The Experts below are selected from a list of 4395 Experts worldwide ranked by ideXlab platform
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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Intron Retention mechanisms that regulate sf3b1 and mitoferrin gene expression during late erythropoiesis
Blood, 2016Co-Authors: Marilyn Parra, Gene W Yeo, John G ConboyAbstract:Intron Retention (IR) regulates hundreds of erythroid genes in a differentiation stage-specific manner during terminal erythropoiesis. Regulated genes include highly expressed RNA binding proteins (RBPs) such as SF3B1, as well as iron transporters (e.g., mitoferrins 1 and 2), and cytoskeletal proteins (e.g., alpha spectrin). Selected IR transcripts are relatively abundant; 25-50% of the above-mentioned transcripts can be polyadenylated, retained in the nucleus, and efficiently spliced at all but one or two Introns, thus limiting the amount of translatable cytoplasmic mRNA. We are studying novel IR regulatory mechanisms involving both splice site strength and deeper Intronic elements, using model Introns that are differentially regulated in human erythroblasts. SF3B1, a key pre-RNA splicing factor implicated in MDS, exhibits dynamic regulation of Intron 4, with low IR in proerythroblasts and high IR in mature orthochromatic erythroblasts. Intron 4 sequences include three ultraconserved elements that encode cryptic exons we term 4a, 4b, and 4c, two of which (4a and 4c) encode premature termination codons (PTCs) expected to induce nonsense-mediated decay (NMD) if spliced into SF3B1 transcripts. We hypothesize that these PTC exons can have an additional function as inefficiently spliced decoy(s), that is, their splice sites may interact with splice sites at the boundaries of Intron 4 to form non-productive complexes that do not permit efficient splicing but instead prevent excision of the Intron. This concept represents an extension of decoy models previously proposed by others to explain selected exon skipping events, and is supported by several recent findings: eCLIP (enhanced cross-linking and immunoprecipitation) data indicate strong binding of 39 splice site factors U2AF1 and U2AF2 at these exons; low levels of exon 4a and 4c splicing can be seen in NMD-inhibited cells; and deletion of exon 4c from minigene splicing reporters decreases IR. Besides SF3B1, we identified numerous other dynamically-regulated IR events encompassing cryptic PTC exons that bind 39 splice site factors. In contrast to the dynamic regulation of IR in SF3B1, IR in SLC25A37 (mitoferrin-1) and SLC25A28 (mitoferrin-2) is stably maintained at a high level throughout terminal erythropoiesis. Baseline IR levels for stably retained Introns correlate with splice site strength, but are also influenced by deeper Intronic elements. For example, SLC25A28 Intron 2 contains Intronic elements that appear to reduce IR, since blocking them with antisense morpholinos leads to substantially increased IR levels. These studies demonstrate that IR in major erythroid genes is regulated by sequences within the retained Introns that can either increase or decrease Retention, suggesting that multiple IR pathways are employed during terminal erythropoiesis to regulate gene expression. Disclosures No relevant conflicts of interest to declare.
John E.j. Rasko - One of the best experts on this subject based on the ideXlab platform.
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holding on to junk bonds Intron Retention in cancer and therapy
Cancer Research, 2021Co-Authors: Geoffray Monteuuis, John E.j. Rasko, Ulf Schmitz, Veronika Petrova, Padraic Scott KearneyAbstract:Intron Retention (IR) in cancer was for a long time overlooked by the scientific community, as it was previously considered to be an artifact of a dysfunctional spliceosome. Technological advancements made in the last decade offer unique opportunities to explore the role of IR as a widespread phenomenon that contributes to the transcriptional diversity of many cancers. Numerous studies in cancer have shed light on dysregulation of cellular mechanisms that lead to aberrant and pathologic IR. IR is not merely a mechanism of gene regulation, but rather it can mediate cancer pathogenesis and therapeutic resistance in various human diseases. The burden of IR in cancer is governed by perturbations to mechanisms known to regulate this phenomenon and include epigenetic variation, mutations within the gene body, and splicing factor dysregulation. This review summarizes possible causes for aberrant IR and discusses the role of IR in therapy or as a consequence of disease treatment. As neoepitopes originating from retained Introns can be presented on the cancer cell surface, the development of personalized cancer vaccines based on IR-derived neoepitopes should be considered. Ultimately, a deeper comprehension about the origins and consequences of aberrant IR may aid in the development of such personalized cancer vaccines.
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the changing paradigm of Intron Retention regulation ramifications and recipes
Nucleic Acids Research, 2019Co-Authors: Geoffray Monteuuis, Charles G. Bailey, John E.j. Rasko, Ulf Schmitz, Justin J.-l. WongAbstract:Intron Retention (IR) is a form of alternative splicing that has long been neglected in mammalian systems although it has been studied for decades in non-mammalian species such as plants, fungi, insects and viruses. It was generally assumed that mis-splicing, leading to the Retention of Introns, would have no physiological consequence other than reducing gene expression by nonsense-mediated decay. Relatively recent landmark discoveries have highlighted the pivotal role that IR serves in normal and disease-related human biology. Significant technical hurdles have been overcome, thereby enabling the robust detection and quantification of IR. Still, relatively little is known about the cis- and trans-acting modulators controlling this phenomenon. The fate of an Intron to be, or not to be, retained in the mature transcript is the direct result of the influence exerted by numerous intrinsic and extrinsic factors at multiple levels of regulation. These factors have altered current biological paradigms and provided unexpected insights into the transcriptional landscape. In this review, we discuss the regulators of IR and methods to identify them. Our focus is primarily on mammals, however, we broaden the scope to non-mammalian organisms in which IR has been shown to be biologically relevant.
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Ctcf Haploinsufficiency Mediates Intron Retention in A Tissue-specific Manner
2019Co-Authors: Adel B Alharbi, Amy D. Marshall, Charles G. Bailey, M Vellozzi, Ulf Schmitz, Darya Vanichkina, Rajini Nagarajah, Justin J.-l. Wong, John E.j. RaskoAbstract:CTCF is a master regulator of gene transcription and chromatin organization with occupancy at thousands of DNA target sites. CTCF is essential for embryonic development and somatic cell viability and has been characterized as a haploinsufficient tumor suppressor. Increasing evidence demonstrates CTCF as a key player in several alternative splicing (AS) regulatory mechanisms, including transcription elongation, regulation of splicing factors, and epigenetic regulation. However, the genome-wide impact of Ctcf dosage on AS has not been investigated. We examined the effect of Ctcf haploinsufficiency on gene expression and AS in multiple tissues from Ctcf hemizygous (Ctcf+/-) mice. Distinct tissue-specific differences in gene expression and AS were observed in Ctcf+/- mice compared to wildtype mice. We observed a surprisingly large number of increased Intron Retention events in Ctcf+/- liver and kidney, specifically in genes associated with cytoskeletal organization, splicing and metabolism. This study provides further evidence for Ctcf dose-dependent and tissue-specific regulation of gene expression and AS. Our data provide a strong foundation for elucidating the mechanistic role of CTCF in AS regulation and its biological consequences.
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Intron Retention enhances gene regulatory complexity in vertebrates
Genome Biology, 2017Co-Authors: Ulf Schmitz, Justin J.-l. Wong, Natalia Pinello, William Ritchie, Fangzhi Jia, Sultan Alasmari, Maria-cristina Keightley, Shaniko Shini, Graham Lieschke, John E.j. RaskoAbstract:While Intron Retention (IR) is now widely accepted as an important mechanism of mammalian gene expression control, it remains the least studied form of alternative splicing. To delineate conserved features of IR, we performed an exhaustive phylogenetic analysis in a highly purified and functionally defined cell type comprising neutrophilic granulocytes from five vertebrate species spanning 430 million years of evolution.
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Intron Retention enhances gene regulatory complexity in vertebrates
Genome Biology, 2017Co-Authors: Ulf Schmitz, Justin J.-l. Wong, Natalia Pinello, William Ritchie, Fangzhi Jia, Sultan Alasmari, Maria-cristina Keightley, Shaniko Shini, Graham Lieschke, John E.j. RaskoAbstract:While Intron Retention (IR) is now widely accepted as an important mechanism of mammalian gene expression control, it remains the least studied form of alternative splicing. To delineate conserved features of IR, we performed an exhaustive phylogenetic analysis in a highly purified and functionally defined cell type comprising neutrophilic granulocytes from five vertebrate species spanning 430 million years of evolution. Our RNA-sequencing-based analysis suggests that IR increases gene regulatory complexity, which is indicated by a strong anti-correlation between the number of genes affected by IR and the number of protein-coding genes in the genome of individual species. Our results confirm that IR affects many orthologous or functionally related genes in granulocytes. Further analysis uncovers new and unanticipated conserved characteristics of Intron-retaining transcripts. We find that Intron-retaining genes are transcriptionally co-regulated from bidirectional promoters. Intron-retaining genes have significantly longer 3′ UTR sequences, with a corresponding increase in microRNA binding sites, some of which include highly conserved sequence motifs. This suggests that Intron-retaining genes are highly regulated post-transcriptionally. Our study provides unique insights concerning the role of IR as a robust and evolutionarily conserved mechanism of gene expression regulation. Our findings enhance our understanding of gene regulatory complexity by adding another contributor to evolutionary adaptation.
William Ritchie - One of the best experts on this subject based on the ideXlab platform.
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Intron Retention and its impact on gene expression and protein diversity a review and a practical guide
Wiley Interdisciplinary Reviews - Rna, 2021Co-Authors: David F Grabski, Lucile Broseus, Marielouise Hammarskjold, David Rekosh, Bandana Kumari, William RitchieAbstract:Intron Retention (IR) occurs when a complete and unspliced Intron remains in mature mRNA. An increasing body of literature has demonstrated a major role for IR in numerous biological functions, including several that impact human health and disease. Although experimental technologies used to study other forms of mRNA splicing can also be used to investigate IR, a specialized downstream computational analysis is optimal for IR discovery and analysis. Here we provide a review of IR and its biological implications, as well as a practical guide for how to detect and analyze it. Several methods, including long read third generation direct RNA sequencing, are described. We have developed an R package, FakIR, to facilitate the execution of the bioinformatic tasks recommended in this review and a tutorial on how to fit them to users aims. Additionally, we provide guidelines and experimental protocols to validate IR discovery and to evaluate the potential impact of IR on gene expression and protein output. This article is categorized under: RNA Evolution and Genomics > Computational Analyses of RNA RNA Processing > Splicing Regulation/Alternative Splicing RNA Methods > RNA Analyses in vitro and In Silico.
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s irfinder stable and accurate measurement of Intron Retention
bioRxiv, 2020Co-Authors: Lucile Bros Eacuteus, William RitchieAbstract:Accurate and reliable quantification of Intron Retention levels is currently the crux for detecting and interpreting the function of retained Introns. Using both simulated and real RNA-seq datasets, we show that current methods suffer from several biases and artifacts, which impair the analysis of Intron Retention. We designed a new approach to measuring Intron Retention called the Stable Intron Retention ratio that we have implemented in a novel algorithm to detect and measure Intron Retention called S-IRFindeR. We demonstrate that it provides a significant improvement in accuracy, higher consistency between replicates and agreement with IR-levels computed from long-read sequencing data. S-IRFindeR is freely available at: https://github.com/lbroseus/SIRFindeR/.
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challenges in detecting and quantifying Intron Retention from next generation sequencing data
Computational and structural biotechnology journal, 2020Co-Authors: Lucile Broseus, William RitchieAbstract:Intron Retention (IR) occurs when an Intron is transcribed into pre-mRNA and remains in the final mRNA. An increasing body of literature has demonstrated a major role for IR in numerous biological functions and in disease. Here we give an overview of the different computational approaches for detecting IR events from sequencing data. We show that these are based on different biological and computational assumptions that may lead to dramatically different results. We describe the various approaches for mitigating errors in detecting Intron Retention and for discovering IR signatures between different conditions.
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Intron Retention enhances gene regulatory complexity in vertebrates
Genome Biology, 2017Co-Authors: Ulf Schmitz, Justin J.-l. Wong, Natalia Pinello, William Ritchie, Fangzhi Jia, Sultan Alasmari, Maria-cristina Keightley, Shaniko Shini, Graham Lieschke, John E.j. RaskoAbstract:While Intron Retention (IR) is now widely accepted as an important mechanism of mammalian gene expression control, it remains the least studied form of alternative splicing. To delineate conserved features of IR, we performed an exhaustive phylogenetic analysis in a highly purified and functionally defined cell type comprising neutrophilic granulocytes from five vertebrate species spanning 430 million years of evolution.
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Intron Retention enhances gene regulatory complexity in vertebrates
Genome Biology, 2017Co-Authors: Ulf Schmitz, Justin J.-l. Wong, Natalia Pinello, William Ritchie, Fangzhi Jia, Sultan Alasmari, Maria-cristina Keightley, Shaniko Shini, Graham Lieschke, John E.j. RaskoAbstract:While Intron Retention (IR) is now widely accepted as an important mechanism of mammalian gene expression control, it remains the least studied form of alternative splicing. To delineate conserved features of IR, we performed an exhaustive phylogenetic analysis in a highly purified and functionally defined cell type comprising neutrophilic granulocytes from five vertebrate species spanning 430 million years of evolution. Our RNA-sequencing-based analysis suggests that IR increases gene regulatory complexity, which is indicated by a strong anti-correlation between the number of genes affected by IR and the number of protein-coding genes in the genome of individual species. Our results confirm that IR affects many orthologous or functionally related genes in granulocytes. Further analysis uncovers new and unanticipated conserved characteristics of Intron-retaining transcripts. We find that Intron-retaining genes are transcriptionally co-regulated from bidirectional promoters. Intron-retaining genes have significantly longer 3′ UTR sequences, with a corresponding increase in microRNA binding sites, some of which include highly conserved sequence motifs. This suggests that Intron-retaining genes are highly regulated post-transcriptionally. Our study provides unique insights concerning the role of IR as a robust and evolutionarily conserved mechanism of gene expression regulation. Our findings enhance our understanding of gene regulatory complexity by adding another contributor to evolutionary adaptation.
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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Intron Retention mechanisms that regulate sf3b1 and mitoferrin gene expression during late erythropoiesis
Blood, 2016Co-Authors: Marilyn Parra, Gene W Yeo, John G ConboyAbstract:Intron Retention (IR) regulates hundreds of erythroid genes in a differentiation stage-specific manner during terminal erythropoiesis. Regulated genes include highly expressed RNA binding proteins (RBPs) such as SF3B1, as well as iron transporters (e.g., mitoferrins 1 and 2), and cytoskeletal proteins (e.g., alpha spectrin). Selected IR transcripts are relatively abundant; 25-50% of the above-mentioned transcripts can be polyadenylated, retained in the nucleus, and efficiently spliced at all but one or two Introns, thus limiting the amount of translatable cytoplasmic mRNA. We are studying novel IR regulatory mechanisms involving both splice site strength and deeper Intronic elements, using model Introns that are differentially regulated in human erythroblasts. SF3B1, a key pre-RNA splicing factor implicated in MDS, exhibits dynamic regulation of Intron 4, with low IR in proerythroblasts and high IR in mature orthochromatic erythroblasts. Intron 4 sequences include three ultraconserved elements that encode cryptic exons we term 4a, 4b, and 4c, two of which (4a and 4c) encode premature termination codons (PTCs) expected to induce nonsense-mediated decay (NMD) if spliced into SF3B1 transcripts. We hypothesize that these PTC exons can have an additional function as inefficiently spliced decoy(s), that is, their splice sites may interact with splice sites at the boundaries of Intron 4 to form non-productive complexes that do not permit efficient splicing but instead prevent excision of the Intron. This concept represents an extension of decoy models previously proposed by others to explain selected exon skipping events, and is supported by several recent findings: eCLIP (enhanced cross-linking and immunoprecipitation) data indicate strong binding of 39 splice site factors U2AF1 and U2AF2 at these exons; low levels of exon 4a and 4c splicing can be seen in NMD-inhibited cells; and deletion of exon 4c from minigene splicing reporters decreases IR. Besides SF3B1, we identified numerous other dynamically-regulated IR events encompassing cryptic PTC exons that bind 39 splice site factors. In contrast to the dynamic regulation of IR in SF3B1, IR in SLC25A37 (mitoferrin-1) and SLC25A28 (mitoferrin-2) is stably maintained at a high level throughout terminal erythropoiesis. Baseline IR levels for stably retained Introns correlate with splice site strength, but are also influenced by deeper Intronic elements. For example, SLC25A28 Intron 2 contains Intronic elements that appear to reduce IR, since blocking them with antisense morpholinos leads to substantially increased IR levels. These studies demonstrate that IR in major erythroid genes is regulated by sequences within the retained Introns that can either increase or decrease Retention, suggesting that multiple IR pathways are employed during terminal erythropoiesis to regulate gene expression. Disclosures No relevant conflicts of interest to declare.
Dingxiao Zhang - One of the best experts on this subject based on the ideXlab platform.
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Intron Retention is a hallmark and spliceosome represents a therapeutic vulnerability in aggressive prostate cancer
Nature Communications, 2020Co-Authors: Dingxiao Zhang, Hsueh Ping Chao, Amanda Tracz, Jason Kirk, Silvia Buonamici, Xiaozhuo Liu, Yan Liu, Ping ZhuAbstract:The role of dysregulation of mRNA alternative splicing (AS) in the development and progression of solid tumors remains to be defined. Here we describe the first comprehensive AS landscape in the spectrum of human prostate cancer (PCa) evolution. We find that the severity of splicing dysregulation correlates with disease progression and establish Intron Retention as a hallmark of PCa stemness and aggressiveness. Systematic interrogation of 274 splicing-regulatory genes (SRGs) uncovers prevalent genomic copy number variations (CNVs), leading to mis-expression of ~68% of SRGs during PCa development and progression. Consequently, many SRGs are prognostic. Surprisingly, androgen receptor controls a splicing program distinct from its transcriptional regulation. The spliceosome modulator, E7107, reverses cancer aggressiveness and inhibits castration-resistant PCa (CRPC) in xenograft and autochthonous PCa models. Altogether, our studies establish aberrant AS landscape caused by dysregulated SRGs as a hallmark of PCa aggressiveness and the spliceosome as a therapeutic vulnerability for CRPC.
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dysregulated alternative splicing landscape identifies Intron Retention as a hallmark and spliceosome as a therapeutic vulnerability in aggressive prostate cancer
bioRxiv, 2019Co-Authors: Dingxiao Zhang, Hsueh Ping Chao, Amanda Tracz, Jason Kirk, Silvia Buonamici, Ping Zhu, Jianmin Wang, Song Liu, Dean G TangAbstract:ABSTRACT Dysregulation of mRNA alternative splicing (AS) has been implicated in development and progression of hematological malignancies. Here we describe the first comprehensive AS landscape in the spectrum of human prostate cancer (PCa) development, progression and therapy resistance. We find that the severity of splicing dysregulation correlates with disease progression and establish Intron Retention (IR) as a hallmark of PCa stemness and aggressiveness. Systematic interrogation of 274 splicing-regulatory genes (SRGs) uncovers prevalent SRG mutations associated with, mainly, copy number variations leading to mis-expression of ~68% of SRGs during PCa evolution. Consequently, we identify many SRGs as prognostic markers associated with splicing disruption and patient outcome. Interestingly, androgen receptor (AR) controls a splicing program distinct from its transcriptional regulation. The spliceosome modulator, E7107, reverses cancer aggressiveness and abolishes the growth of castration-resistant PCa (CRPC) models. Altogether, we establish aberrant AS landscape caused by dysregulated SRGs as a novel therapeutic vulnerability for CRPC. Statement of significance We present the first comprehensive AS landscape during PCa evolution and link genomic and transcriptional alterations in SRGs to global splicing dysregulation. AR regulates splicing in pri-PCa and CRPC distinct from its transcriptional regulation. Intron Retention is a hallmark for and spliceosome represents a therapeutic vulnerability in aggressive PCa.
