):  
Andrés Cano-Galiano ◽  
Anais Oudin ◽  
Fred Fack ◽  
Maria-Francesca Allega ◽  
David Sumpton ◽  
...  
Keyword(s):  
Antioxidant Defense ◽  
De Novo ◽  
Idh1 Mutation ◽  
Wild Type ◽  
Isocitrate Dehydrogenase 1 ◽  
Astrocytic Gliomas ◽  
The Impact ◽  
Reducing Potential

Abstract Mutations in isocitrate dehydrogenase 1 or 2 (IDH1/2) define glioma subtypes and are considered primary events in gliomagenesis, impacting tumor epigenetics and metabolism. IDH enzymes are crucial for the generation of reducing potential, yet the impact of the mutation on the cellular antioxidant system is not understood. Here, we investigate how glutathione (GSH) levels are maintained in IDH1 mutant gliomas, despite an altered NADPH/NADP balance. We find that IDH1 mutant astrocytomas specifically upregulate cystathionine γ-lyase (CSE), the enzyme responsible for cysteine production upstream of GSH biosynthesis. Genetic and chemical interference with CSE in patient-derived glioma cells carrying the endogenous IDH1 mutation, sensitized tumor cells to cysteine depletion, an effect not observed in IDH1 wild-type gliomas. This correlated with reduced GSH synthesis as shown by in vitro and in vivo serine tracing and led to delayed tumor growth in mice. Thus we show that IDH1 mutant astrocytic gliomas critically rely on NADPH-independent de novo GSH synthesis to maintain the antioxidant defense, which uncovers a novel metabolic vulnerability in this dismal disease.

scholarly journals Overexpression of Wild-Type Aspartokinase Increases l-Lysine Production in the Thermotolerant Methylotrophic Bacterium Bacillus methanolicus

2008 ◽  
Vol 75 (3) ◽  
pp. 652-661 ◽  
Author(s):  
�yvind M. Jakobsen ◽  
Trygve Brautaset ◽  
Kristin F. Degnes ◽  
Tonje M. B. Heggeset ◽  
Simone Balzer ◽  
...  
Keyword(s):  
Amino Acids ◽  
Methylotrophic Bacterium ◽  
Wild Type ◽  
Lysine Production ◽  
Bacillus Methanolicus ◽  
Semialdehyde Dehydrogenase ◽  
Aspartate Pathway ◽  
The Impact

ABSTRACT Aspartokinase (AK) controls the carbon flow into the aspartate pathway for the biosynthesis of the amino acids l-methionine, l-threonine, l-isoleucine, and l-lysine. We report here the cloning of four genes (asd, encoding aspartate semialdehyde dehydrogenase; dapA, encoding dihydrodipicolinate synthase; dapG, encoding AKI; and yclM, encoding AKIII) of the aspartate pathway in Bacillus methanolicus MGA3. Together with the known AKII gene lysC, dapG and yclM form a set of three AK genes in this organism. Overexpression of dapG, lysC, and yclM increased l-lysine production in wild-type B. methanolicus strain MGA3 2-, 10-, and 60-fold (corresponding to 11 g/liter), respectively, without negatively affecting the specific growth rate. The production levels of l-methionine (less than 0.5 g/liter) and l-threonine (less than 0.1 g/liter) were low in all recombinant strains. The AK proteins were purified, and biochemical analyses demonstrated that they have similar V max values (between 47 and 58 μmol/min/mg protein) and Km values for l-aspartate (between 1.9 and 5.0 mM). AKI and AKII were allosterically inhibited by meso-diaminopimelate (50% inhibitory concentration [IC50], 0.1 mM) and by l-lysine (IC50, 0.3 mM), respectively. AKIII was inhibited by l-threonine (IC50, 4 mM) and by l-lysine (IC50, 5 mM), and this enzyme was synergistically inhibited in the presence of both of these amino acids at low concentrations. The correlation between the impact on l-lysine production in vivo and the biochemical properties in vitro of the individual AK proteins is discussed. This is the first example of improving l-lysine production by metabolic engineering of B. methanolicus and also the first documentation of considerably increasing l-lysine production by overexpression of a wild-type AK.

scholarly journals CXCR4 and CXCR7 Inhibition Ameliorates the Formation of Platelet–Neutrophil Complexes and Neutrophil Extracellular Traps through Adora2b Signaling

2021 ◽  
Vol 22 (24) ◽  
pp. 13576
Author(s):  
Kristian-Christos Ngamsri ◽  
Rizki A. Putri ◽  
Christoph Jans ◽  
Katharina Schindler ◽  
Anika Fuhr ◽  
...  
Keyword(s):  
Chemokine Receptors ◽  
Acute Inflammation ◽  
Neutrophil Extracellular Traps ◽  
Polymorphonuclear Neutrophils ◽  
Protective Effects ◽  
Wild Type ◽  
Extracellular Traps ◽  
The Impact

Peritonitis and peritonitis-associated sepsis are characterized by an increased formation of platelet–neutrophil complexes (PNCs), which contribute to an excessive migration of polymorphonuclear neutrophils (PMN) into the inflamed tissue. An important neutrophilic mechanism to capture and kill invading pathogens is the formation of neutrophil extracellular traps (NETs). Formation of PNCs and NETs are essential to eliminate pathogens, but also lead to aggravated tissue damage. The chemokine receptors CXCR4 and CXCR7 on platelets and PMNs have been shown to play a pivotal role in inflammation. Thereby, CXCR4 and CXCR7 were linked with functional adenosine A2B receptor (Adora2b) signaling. We evaluated the effects of selective CXCR4 and CXCR7 inhibition on PNCs and NETs in zymosan- and fecal-induced sepsis. We determined the formation of PNCs in the blood and, in addition, their infiltration into various organs in wild-type and Adora2b−/− mice by flow cytometry and histological methods. Further, we evaluated NET formation in both mouse lines and the impact of Adora2b signaling on it. We hypothesized that the protective effects of CXCR4 and CXCR7 antagonism on PNC and NET formation are linked with Adora2b signaling. We observed an elevated CXCR4 and CXCR7 expression in circulating platelets and PMNs during acute inflammation. Specific CXCR4 and CXCR7 inhibition reduced PNC formation in the blood, respectively, in the peritoneal, lung, and liver tissue in wild-type mice, while no protective anti-inflammatory effects were observed in Adora2b−/− animals. In vitro, CXCR4 and CXCR7 antagonism dampened PNC and NET formation with human platelets and PMNs, confirming our in vivo data. In conclusion, our study reveals new protective aspects of the pharmacological modulation of CXCR4 and CXCR7 on PNC and NET formation during acute inflammation.

scholarly journals Mycobacterium tuberculosis embBCodon 306 Mutations Confer Moderately Increased Resistance to Ethambutol In Vitro and In Vivo

2011 ◽  
Vol 55 (6) ◽  
pp. 2891-2896 ◽  
Author(s):  
Claudia Plinke ◽  
Kerstin Walter ◽  
Sahar Aly ◽  
Stefan Ehlers ◽  
Stefan Niemann
Keyword(s):  
Resistance Mechanism ◽  
Point Mutations ◽  
Fold Increase ◽  
Wild Type ◽  
Content Type ◽  
First Line Therapy ◽  
The One ◽  
The Impact

ABSTRACTEthambutol (EMB) is a major component of the first-line therapy of tuberculosis. Mutations in codon 306 ofembB(embB306) were suggested as a major resistance mechanism in clinical isolates. To directly analyze the impact of individualembB306 mutations on EMB resistance, we used allelic exchange experiments to generateembB306 mutants ofM. tuberculosisH37Rv. The level of EMB resistance conferred by particular mutations was measuredin vitroandin vivoafter EMB therapy by daily gavage in a mouse model of aerogenic tuberculosis. The wild-typeembB306 ATG codon was replaced byembB306 ATC, ATA, or GTG, respectively. All of the obtainedembB306 mutants exhibited a 2- to 4-fold increase in EMB MIC compared to the wild-type H37Rv.In vivo, the one selectedembB306 GTG mutant required a higher dose of ethambutol to restrict its growth in the lung compared to wild-type H37Rv. These experiments demonstrate thatembB306 point mutations enhance the EMB MICin vitroto a moderate, but significant extent, and reduce the efficacy of EMB treatment in the animal model. We propose that conventional EMB susceptibility testing, in combination withembB306 genotyping, may guide dose adjustment to avoid clinical treatment failure in these low-level resistant strains.

scholarly journals Evaluation of Regulated Delayed Attenuation Strategies for Salmonella enterica Serovar Typhi Vaccine Vectors in Neonatal and Infant Mice

2013 ◽  
Vol 20 (6) ◽  
pp. 931-944 ◽  
Author(s):  
Huoying Shi ◽  
Shifeng Wang ◽  
Roy Curtiss
Keyword(s):  
Salmonella Enterica ◽  
Peyer's Patches ◽  
Interleukin 4 ◽  
Peyer’S Patches ◽  
Wild Type ◽  
Vaccine Vectors ◽  
Content Type ◽  
The Impact

ABSTRACTWe developed regulated delayed attenuation strategies forSalmonellavaccine vectors. In this study, we evaluated the combination of these strategies in recombinant attenuatedSalmonella entericaserovar Typhi andSalmonella entericaserovar Typhimurium vaccine vectors with similar genetic backgroundsin vitroandin vivo. Our goal is to develop a vaccine to preventStreptococcus pneumoniaeinfection in newborns; thus, all strains delivered a pneumococcal antigen PspA and the impact of maternal antibodies was evaluated. The results showed that all strains with the regulated delayed attenuated phenotype (RDAP) displayed an invasive ability stronger than that of theS.Typhi vaccine strain, Ty21a, but weaker than that of their corresponding wild-type parental strains. The survival curves of different RDAP vaccine vectorsin vitroandin vivoexhibited diverse regulated delayed attenuation kinetics, which was different fromS.Typhi Ty21a and the wild-type parental strains. Under the influence of maternal antibody, the persistence of theS.Typhimurium RDAP strain displayed a regulated delayed attenuation trend in nasal lymphoid tissue (NALT), lung, and Peyer's patches, while the persistence ofS.Typhi RDAP strains followed the curve only in NALT. The bacterial loads ofS.Typhi RDAP strains were lower in NALT, lung, and Peyer's patches in mice born to immune mothers than in those born to naive mothers. In accordance with these results, RDAP vaccine strains induced high titers of IgG antibodies against PspA and againstSalmonellalipopolysaccharides. Immunization of mothers withS.Typhi RDAP strains enhanced the level of vaginal mucosal IgA, gamma interferon (IFN-γ), and interleukin 4 (IL-4) and resulted in a higher level of protection againstS. pneumoniaechallenge.

scholarly journals Acute, reproductive, and developmental toxicity of essential oils assessed with alternative in vitro and in vivo systems

2020 ◽  
Author(s):  
Peter Lanzerstorfer ◽  
Georg Sandner ◽  
Johannes Pitsch ◽  
Bianca Mascher ◽  
Tobias Aumiller ◽  
...  
Keyword(s):  
Essential Oils ◽  
Mucous Membrane ◽  
Relative Concentration ◽  
Feed Additives ◽  
Wild Type ◽  
Food Preservatives ◽  
C Elegans ◽  
The Impact

Abstract Essential oils (EOs) have attracted increased interest for different applications such as food preservatives, feed additives and ingredients in cosmetics. Due to their reported variable composition of components, they might be acutely toxic to humans and animals in small amounts. Despite the necessity, rigorous toxicity testing in terms of safety evaluation has not been reported so far, especially using alternatives to animal models. Here, we provide a strategy by use of alternative in vitro (cell cultures) and in vivo (Caenorhabditis elegans, hen’s egg test) approaches for detailed investigation of the impact of commonly used rosemary, citrus and eucalyptus essential oil on acute, developmental and reproductive toxicity as well as on mucous membrane irritation. In general, all EOs under study exhibited a comparable impact on measured parameters, with a slightly increased toxic potential of rosemary oil. In vitro cell culture results indicated a concentration-dependent decrease of cell viability for all EOs, with mean IC50 values ranging from 0.08 to 0.17% [v/v]. Similar results were obtained for the C. elegans model when using a sensitized bus-5 mutant strain, with a mean LC50 value of 0.42% [v/v]. In wild-type nematodes, approximately tenfold higher LC50 values were detected. C. elegans development and reproduction was already significantly inhibited at concentrations of 0.5% (wild-type) and 0.1% (bus-5) [v/v] of EO, respectively. Gene expression analysis revealed a significant upregulation of xenobiotic and oxidative stress genes such as cyp-14a3, gst-4, gpx-6 and sod-3. Furthermore, all three EOs under study showed an increased short-time mucous membrane irritation potential, already at 0.5% [v/v] of EO. Finally, GC–MS analysis was performed to quantitate the relative concentration of the most prominent EO compounds. In conclusion, our results demonstrate that EOs can exhibit severe toxic properties, already at low concentrations. Therefore, a detailed toxicological assessment is highly recommended for each EO and single intended application.

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