The Experts below are selected from a list of 19884 Experts worldwide ranked by ideXlab platform
Amarda Shehu - One of the best experts on this subject based on the ideXlab platform.
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Decoy selection for protein structure prediction via extreme gradient boosting and ranking
BMC Bioinformatics, 2020Co-Authors: Nasrin Akhter, Gopinath Chennupati, Hristo Djidjev, Amarda ShehuAbstract:Identifying one or more biologically-active/native Decoys from millions of non-native Decoys is one of the major challenges in computational structural biology. The extreme lack of balance in positive and negative samples (native and non-native Decoys) in a Decoy set makes the problem even more complicated. Consensus methods show varied success in handling the challenge of Decoy selection despite some issues associated with clustering large Decoy sets and Decoy sets that do not show much structural similarity. Recent investigations into energy landscape-based Decoy selection approaches show promises. However, lack of generalization over varied test cases remains a bottleneck for these methods. We propose a novel Decoy selection method, ML-Select, a machine learning framework that exploits the energy landscape associated with the structure space probed through a template-free Decoy generation. The proposed method outperforms both clustering and energy ranking-based methods, all the while consistently offering better performance on varied test-cases. Moreover, ML-Select shows promising results even for the Decoy sets consisting of mostly low-quality Decoys. ML-Select is a useful method for Decoy selection. This work suggests further research in finding more effective ways to adopt machine learning frameworks in achieving robust performance for Decoy selection in template-free protein structure prediction.
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balancing multiple objectives in conformation sampling to control Decoy diversity in template free protein structure prediction
BMC Bioinformatics, 2019Co-Authors: Ahmed Bin Zaman, Amarda ShehuAbstract:Computational approaches for the determination of biologically-active/native three-dimensional structures of proteins with novel sequences have to handle several challenges. The (conformation) space of possible three-dimensional spatial arrangements of the chain of amino acids that constitute a protein molecule is vast and high-dimensional. Exploration of the conformation spaces is performed in a sampling-based manner and is biased by the internal energy that sums atomic interactions. Even state-of-the-art energy functions that quantify such interactions are inherently inaccurate and associate with protein conformation spaces overly rugged energy surfaces riddled with artifact local minima. The response to these challenges in template-free protein structure prediction is to generate large numbers of low-energy conformations (also referred to as Decoys) as a way of increasing the likelihood of having a diverse Decoy dataset that covers a sufficient number of local minima possibly housing near-native conformations. In this paper we pursue a complementary approach and propose to directly control the diversity of generated Decoys. Inspired by hard optimization problems in high-dimensional and non-linear variable spaces, we propose that conformation sampling for Decoy generation is more naturally framed as a multi-objective optimization problem. We demonstrate that mechanisms inherent to evolutionary search techniques facilitate such framing and allow balancing multiple objectives in protein conformation sampling. We showcase here an operationalization of this idea via a novel evolutionary algorithm that has high exploration capability and is also able to access lower-energy regions of the energy landscape of a given protein with similar or better proximity to the known native structure than several state-of-the-art Decoy generation algorithms. The presented results constitute a promising research direction in improving Decoy generation for template-free protein structure prediction with regards to balancing of multiple conflicting objectives under an optimization framework. Future work will consider additional optimization objectives and variants of improvement and selection operators to apportion a fixed computational budget. Of particular interest are directions of research that attenuate dependence on protein energy models.
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from extraction of local structures of protein energy landscapes to improved Decoy selection in template free protein structure prediction
Molecules, 2018Co-Authors: Nasrin Akhter, Amarda ShehuAbstract:Due to the essential role that the three-dimensional conformation of a protein plays in regulating interactions with molecular partners, wet and dry laboratories seek biologically-active conformations of a protein to decode its function. Computational approaches are gaining prominence due to the labor and cost demands of wet laboratory investigations. Template-free methods can now compute thousands of conformations known as Decoys, but selecting native conformations from the generated Decoys remains challenging. Repeatedly, research has shown that the protein energy functions whose minima are sought in the generation of Decoys are unreliable indicators of nativeness. The prevalent approach ignores energy altogether and clusters Decoys by conformational similarity. Complementary recent efforts design protein-specific scoring functions or train machine learning models on labeled Decoys. In this paper, we show that an informative consideration of energy can be carried out under the energy landscape view. Specifically, we leverage local structures known as basins in the energy landscape probed by a template-free method. We propose and compare various strategies of basin-based Decoy selection that we demonstrate are superior to clustering-based strategies. The presented results point to further directions of research for improving Decoy selection, including the ability to properly consider the multiplicity of native conformations of proteins.
John G Conboy - One of the best experts on this subject based on the ideXlab platform.
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antisense targeting of Decoy exons can reduce intron retention and increase protein expression in human erythroblasts
RNA, 2020Co-Authors: Marilyn Parra, Weiguo Zhang, Mark A Dewitt, John G ConboyAbstract:The Decoy exon model has been proposed to regulate a subset of intron retention (IR) events involving predominantly larger introns (>1 kb). Splicing reporter studies have shown that Decoy splice sites are essential for activity, suggesting that Decoys act by engaging intron-terminal splice sites and competing with cross-intron interactions required for intron excision. The Decoy model predicts that antisense oligonucleotides may be able to block Decoy splice sites in endogenous pre-mRNA, thereby reducing IR and increasing productive gene expression. Indeed, we now demonstrate that targeting a Decoy 5' splice site in the O-GlcNAc transferase (OGT) gene reduced IR from ∼80% to ∼20% in primary human erythroblasts, accompanied by increases in spliced OGT RNA and OGT protein expression. The remaining OGT IR was refractory to antisense treatment and might be mediated by independent mechanism(s). In contrast, other retained introns were strongly dependent on Decoy function, since antisense targeting of Decoy 5' splice sites greatly reduced (SNRNP70) or nearly eliminated (SF3B1) IR in two widely expressed splicing factors, and also greatly reduced IR in transcripts encoding the erythroid-specific structural protein, α-spectrin (SPTA1). These results show that modulating Decoy exon function can dramatically alter IR and suggest that dynamic regulation of Decoy exons could be a mechanism to fine-tune gene expression post-transcriptionally in many cell types.
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antisense targeting of Decoy exons can reduce intron retention and increase protein expression in human erythroblasts
bioRxiv, 2020Co-Authors: Marilyn Parra, Weiguo Zhang, Mark A Dewitt, John G ConboyAbstract:The Decoy exon model has been proposed to regulate a subset of intron retention (IR) events involving predominantly larger introns (>1kb). Splicing reporter studies have shown that Decoy splice sites are essential for activity, suggesting that Decoys act by engaging intron-terminal splice sites and competing with cross-intron interactions required for intron excision. The Decoy model predicts that antisense oligonucleotides blocking Decoy splice sites in endogenous pre-mRNA should increase productive gene expression by reducing IR. Indeed, we now demonstrate that targeting a Decoy 5' splice site in the O-GlcNAc transferase (OGT) gene reduced IR from [~]80% to [~]20% in primary human erythroblasts, accompanied by increases in spliced OGT RNA and OGT protein expression. The remaining OGT IR was refractory to antisense treatment and might be mediated by independent mechanism(s). In contrast, other retained introns were strongly dependent on Decoy function, since IR was nearly eliminated by antisense targeting of 5' splice sites. Genes in the latter group encode the widely expressed splicing factor (SF3B1), and the erythroid-specific structural protein, alpha-spectrin (SPTA1). These results show that modulating Decoy exon function can dramatically alter IR, and suggest that dynamic regulation of Decoy exons could be a mechanism to fine tune gene expression post-transcriptionally in many cell types.
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an important class of intron retention events in human erythroblasts is regulated by cryptic exons proposed to function as splicing Decoys
RNA, 2018Co-Authors: Marilyn Parra, Ben W Booth, Richard Weiszmann, Brian A Yee, Gene W Yeo, James B Brown, Susan E Celniker, John G ConboyAbstract:During terminal erythropoiesis, the splicing machinery in differentiating erythroblasts executes a robust intron retention (IR) program that impacts expression of hundreds of genes. We studied IR mechanisms in the SF3B1 splicing factor gene, which expresses ∼50% of its transcripts in late erythroblasts as a nuclear isoform that retains intron 4. RNA-seq analysis of nonsense-mediated decay (NMD)-inhibited cells revealed previously undescribed splice junctions, rare or not detected in normal cells, that connect constitutive exons 4 and 5 to highly conserved cryptic cassette exons within the intron. Minigene splicing reporter assays showed that these cassettes promote IR. Genome-wide analysis of splice junction reads demonstrated that cryptic noncoding cassettes are much more common in large (>1 kb) retained introns than they are in small retained introns or in nonretained introns. Functional assays showed that heterologous cassettes can promote retention of intron 4 in the SF3B1 splicing reporter. Although many of these cryptic exons were spliced inefficiently, they exhibited substantial binding of U2AF1 and U2AF2 adjacent to their splice acceptor sites. We propose that these exons function as Decoys that engage the intron-terminal splice sites, thereby blocking cross-intron interactions required for excision. Developmental regulation of Decoy function underlies a major component of the erythroblast IR program.
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an important class of intron retention events in human erythroblasts is regulated by cryptic exons proposed to function as splicing Decoys
bioRxiv, 2018Co-Authors: Marilyn Parra, Ben W Booth, Richard Weiszmann, Brian A Yee, Gene W Yeo, James B Brown, Susan E Celniker, John G ConboyAbstract:During terminal erythropoiesis, differentiating erythroblasts execute a robust program of intron retention (IR). We studied IR mechanisms in the SF3B1 splicing factor gene, which expresses ~50% of its transcripts in late erythroblasts as a nuclear isoform that retains intron 4. RNA-seq splice junction reads from nonsense-mediated decay (NMD)-inhibited cells revealed that highly conserved intron sequences encode cryptic cassette exons, and minigene splicing reporter assays showed that these cassettes function as Decoys that promote IR. Novel Decoy exons were common in large (>1kb) retained introns, and heterologous Decoys promoted retention of intron 4. Although most Decoys were spliced inefficiently, they exhibited substantial binding of U2AF1 and U2AF2 adjacent to their splice acceptor sites. We propose that Decoy exons engage intron-terminal splice sites, blocking cross-intron interactions required for excision, and that developmental regulation of Decoy function underlies a major component of the erythroblast IR program.
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sf3b1 gene expression in erythroid cells is regulated by intron retention via a posttranscriptional mechanism involving cryptic exons proposed to function as splicing Decoys
Blood, 2017Co-Authors: Marilyn Parra, Ben W Booth, Richard Weiszmann, Gene W Yeo, James B Brown, Susan E Celniker, John G ConboyAbstract:Proper expression of the MDS-disease gene, SF3B1, ensures appropriate pre-mRNA splicing in erythroid progenitors and during terminal erythropoiesis. We previously showed that the SF3B1 gene is post-transcriptionally regulated in a differentiation stage-specific manner by intron retention (IR), such that ~50% of its transcripts in mature erythroblasts retain intron 4. Based on new mechanistic studies, we propose a model in which mostly unannotated and noncoding exons within intron 4 function as splicing Decoys; i.e., they promote retention of intron 4 by interacting with, and blocking splice sites of, the adjacent exons 4 and 5. A total of six putative Decoy exons were revealed via RT-PCR and RNA-seq analysis of RNA from erythroblasts treated with inhibitors of nonsense-mediated decay. That Decoy exons have IR-promoting activity is suggested by several criteria. First, the frequency of interaction between constitutive exons 4 and 5 and putative Decoy exons within intron 4, measured by the abundance of splice junctions in RNA-seq read data, is temporally correlated with levels of intron 4 retention during terminal erythropoiesis. Both IR and Decoy splice junctions were low in early stage erythroblasts and much higher in mature erythroblasts. Second, selected Decoy exons exhibited IR-promoting activity in the context of minigene splicing reporters expressing the exon 3-6 region of SF3B1 in transfected K562 cells. The wild type minigene reproduced the intron-specific retention phenotype, since it was fully spliced at introns 3 and 5 but exhibited substantial retention of intron 4, whereas deletion of Decoy exon 4e, or mutation of its splice sites, substantially decreased IR. Third, RBP (RNA binding protein) cross-linking data from K562 cells show that 39 splice site factors including U2AF1 and U2AF2 can bind specifically to 39 splice sites of intron 49s Decoy exons. Finally, several experiments showed that IR-promoting activity of Decoy exons is a more general phenomenon that likely governs IR in other erythroid genes. We observed not only that SF3B1 intron 4 Decoy exons could promote IR in heterologous contexts, but also that predicted Decoy exons from other erythroblast transcripts could promote IR in the SF3B1 minigene. Apart from this experimental data, comparative genomics revealed that the SF3B1 Decoy exons are extremely conserved among vertebrate genomes, with two of the exons being essentially identical from fish to humans. Together this data supports the hypothesis that a subset of up-regulated IR events in late erythroblasts are controlled by Decoy exons that block productive splicing at the flanking exons. We propose that regulated IR is an important post-transcriptional mechanism for adjusting cellular splicing capacity during terminal erythropoiesis by regulating expression of key splicing factors such as SF3B1. Disclosures No relevant conflicts of interest to declare.
Rosa Carnuccio - One of the best experts on this subject based on the ideXlab platform.
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transcription factor Decoy oligodeoxynucleotides to nuclear factor kappab inhibit reverse passive arthus reaction in rat
Naunyn-schmiedebergs Archives of Pharmacology, 2001Co-Authors: Fulvio Dacquisto, Angela Ianaro, Armando Ialenti, Pasquale Maffia, Maria Chiara Maiuri, Rosa CarnuccioAbstract:In the present study we investigated in the reverse passive Arthus reaction elicited in the rat skin the anti-inflammatory effect of double-stranded oligodeoxynucleotides (ODN) with consensus nuclear factor-κB (NF-κB) sequence as transcription factor Decoys (TFD) to inhibit NF-κB binding to native DNA sites. Local administration of wild-type-, but not mutant-Decoy ODN, dose-dependently reduced both plasma leakage and neutrophil infiltration in rat skin. Molecular analysis performed on soft tissue obtained from rat skin demonstrated: (1) an inhibition of NF-κB/DNA binding activity; (2) a decreased nuclear level of p50 and p65 NF-κB subunits; (3) an inhibition of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) protein expression, two inflammatory enzymes transcriptionally controlled by NF-κB. Furthermore, SN-50, a cell-permeable peptide capable of inhibiting the nuclear translocation of NF-κB complexes, as well as ammonium pyrrolidine dithiocarbamate, an inhibitor of NF-κB activation, exhibited a similar profile of activity of Decoy ODN. Our results indicate that Decoy ODN, acting as an in vivo competitor for the transcription factor's ability to bind to cognate recognition sequence, may represent a novel strategy to modulate immune reactions.
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local administration of transcription factor Decoy oligonucleotides to nuclear factor kappab prevents carrageenin induced inflammation in rat hind paw
Gene Therapy, 2000Co-Authors: Fulvio Dacquisto, Angela Ianaro, Armando Ialenti, R Di Vaio, Rosa CarnuccioAbstract:The transcription factor nuclear factor-kappaB (NF-kappaB) plays a key role in the expression of several genes involved in the inflammatory process. In the present study we investigated in an acute model of inflammation, the carrageenin-induced hind paw edema, the anti-inflammatory effect of double stranded oligodeoxynucleotides (ODN) with consensus nuclear factor-kappaB (NF-kappaB) sequence as transcription factor Decoys (TFD) to inhibit NF-kappaB binding to native DNA sites. Local administration of wild-type, but not mutant-ODN Decoy, dose-dependently inhibited edema formation induced by carrageenin in rat paw. Molecular analysis performed on soft tissue obtained from inflamed paw demonstrated: (1) an inhibition of NF-kappaB DNA binding activity; (2) a decreased nuclear level of p50 and p65 NF-kappaB subunits; (3) an inhibition of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) protein expression, two inflammatory enzymes transcriptionally controlled by NF-kappaB. Furthermore, SN-50, a cell-permeable peptide capable of inhibiting the nuclear translocation of NF-kappaB complexes, exhibited a similar profile of activity of ODN Decoy. Our results indicate for the first time that ODN Decoy, acting as an in vivo competitor for the transcription factor's ability to bind to cognate recognition sequence, may represent a novel strategy to modulate acute inflammation.
Marilyn Parra - One of the best experts on this subject based on the ideXlab platform.
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antisense targeting of Decoy exons can reduce intron retention and increase protein expression in human erythroblasts
RNA, 2020Co-Authors: Marilyn Parra, Weiguo Zhang, Mark A Dewitt, John G ConboyAbstract:The Decoy exon model has been proposed to regulate a subset of intron retention (IR) events involving predominantly larger introns (>1 kb). Splicing reporter studies have shown that Decoy splice sites are essential for activity, suggesting that Decoys act by engaging intron-terminal splice sites and competing with cross-intron interactions required for intron excision. The Decoy model predicts that antisense oligonucleotides may be able to block Decoy splice sites in endogenous pre-mRNA, thereby reducing IR and increasing productive gene expression. Indeed, we now demonstrate that targeting a Decoy 5' splice site in the O-GlcNAc transferase (OGT) gene reduced IR from ∼80% to ∼20% in primary human erythroblasts, accompanied by increases in spliced OGT RNA and OGT protein expression. The remaining OGT IR was refractory to antisense treatment and might be mediated by independent mechanism(s). In contrast, other retained introns were strongly dependent on Decoy function, since antisense targeting of Decoy 5' splice sites greatly reduced (SNRNP70) or nearly eliminated (SF3B1) IR in two widely expressed splicing factors, and also greatly reduced IR in transcripts encoding the erythroid-specific structural protein, α-spectrin (SPTA1). These results show that modulating Decoy exon function can dramatically alter IR and suggest that dynamic regulation of Decoy exons could be a mechanism to fine-tune gene expression post-transcriptionally in many cell types.
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antisense targeting of Decoy exons can reduce intron retention and increase protein expression in human erythroblasts
bioRxiv, 2020Co-Authors: Marilyn Parra, Weiguo Zhang, Mark A Dewitt, John G ConboyAbstract:The Decoy exon model has been proposed to regulate a subset of intron retention (IR) events involving predominantly larger introns (>1kb). Splicing reporter studies have shown that Decoy splice sites are essential for activity, suggesting that Decoys act by engaging intron-terminal splice sites and competing with cross-intron interactions required for intron excision. The Decoy model predicts that antisense oligonucleotides blocking Decoy splice sites in endogenous pre-mRNA should increase productive gene expression by reducing IR. Indeed, we now demonstrate that targeting a Decoy 5' splice site in the O-GlcNAc transferase (OGT) gene reduced IR from [~]80% to [~]20% in primary human erythroblasts, accompanied by increases in spliced OGT RNA and OGT protein expression. The remaining OGT IR was refractory to antisense treatment and might be mediated by independent mechanism(s). In contrast, other retained introns were strongly dependent on Decoy function, since IR was nearly eliminated by antisense targeting of 5' splice sites. Genes in the latter group encode the widely expressed splicing factor (SF3B1), and the erythroid-specific structural protein, alpha-spectrin (SPTA1). These results show that modulating Decoy exon function can dramatically alter IR, and suggest that dynamic regulation of Decoy exons could be a mechanism to fine tune gene expression post-transcriptionally in many cell types.
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an important class of intron retention events in human erythroblasts is regulated by cryptic exons proposed to function as splicing Decoys
RNA, 2018Co-Authors: Marilyn Parra, Ben W Booth, Richard Weiszmann, Brian A Yee, Gene W Yeo, James B Brown, Susan E Celniker, John G ConboyAbstract:During terminal erythropoiesis, the splicing machinery in differentiating erythroblasts executes a robust intron retention (IR) program that impacts expression of hundreds of genes. We studied IR mechanisms in the SF3B1 splicing factor gene, which expresses ∼50% of its transcripts in late erythroblasts as a nuclear isoform that retains intron 4. RNA-seq analysis of nonsense-mediated decay (NMD)-inhibited cells revealed previously undescribed splice junctions, rare or not detected in normal cells, that connect constitutive exons 4 and 5 to highly conserved cryptic cassette exons within the intron. Minigene splicing reporter assays showed that these cassettes promote IR. Genome-wide analysis of splice junction reads demonstrated that cryptic noncoding cassettes are much more common in large (>1 kb) retained introns than they are in small retained introns or in nonretained introns. Functional assays showed that heterologous cassettes can promote retention of intron 4 in the SF3B1 splicing reporter. Although many of these cryptic exons were spliced inefficiently, they exhibited substantial binding of U2AF1 and U2AF2 adjacent to their splice acceptor sites. We propose that these exons function as Decoys that engage the intron-terminal splice sites, thereby blocking cross-intron interactions required for excision. Developmental regulation of Decoy function underlies a major component of the erythroblast IR program.
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an important class of intron retention events in human erythroblasts is regulated by cryptic exons proposed to function as splicing Decoys
bioRxiv, 2018Co-Authors: Marilyn Parra, Ben W Booth, Richard Weiszmann, Brian A Yee, Gene W Yeo, James B Brown, Susan E Celniker, John G ConboyAbstract:During terminal erythropoiesis, differentiating erythroblasts execute a robust program of intron retention (IR). We studied IR mechanisms in the SF3B1 splicing factor gene, which expresses ~50% of its transcripts in late erythroblasts as a nuclear isoform that retains intron 4. RNA-seq splice junction reads from nonsense-mediated decay (NMD)-inhibited cells revealed that highly conserved intron sequences encode cryptic cassette exons, and minigene splicing reporter assays showed that these cassettes function as Decoys that promote IR. Novel Decoy exons were common in large (>1kb) retained introns, and heterologous Decoys promoted retention of intron 4. Although most Decoys were spliced inefficiently, they exhibited substantial binding of U2AF1 and U2AF2 adjacent to their splice acceptor sites. We propose that Decoy exons engage intron-terminal splice sites, blocking cross-intron interactions required for excision, and that developmental regulation of Decoy function underlies a major component of the erythroblast IR program.
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sf3b1 gene expression in erythroid cells is regulated by intron retention via a posttranscriptional mechanism involving cryptic exons proposed to function as splicing Decoys
Blood, 2017Co-Authors: Marilyn Parra, Ben W Booth, Richard Weiszmann, Gene W Yeo, James B Brown, Susan E Celniker, John G ConboyAbstract:Proper expression of the MDS-disease gene, SF3B1, ensures appropriate pre-mRNA splicing in erythroid progenitors and during terminal erythropoiesis. We previously showed that the SF3B1 gene is post-transcriptionally regulated in a differentiation stage-specific manner by intron retention (IR), such that ~50% of its transcripts in mature erythroblasts retain intron 4. Based on new mechanistic studies, we propose a model in which mostly unannotated and noncoding exons within intron 4 function as splicing Decoys; i.e., they promote retention of intron 4 by interacting with, and blocking splice sites of, the adjacent exons 4 and 5. A total of six putative Decoy exons were revealed via RT-PCR and RNA-seq analysis of RNA from erythroblasts treated with inhibitors of nonsense-mediated decay. That Decoy exons have IR-promoting activity is suggested by several criteria. First, the frequency of interaction between constitutive exons 4 and 5 and putative Decoy exons within intron 4, measured by the abundance of splice junctions in RNA-seq read data, is temporally correlated with levels of intron 4 retention during terminal erythropoiesis. Both IR and Decoy splice junctions were low in early stage erythroblasts and much higher in mature erythroblasts. Second, selected Decoy exons exhibited IR-promoting activity in the context of minigene splicing reporters expressing the exon 3-6 region of SF3B1 in transfected K562 cells. The wild type minigene reproduced the intron-specific retention phenotype, since it was fully spliced at introns 3 and 5 but exhibited substantial retention of intron 4, whereas deletion of Decoy exon 4e, or mutation of its splice sites, substantially decreased IR. Third, RBP (RNA binding protein) cross-linking data from K562 cells show that 39 splice site factors including U2AF1 and U2AF2 can bind specifically to 39 splice sites of intron 49s Decoy exons. Finally, several experiments showed that IR-promoting activity of Decoy exons is a more general phenomenon that likely governs IR in other erythroid genes. We observed not only that SF3B1 intron 4 Decoy exons could promote IR in heterologous contexts, but also that predicted Decoy exons from other erythroblast transcripts could promote IR in the SF3B1 minigene. Apart from this experimental data, comparative genomics revealed that the SF3B1 Decoy exons are extremely conserved among vertebrate genomes, with two of the exons being essentially identical from fish to humans. Together this data supports the hypothesis that a subset of up-regulated IR events in late erythroblasts are controlled by Decoy exons that block productive splicing at the flanking exons. We propose that regulated IR is an important post-transcriptional mechanism for adjusting cellular splicing capacity during terminal erythropoiesis by regulating expression of key splicing factors such as SF3B1. Disclosures No relevant conflicts of interest to declare.
Giulio Cabrini - One of the best experts on this subject based on the ideXlab platform.
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effects of Decoy molecules targeting nf kappab transcription factors in cystic fibrosis ib3 1 cells recruitment of nf kappab to the il 8 gene promoter and transcription of the il 8 gene
Artificial DNA PNA & XNA, 2012Co-Authors: Alessia Finotti, Monica Borgatti, Valentino Bezzerri, Elena Nicolis, Ilaria Lampronti, Maria Cristina Dechecchi, Irene Mancini, Giulio Cabrini, Michele Saviano, Concetta AvitabileAbstract:One of the clinical features of cystic fibrosis (CF) is a deep inflammatory process, which is characterized by production and release of cytokines and chemokines, among which interleukin 8 (IL-8) represents one of the most important. Accordingly, there is a growing interest in developing therapies against CF to reduce the excessive inflammatory response in the airways of CF patients. Since transcription factor NF-kappaB plays a critical role in IL-8 expression, the transcription factor Decoy (TFD) strategy might be of interest. In order to demonstrate that TFD against NF-kappaB interferes with the NF-kappaB pathway we proved, by chromatin immunoprecipitation (ChIP) that treatment with TFD oligodeoxyribonucleotides of cystic fibrosis IB3–1 cells infected with Pseudomonas aeruginosa leads to a decrease occupancy of the Il-8 gene promoter by NF-kappaB factors. In order to develop more stable therapeutic molecules, peptide nucleic acids (PNAs) based agents were considered. In this respect PNA-DNA-PNA (PDP) chimeras are molecules of great interest from several points of view: (1) they can be complexed with liposomes and microspheres; (2) they are resistant to DNases, serum and cytoplasmic extracts; (3) they are potent Decoy molecules. By using electrophoretic mobility shift assay and RT-PCR analysis we have demonstrated that (1) the effects of PDP/PDP NF-kappaB Decoy chimera on accumulation of pro-inflammatory mRNAs in P.aeruginosa infected IB3–1 cells reproduce that of Decoy oligonucleotides; in particular (2) the PDP/PDP chimera is a strong inhibitor of IL-8 gene expression; (3) the effect of PDP/PDP chimeras, unlike those of ODN-based Decoys, are observed even in the absence of protection with lipofectamine. These informations are of great impact, in our opinion, for the development of stable molecules to be used in non-viral gene therapy of cystic fibrosis.
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Effects of Decoy molecules targeting NFkappaBtranscription factors in cystic fibrosis IB3-1 cellsRecruitment of NFkappaB to the IL-8 gene promoterand transcription of the IL-8 gene
'Informa UK Limited', 2012Co-Authors: Alessia Finotti, Monica Borgatti, Valentino Bezzerri, Elena Nicolis, Ilaria Lampronti, Maria Cristina Dechecchi, Irene Mancini, Giulio Cabrini, Michele Saviano, Concetta AvitabileAbstract:One of the clinical feature of cystic fibrosis (CF) is a deep inflammatory process, which is characterized by production and release of cytokines and chemokines, among which interleukin 8 (IL-8) represents one of the most important. Accordingly, there is a growing interest in developing therapies against CF to reduce the excessive inflammatory response in the airways of CF patients. Since transcription factor NFkappaB plays a critical role in IL-8 expression, the transcription factor Decoy (TFD) strategy might be of interest. In order to demonstrate that TFD against NFkappaB interferes with the NFkappaB pathway we proved, by chromatin immunoprecipitation (ChIP) that treatment with TFD oligodeoxyribonucleotides of cystic fibrosis IB3-1 cells infected with Pseudomonas aeruginosa leads to a decrease occupancy of the Il-8 gene promoter by NFkappaB factors. In order to develop more stable therapeutic molecules, peptide nucleic acids (PNAs) based agents were considered. In this respect PNA-DNA-PNA (PDP) chimeras are molecules of great interest from several points of view: (1) they can be complexed with liposomes and microspheres; (2) they are resistant to DNases, serum and cytoplasmic extracts; (3) they are potent Decoy molecules. By using electrophoretic mobility shift assay and RT-PCR analysis we have demonstrated that (1) the effects of PDP/PDP NFkappaB Decoy chimera on accumulation of pro-inflammatory mRNAs in P. aeruginosa infected IB3-1 cells reproduce that of Decoy oligonucleotides; in particular (2) the PDP/PDP chimera is a strong inhibitor of IL-8 gene expression; (3) the effect of PDP/PDP chimeras, unlike those of ODN-based Decoys, are observed even in the absence of protection with lipofectamine. These informations are of great impact, in our opinion, for the development of stable molecules to be used in non-viral gene therapy of cystic fibrosis
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Effects of Decoy molecules targeting NFkappaB transcription factors in Cystic fibrosis IB3-1 cells : recruitment of NFkappaB to the IL-8 gene promoter and transcription of the IL-8 gene
'Informa UK Limited', 2012Co-Authors: Alessia Finotti, Monica Borgatti, Valentino Bezzerri, Elena Nicolis, Ilaria Lampronti, Maria Cristina Dechecchi, Irene Mancini, Giulio Cabrini, Michele Saviano, Concetta AvitabileAbstract:One of the clinical feature of cystic fibrosis (CF) is a deep inflammatory process, which is characterized by production and release of cytokines and chemokines, among which interleukin 8 (IL-8) represents one of the most important. Accordingly, there is a growing interest in developing therapies against CF to reduce the excessive inflammatory response in the airways of CF patients. Since transcription factor NFkappaB plays a critical role in IL-8 expression, the transcription factor Decoy (TFD) strategy might be of interest. In order to demonstrate that TFD against NFkappaB interferes with the NFkappaB pathway we proved, by chromatin immunoprecipitation (ChIP) that treatment with TFD oligodeoxyribonucleotides of cystic fibrosis IB3-1 cells infected with Pseudomonas aeruginosa leads to a decrease occupancy of the Il-8 gene promoter by NFkappaB factors. In order to develop more stable therapeutic molecules, peptide nucleic acids (PNAs) based agents were considered. In this respect PNA-DNA-PNA (PDP) chimeras are molecules of great interest from several points of view: (1) they can be complexed with liposomes and microspheres; (2) they are resistant to DNases, serum and cytoplasmic extracts; (3) they are potent Decoy molecules. By using electrophoretic mobility shift assay and RT-PCR analysis we have demonstrated that (1) the effects of PDP/PDP NFkappaB Decoy chimera on accumulation of pro-inflammatory mRNAs in P. aeruginosa infected IB3-1 cells reproduce that of Decoy oligonucleotides; in particular (2) the PDP/PDP chimera is a strong inhibitor of IL-8 gene expression; (3) the effect of PDP/PDP chimeras, unlike those of ODN-based Decoys, are observed even in the absence of protection with lipofectamine. These informations are of great impact, in our opinion, for the development of stable molecules to be used in non-viral gene therapy of cystic fibrosis
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Decoy oligodeoxyribonucleotides and peptide nucleic acids dna chimeras targeting nuclear factor kappa b inhibition of il 8 gene expression in cystic fibrosis cells infected with pseudomonas aeruginosa
Biochemical Pharmacology, 2010Co-Authors: Roberto Gambari, Monica Borgatti, Valentino Bezzerri, Elena Nicolis, Ilaria Lampronti, Maria Cristina Dechecchi, Irene Mancini, Anna Tamanini, Giulio CabriniAbstract:Abstract Cystic fibrosis (CF) is characterized by a deep inflammatory process, with production and release of cytokines and chemokines, among which interleukin 8 (IL-8) represents one of the most important. Accordingly, there is a growing interest in developing therapies against IL-8, with the aim of reducing the excessive inflammatory response in the airways of CF patients. Since transcription factor NF-kappaB plays a critical role in IL-8 expression, the transcription factor Decoy (TFD) strategy might be of interest. TFD is based on biomolecules mimicking the target sites of transcription factors (TFs) and able to interfere with TF activity when delivered to target cells. Here, we review the inhibitory effects of Decoy oligodeoxyribonucleotides (ODNs) on expression of IL-8 gene and secretion of IL-8 by cystic fibrosis cells infected by Pseudomonas aeruginosa . In addition, the effects of Decoy molecules based on peptide nucleic acids (PNAs) are discussed. In this respect PNA–DNA–PNA (PDP) chimeras are interesting: (a) unlike PNAs, they can be complexed with liposomes and microspheres; (b) unlike oligodeoxyribonucleotides (ODNs), they are resistant to DNAses, serum and cytoplasmic extracts; (c) unlike PNA/PNA and PNA/DNA hybrids, they are potent Decoy molecules. Interestingly, PDP/PDP NF-kappaB Decoy chimeras inhibit accumulation of pro-inflammatory mRNAs (including IL-8 mRNA) in P. aeruginosa infected IB3-1, cells reproducing the effects of Decoy oligonucleotides. The effects of PDP/PDP chimeras, unlike ODN-based Decoys, are observed even in absence of protection with lipofectamine. Since IL-8 is pivotal in pro-inflammatory processes affecting cystic fibrosis, inhibition of its functions might have a clinical relevance.
