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Joseph A. Adams - One of the best experts on this subject based on the ideXlab platform.
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molecular interactions connecting the function of the serine arginine rich Protein SRsf1 to Protein phosphatase 1
Journal of Biological Chemistry, 2018Co-Authors: Brandon E. Aubol, Laurent Fattet, P Serrano, Kurt Wuthrich, Joseph A. AdamsAbstract:Splicing generates many mRNA strands from a single precursor mRNA, expanding the proteome and enhancing intracellular diversity. Both initial assembly and activation of the spliceosome require an essential family of splicing factors called serine-arginine (SR) Proteins. Protein phosphatase 1 (PP1) regulates the SR Proteins by controlling phosphorylation of a C-terminal arginine-serine–rich (RS) domain. These modifications are vital for the subcellular localization and mRNA splicing function of the SR Protein. Although PP1 has been shown to dephosphorylate the prototype SR Protein splicing factor 1 (SRSF1), the molecular nature of this interaction is not understood. Here, using NMR spectroscopy, we identified two electrostatic residues in helix α2 and a hydrophobic residue in helix α1 in the RNA recognition motif 1 (RRM1) of SRSF1 that constitute a binding surface for PP1. Substitution of these residues dissociated SRSF1 from PP1 and enhanced phosphatase activity, reducing phosphorylation in the RS domain. These effects lead to shifts in alternative splicing patterns that parallel increases in SRSF1 diffusion from speckles to the nucleoplasm brought on by regiospecific decreases in RS domain phosphorylation. Overall, these findings establish a molecular and biological connection between PP1-targeted amino acids in an RRM with the phosphorylation state and mRNA-processing function of an SR Protein.
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Redirecting SR Protein Nuclear Trafficking through an Allosteric Platform.
Journal of molecular biology, 2017Co-Authors: Brandon E. Aubol, Kendra L. Hailey, Laurent Fattet, Patricia A. Jennings, Joseph A. AdamsAbstract:Abstract Although phosphorylation directs serine-arginine (SR) Proteins from nuclear storage speckles to the nucleoplasm for splicing function, dephosphorylation paradoxically induces similar movement, raising the question of how such chemical modifications are balanced in these essential splicing factors. In this new study, we investigated the interaction of Protein phosphatase 1 (PP1) with the SR Protein splicing factor (SRSF1) to understand the foundation of these opposing effects in the nucleus. We found that RNA recognition motif 1 (RRM1) in SRSF1 binds PP1 and represses its catalytic function through an allosteric mechanism. DiSRuption of RRM1–PP1 interactions reduces the phosphorylation status of the RS domain in vitro and in cells, redirecting SRSF1 in the nucleus. The data imply that an allosteric SR Protein-phosphatase platform balances phosphorylation levels in a “goldilocks” region for the proper subnuclear storage of an SR Protein splicing factor.
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Conserved proline-directed phosphorylation regulates SR Protein conformation and splicing function.
The Biochemical journal, 2015Co-Authors: Malik M. Keshwani, Brandon E. Aubol, Laurent Fattet, Patricia A. Jennings, Jinsong Qiu, Joseph A. AdamsAbstract:The alternative splicing of human genes is dependent on SR Proteins, a family of essential splicing factors whose name derives from a signature C-terminal domain rich in arginine–serine dipeptide repeats (RS domains). Although the SRPKs (SR-specific Protein kinases) phosphorylate these repeats, RS domains also contain prolines with flanking serines that are phosphorylated by a second family of Protein kinases known as the CLKs (Cdc2-like kinases). The role of specific serine–proline phosphorylation within the RS domain has been difficult to assign since CLKs also phosphorylate arginine–serine dipeptides and, thus, display overlapping residue specificities with the SRPKs. In the present study, we address the effects of discrete serine–proline phosphorylation on the conformation and cellular function of the SR Protein SRSF1 (SR Protein splicing factor 1). Using chemical tagging and dephosphorylation experiments, we show that modification of serine–proline dipeptides broadly amplifies the conformational ensemble of SRSF1. The induction of these new structural forms triggers SRSF1 mobilization in the nucleus and alters its binding mechanism to an exonic splicing enhancer in precursor mRNA. These physical events correlate with changes in the alternative splicing of over 100 human genes based on a global splicing assay. Overall, these studies draw a direct causal relationship between a specific type of chemical modification in an SR Protein and the regulation of alternative gene splicing programmes. Abbreviations: CLK1, Cdc2-like kinase 1; ESE, exonic splicing enhancer; PP1, Protein phosphatase-1; RRM, RNA recognition motif; RS, domain, domain rich in arginine–serine repeats; SR, Protein, splicing factor containing a C-terminal RS domain; SRPK1, SR-specific Protein kinase 1; SRSF1, SR Protein splicing factor 1 (aka ASF/SF2)
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N-terminus of the Protein kinase CLK1 induces SR Protein hyperphosphorylation.
The Biochemical journal, 2014Co-Authors: Brandon E. Aubol, Malik M. Keshwani, Ryan M. Plocinik, Maria L. Mcglone, Jonathan C. Hagopian, Gourisankar Ghosh, Joseph A. AdamsAbstract:SR Proteins are essential splicing factors that are regulated through multisite phosphorylation of their RS (arginine/serine-rich) domains by two major families of Protein kinases. The SRPKs (SR-specific Protein kinases) efficiently phosphorylate the arginine/serine dipeptides in the RS domain using a conserved docking groove in the kinase domain. In contrast, CLKs (Cdc2-like kinases) lack a docking groove and phosphorylate both arginine/serine and serine–proline dipeptides, modifications that generate a hyperphosphorylated state important for unique SR Protein-dependent splicing activities. All CLKs contain long flexible N-terminal extensions (140–300 residues) that resemble the RS domains present in their substrate SR Proteins. We showed that the N-terminus in CLK1 contacts both the kinase domain and the RS domain of the SR Protein SRSF1 (SR Protein splicing factor 1). This interaction not only is essential for facilitating hyperphosphorylation, but also induces co-operative binding of SRSF1 to RNA. The N-terminus of CLK1 enhances the total phosphoryl contents of a panel of physiological substrates including SRSF1, SRSF2, SRSF5 and Tra2β1 (transformer 2β1) by 2–3-fold. These findings suggest that CLK1-dependent hyperphosphorylation is the result of a general mechanism in which the N-terminus acts as a bridge connecting the kinase domain and the RS domain of the SR Protein.
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Splicing kinase SRPK1 conforms to the landscape of its SR Protein substrate.
Biochemistry, 2013Co-Authors: Brandon E. Aubol, Maria L. Mcglone, Michael A. Jamros, Joseph A. AdamsAbstract:The splicing function of SR Proteins is regulated by multisite phosphorylation of their C-terminal RS (arginine–serine rich) domains. SRPK1 has been shown to phosphorylate the prototype SR Protein SRSF1 using a directional mechanism in which 11 serines flanked by arginines are sequentially fed from a docking groove in the large lobe of the kinase domain to the active site. Although this process is expected to operate on lengthy arginine–serine repeats (≥8), many SR Proteins contain smaller repeats of only 1–4 dipeptides, raising the question of how alternate RS domain configurations are phosphorylated. To address this, we studied a splice variant of Tra2β that contains a C-terminal RS domain with short arginine–serine repeats [Tra2β(ΔN)]. We showed that SRPK1 selectively phosphorylates several serines near the C-terminus of the RS domain. SRPK1 uses a distributive mechanism for Tra2β(ΔN) where the rate-limiting step is the dissociation of the Protein substrate rather than nucleotide exchange as in the cas...
Jamal Tazi - One of the best experts on this subject based on the ideXlab platform.
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A Role for the Serine/Arginine-Rich (SR) Protein B52/SRSF6 in Cell Growth and Myc Expression in Drosophila
Genetics, 2015Co-Authors: Celine Fernando, Jamal Tazi, Agnes Audibert, Francoise Simon, Francois JugeAbstract:Serine-/arginine-rich (SR) Proteins are RNA-binding Proteins that are primarily involved in alternative splicing. Expression of some SR Proteins is frequently upregulated in tumors, and previous reports have demonstrated that these Proteins can directly participate in cell transformation. Identifying factors that can rescue the effects of SR overexpression in vivo is, therefore, of potential therapeutic interest. Here, we analyzed phenotypes induced by overexpression of the SR Protein B52 during Drosophila development and identified several Proteins that can rescue these phenotypes. Using the mechanosensory bristle lineage as a developmental model, we show that B52 expression level influences cell growth, but not differentiation, in this lineage. In particular, B52 overexpression increases cell growth, upregulates myc transcription, and gives rise to flies lacking thoracic bristles. Using a genetic screen, we identified several suppressors of the phenotypes induced by overexpression of B52 in vivo in two different organs. We show that upregulation of brain tumor (brat), a tumor suppressor and post-transcriptional repressor of myc, and downregulation of lilliputian (lilli), a subunit of the superelongation complex involved in transcription elongation, efficiently rescue the phenotypes induced by B52 overexpression. Our results demonstrate a role of this SR Protein in cell growth and identify candidate Proteins that may overcome the effects of SR Protein overexpression in mammals.
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a role for the serine arginine rich SR Protein b52 SRsf6 in cell growth and myc expression in drosophila
Genetics, 2015Co-Authors: Celine Fernando, Jamal Tazi, Agnes Audibert, Francoise Simon, Francois JugeAbstract:Serine-/arginine-rich (SR) Proteins are RNA-binding Proteins that are primarily involved in alternative splicing. Expression of some SR Proteins is frequently upregulated in tumors, and previous reports have demonstrated that these Proteins can directly participate in cell transformation. Identifying factors that can rescue the effects of SR overexpression in vivo is, therefore, of potential therapeutic interest. Here, we analyzed phenotypes induced by overexpression of the SR Protein B52 during Drosophila development and identified several Proteins that can rescue these phenotypes. Using the mechanosensory bristle lineage as a developmental model, we show that B52 expression level influences cell growth, but not differentiation, in this lineage. In particular, B52 overexpression increases cell growth, upregulates myc transcription, and gives rise to flies lacking thoracic bristles. Using a genetic screen, we identified several suppressors of the phenotypes induced by overexpression of B52 in vivo in two different organs. We show that upregulation of brain tumor (brat), a tumor suppressor and post-transcriptional repressor of myc, and downregulation of lilliputian (lilli), a subunit of the superelongation complex involved in transcription elongation, efficiently rescue the phenotypes induced by B52 overexpression. Our results demonstrate a role of this SR Protein in cell growth and identify candidate Proteins that may overcome the effects of SR Protein overexpression in mammals.
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the SR Protein b52 SRp55 is required for dna topoisomerase i recruitment to chromatin mrna release and transcription shutdown
PLOS Genetics, 2010Co-Authors: Francois Juge, Celine Fernando, W. Fic, Jamal TaziAbstract:DNA- and RNA-processing pathways are integrated and interconnected in the eukaryotic nucleus to allow efficient gene expression and to maintain genomic stability. The recruitment of DNA Topoisomerase I (Topo I), an enzyme controlling DNA supercoiling and acting as a specific kinase for the SR-Protein family of splicing factors, to highly transcribed loci represents a mechanism by which transcription and processing can be coordinated and genomic instability avoided. Here we show that Drosophila Topo I associates with and phosphorylates the SR Protein B52. Surprisingly, expression of a high-affinity binding site for B52 in transgenic flies restricted localization, not only of B52, but also of Topo I to this single transcription site, whereas B52 RNAi knockdown induced mis-localization of Topo I in the nucleolus. Impaired delivery of Topo I to a heat shock gene caused retention of the mRNA at its site of transcription and delayed gene deactivation after heat shock. Our data show that B52 delivers Topo I to RNA polymerase II-active chromatin loci and provide the first evidence that DNA topology and mRNA release can be coordinated to control gene expression.
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Protection against retrovirus pathogenesis by SR Protein inhibitors.
PLoS ONE, 2009Co-Authors: Anne Keriel, Florence Mahuteau-betzer, Chantal Jacquet, Marc Plays, David Grierson, Marc Sitbon, Jamal TaziAbstract:Indole derivatives compounds (IDC) are a new class of splicing inhibitors that have a selective action on exonic splicing enhancers (ESE)-dependent activity of individual serine-arginine-rich (SR) Proteins. Some of these molecules have been shown to compromise assembly of HIV infectious particles in cell cultures by interfering with the activity of the SR Protein SF2/ASF and by subsequently suppressing production of splicing-dependent retroviral accessory Proteins. For all replication-competent retroviruses, a limiting requirement for infection and pathogenesis is the expression of the envelope glycoProtein which strictly depends on the host splicing machinery. Here, we have evaluated the efficiency of IDC on an animal model of retroviral pathogenesis using a fully replication-competent retrovirus. In this model, all newborn mice infected with a fully replicative murine leukemia virus (MLV) develop erythroleukemia within 6 to 8 weeks of age. We tested several IDC for their ability to interfere ex vivo with MLV splicing and virus spreading as well as for their protective effect in vivo. We show here that two of these IDC, IDC13 and IDC78, selectively altered splicing-dependent production of the retroviral envelope gene, thus inhibiting early viral replication in vivo, sufficiently to protect mice from MLV-induced pathogenesis. The apparent specificity and clinical safety observed here for both IDC13 and IDC78 strongly support further assessment of inhibitors of SR Protein splicing factors as a new class of antiretroviral therapeutic agents.
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a novel role for pa28γ proteasome in nuclear speckle organization and SR Protein trafficking
Molecular Biology of the Cell, 2008Co-Authors: Veronique Baldin, W. Fic, Muriel Militello, Yann Thomas, Christine Doucet, Stephanie Boireau, Isabelle Jarielencontre, Marc Piechaczyk, Edouard Bertrand, Jamal TaziAbstract:In eukaryotic cells, proteasomes play an essential role in intracellular proteolysis and are involved in the control of most biological processes through regulated degradation of key Proteins. Analysis of 20S proteasome localization in human cell lines, using ectopic expression of its CFP-tagged α7 subunit, revealed the presence in nuclear foci of a specific and proteolytically active complex made by association of the 20S proteasome with its PA28γ regulator. Identification of these foci as the nuclear speckles (NS), which are dynamic subnuclear structures enriched in splicing factors (including the SR Protein family), prompted us to analyze the role(s) of proteasome-PA28γ complexes in the NS. Here, we show that knockdown of these complexes by small interfering RNAs directed against PA28γ strongly impacts the organization of the NS. Further analysis of PA28γ-depleted cells demonstrated an alteration of intranuclear trafficking of SR Proteins. Thus, our data identify proteasome-PA28γ complexes as a novel regulator of NS organization and function, acting most likely through selective proteolysis. These results constitute the first demonstration of a role of a specific proteasome complex in a defined subnuclear compartment and suggest that proteolysis plays important functions in the precise control of splicing factors trafficking within the nucleus.
James L. Manley - One of the best experts on this subject based on the ideXlab platform.
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new talents for an old acquaintance the SR Protein splicing factor asf sf2 functions in the maintenance of genome stability
Cell Cycle, 2005Co-Authors: James L. ManleyAbstract:ASF/SF2 is a well-studied SR Protein that plays important roles in premRNA splicing and other aspects of RNA metabolism. Genetic inactivation experiments have revealed the fundamental roles of ASF/SF2 and other SR Proteins in cell viability and animal development. However, the nature of the events triggered by in vivo depletion of ASF/SF2 remained largely elusive. Recently, we have demonstrated a significant function of ASF/SF2 in the maintenance of genome stability by preventing the formation of R loops, which provided new insights into the essential roles of ASF/SF2 in cellular physiology.
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inactivation of the SR Protein splicing factor asf sf2 results in genomic instability
Cell, 2005Co-Authors: James L. ManleyAbstract:SR Proteins constitute a family of pre-mRNA splicing factors now thought to play several roles in mRNA metabolism in metazoan cells. Here we provide evidence that a prototypical SR Protein, ASF/SF2, is unexpectedly required for maintenance of genomic stability. We first show that in vivo depletion of ASF/SF2 results in a hypermutation phenotype likely due to DNA rearrangements, reflected in the rapid appearance of DNA double-strand breaks and high-molecular-weight DNA fragments. Analysis of DNA from ASF/SF2-depleted cells revealed that the nontemplate strand of a transcribed gene was single stranded due to formation of an RNA:DNA hybrid, R loop structure. Stable overexpression of RNase H suppressed the DNA-fragmentation and hypermutation phenotypes. Indicative of a direct role, ASF/SF2 prevented R loop formation in a reconstituted in vitro transcription reaction. Our results support a model by which recruitment of ASF/SF2 to nascent transcripts by RNA polymerase II prevents formation of mutagenic R loop structures.
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Regulation and Substrate Specificity of the SR Protein Kinase Clk/Sty
Molecular and cellular biology, 2003Co-Authors: Jayendra Prasad, James L. ManleyAbstract:SR Proteins constitute a family of splicing factors that play key roles in both constitutive and regulated splicing in metazoan organisms. The Proteins are extensively phosphorylated, and kinases capable of phosphorylating them have been identified. However, little is known about how these kinases function, for example, whether they target specific SR Proteins or whether the kinases themselves are regulated. Here we describe properties of one such kinase, Clk/Sty, the founding member of the Clk/Sty family of dual-specificity kinases. Clk/Sty is autophosphorylated on both Ser/Thr and Thr residues, and using both direct kinase assays and SR Protein-dependent splicing assays, we have analyzed the effects of each type of modification. We find not only that the pattern of phosphorylation on a specific SR Protein substrate, ASF/SF2, is modulated by autophosphorylation but also that the ability of Clk/Sty to recognize different SR Proteins is influenced by the extent and nature of autophosphorylation. Strikingly, phosphorylation of ASF/SF2 is sensitive to changes in Tyr, but not Ser/Thr, autophosphorylation while that of SC35 displays the opposite pattern. In contrast, phosphorylation of a third SR Protein, SRp40, is unaffected by autophosphorylation. We also present biochemical data indicating that as expected for a factor directly involved in splicing control (but in contrast to recent reports), Clk/Sty is found in the nucleus of several different cell types.
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regulation and substrate specificity of the SR Protein kinase clk sty
Molecular and Cellular Biology, 2003Co-Authors: Jayendra Prasad, James L. ManleyAbstract:SR Proteins constitute a family of splicing factors that play key roles in both constitutive and regulated splicing in metazoan organisms. The Proteins are extensively phosphorylated, and kinases capable of phosphorylating them have been identified. However, little is known about how these kinases function, for example, whether they target specific SR Proteins or whether the kinases themselves are regulated. Here we describe properties of one such kinase, Clk/Sty, the founding member of the Clk/Sty family of dual-specificity kinases. Clk/Sty is autophosphorylated on both Ser/Thr and Thr residues, and using both direct kinase assays and SR Protein-dependent splicing assays, we have analyzed the effects of each type of modification. We find not only that the pattern of phosphorylation on a specific SR Protein substrate, ASF/SF2, is modulated by autophosphorylation but also that the ability of Clk/Sty to recognize different SR Proteins is influenced by the extent and nature of autophosphorylation. Strikingly, phosphorylation of ASF/SF2 is sensitive to changes in Tyr, but not Ser/Thr, autophosphorylation while that of SC35 displays the opposite pattern. In contrast, phosphorylation of a third SR Protein, SRp40, is unaffected by autophosphorylation. We also present biochemical data indicating that as expected for a factor directly involved in splicing control (but in contrast to recent reports), Clk/Sty is found in the nucleus of several different cell types.
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The SR Protein SRp38 represses splicing in M phase cells.
Cell, 2002Co-Authors: Chanseok Shin, James L. ManleyAbstract:SR Proteins constitute a family of pre-mRNA splicing factors that play important roles in both constitutive and regulated splicing. Here, we describe one member of the family, which we call SRp38, with unexpected properties. Unlike other SR Proteins, SRp38 cannot activate splicing and is essentially inactive in splicing assays. However, dephosphorylation converts SRp38 to a potent, general repressor that inhibits splicing at an early step. To investigate the cellular function of SRp38, we examined its possible role in cell cycle control. We show first that splicing, like other steps in gene expression, is inhibited in extracts of mitotic cells. Strikingly, SRp38 was found to be dephosphorylated specifically in mitotic cells, and we show that dephosphorylated SRp38 is required for the observed splicing repression.
Brandon E. Aubol - One of the best experts on this subject based on the ideXlab platform.
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molecular interactions connecting the function of the serine arginine rich Protein SRsf1 to Protein phosphatase 1
Journal of Biological Chemistry, 2018Co-Authors: Brandon E. Aubol, Laurent Fattet, P Serrano, Kurt Wuthrich, Joseph A. AdamsAbstract:Splicing generates many mRNA strands from a single precursor mRNA, expanding the proteome and enhancing intracellular diversity. Both initial assembly and activation of the spliceosome require an essential family of splicing factors called serine-arginine (SR) Proteins. Protein phosphatase 1 (PP1) regulates the SR Proteins by controlling phosphorylation of a C-terminal arginine-serine–rich (RS) domain. These modifications are vital for the subcellular localization and mRNA splicing function of the SR Protein. Although PP1 has been shown to dephosphorylate the prototype SR Protein splicing factor 1 (SRSF1), the molecular nature of this interaction is not understood. Here, using NMR spectroscopy, we identified two electrostatic residues in helix α2 and a hydrophobic residue in helix α1 in the RNA recognition motif 1 (RRM1) of SRSF1 that constitute a binding surface for PP1. Substitution of these residues dissociated SRSF1 from PP1 and enhanced phosphatase activity, reducing phosphorylation in the RS domain. These effects lead to shifts in alternative splicing patterns that parallel increases in SRSF1 diffusion from speckles to the nucleoplasm brought on by regiospecific decreases in RS domain phosphorylation. Overall, these findings establish a molecular and biological connection between PP1-targeted amino acids in an RRM with the phosphorylation state and mRNA-processing function of an SR Protein.
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Redirecting SR Protein Nuclear Trafficking through an Allosteric Platform.
Journal of molecular biology, 2017Co-Authors: Brandon E. Aubol, Kendra L. Hailey, Laurent Fattet, Patricia A. Jennings, Joseph A. AdamsAbstract:Abstract Although phosphorylation directs serine-arginine (SR) Proteins from nuclear storage speckles to the nucleoplasm for splicing function, dephosphorylation paradoxically induces similar movement, raising the question of how such chemical modifications are balanced in these essential splicing factors. In this new study, we investigated the interaction of Protein phosphatase 1 (PP1) with the SR Protein splicing factor (SRSF1) to understand the foundation of these opposing effects in the nucleus. We found that RNA recognition motif 1 (RRM1) in SRSF1 binds PP1 and represses its catalytic function through an allosteric mechanism. DiSRuption of RRM1–PP1 interactions reduces the phosphorylation status of the RS domain in vitro and in cells, redirecting SRSF1 in the nucleus. The data imply that an allosteric SR Protein-phosphatase platform balances phosphorylation levels in a “goldilocks” region for the proper subnuclear storage of an SR Protein splicing factor.
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Conserved proline-directed phosphorylation regulates SR Protein conformation and splicing function.
The Biochemical journal, 2015Co-Authors: Malik M. Keshwani, Brandon E. Aubol, Laurent Fattet, Patricia A. Jennings, Jinsong Qiu, Joseph A. AdamsAbstract:The alternative splicing of human genes is dependent on SR Proteins, a family of essential splicing factors whose name derives from a signature C-terminal domain rich in arginine–serine dipeptide repeats (RS domains). Although the SRPKs (SR-specific Protein kinases) phosphorylate these repeats, RS domains also contain prolines with flanking serines that are phosphorylated by a second family of Protein kinases known as the CLKs (Cdc2-like kinases). The role of specific serine–proline phosphorylation within the RS domain has been difficult to assign since CLKs also phosphorylate arginine–serine dipeptides and, thus, display overlapping residue specificities with the SRPKs. In the present study, we address the effects of discrete serine–proline phosphorylation on the conformation and cellular function of the SR Protein SRSF1 (SR Protein splicing factor 1). Using chemical tagging and dephosphorylation experiments, we show that modification of serine–proline dipeptides broadly amplifies the conformational ensemble of SRSF1. The induction of these new structural forms triggers SRSF1 mobilization in the nucleus and alters its binding mechanism to an exonic splicing enhancer in precursor mRNA. These physical events correlate with changes in the alternative splicing of over 100 human genes based on a global splicing assay. Overall, these studies draw a direct causal relationship between a specific type of chemical modification in an SR Protein and the regulation of alternative gene splicing programmes. Abbreviations: CLK1, Cdc2-like kinase 1; ESE, exonic splicing enhancer; PP1, Protein phosphatase-1; RRM, RNA recognition motif; RS, domain, domain rich in arginine–serine repeats; SR, Protein, splicing factor containing a C-terminal RS domain; SRPK1, SR-specific Protein kinase 1; SRSF1, SR Protein splicing factor 1 (aka ASF/SF2)
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N-terminus of the Protein kinase CLK1 induces SR Protein hyperphosphorylation.
The Biochemical journal, 2014Co-Authors: Brandon E. Aubol, Malik M. Keshwani, Ryan M. Plocinik, Maria L. Mcglone, Jonathan C. Hagopian, Gourisankar Ghosh, Joseph A. AdamsAbstract:SR Proteins are essential splicing factors that are regulated through multisite phosphorylation of their RS (arginine/serine-rich) domains by two major families of Protein kinases. The SRPKs (SR-specific Protein kinases) efficiently phosphorylate the arginine/serine dipeptides in the RS domain using a conserved docking groove in the kinase domain. In contrast, CLKs (Cdc2-like kinases) lack a docking groove and phosphorylate both arginine/serine and serine–proline dipeptides, modifications that generate a hyperphosphorylated state important for unique SR Protein-dependent splicing activities. All CLKs contain long flexible N-terminal extensions (140–300 residues) that resemble the RS domains present in their substrate SR Proteins. We showed that the N-terminus in CLK1 contacts both the kinase domain and the RS domain of the SR Protein SRSF1 (SR Protein splicing factor 1). This interaction not only is essential for facilitating hyperphosphorylation, but also induces co-operative binding of SRSF1 to RNA. The N-terminus of CLK1 enhances the total phosphoryl contents of a panel of physiological substrates including SRSF1, SRSF2, SRSF5 and Tra2β1 (transformer 2β1) by 2–3-fold. These findings suggest that CLK1-dependent hyperphosphorylation is the result of a general mechanism in which the N-terminus acts as a bridge connecting the kinase domain and the RS domain of the SR Protein.
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Splicing kinase SRPK1 conforms to the landscape of its SR Protein substrate.
Biochemistry, 2013Co-Authors: Brandon E. Aubol, Maria L. Mcglone, Michael A. Jamros, Joseph A. AdamsAbstract:The splicing function of SR Proteins is regulated by multisite phosphorylation of their C-terminal RS (arginine–serine rich) domains. SRPK1 has been shown to phosphorylate the prototype SR Protein SRSF1 using a directional mechanism in which 11 serines flanked by arginines are sequentially fed from a docking groove in the large lobe of the kinase domain to the active site. Although this process is expected to operate on lengthy arginine–serine repeats (≥8), many SR Proteins contain smaller repeats of only 1–4 dipeptides, raising the question of how alternate RS domain configurations are phosphorylated. To address this, we studied a splice variant of Tra2β that contains a C-terminal RS domain with short arginine–serine repeats [Tra2β(ΔN)]. We showed that SRPK1 selectively phosphorylates several serines near the C-terminus of the RS domain. SRPK1 uses a distributive mechanism for Tra2β(ΔN) where the rate-limiting step is the dissociation of the Protein substrate rather than nucleotide exchange as in the cas...
Masatoshi Hagiwara - One of the best experts on this subject based on the ideXlab platform.
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akt2 regulation of cdc2 like kinases clk sty serine arginine rich SR Protein phosphorylation and insulin induced alternative splicing of pkcβii messenger ribonucleic acid
Endocrinology, 2009Co-Authors: Kun Jiang, Masatoshi Hagiwara, Niketa A Patel, James E Watson, Hercules Apostolatos, Eden Kleiman, Olivia Hanson, Denise R CooperAbstract:Serine/arginine-rich (SR) Proteins play essential roles in the constitutive and regulated splicing of precursor mRNAs. Phosphorylation of the arginine/serine dipeptide-rich (RS) domain by SR Protein kinases such as Cdc2-like kinases (Clk/Sty) modulates their subcellular localization and activation. However, it remains unclear how these kinases and their target SR Proteins are regulated by extracellular signals. Regulation of Protein kinase C βII (PKCβII) pre-mRNA alternative splicing via exon inclusion by Akt2, a central kinase in insulin action, involves phosphorylation of SR Proteins. Here we showed that Akt2, in response to insulin, resulted in phosphorylation of Clk/Sty, which then altered SR Protein phosphorylation in concert with Akt2. Insulin-stimulated PKCβII pre-mRNA splicing was blocked by Clk/Sty and phosphatidylinositol-3-kinase inhibitors, and diabetic Akt2-null mouse tissues had impaired phospho-Clk/Sty, SR Protein phosphorylation, and PKCβII expression. Furthermore, we observed that Akt2 ph...
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Regulation of Binding of Lamin B Receptor to Chromatin by SR Protein Kinase and cdc2 Kinase in Xenopus Egg Extracts
Journal of Biological Chemistry, 2004Co-Authors: Makoto Takano, Satomi Hoshino, Kazuhiro Furukawa, Hiroshi Onogi, Masatoshi Hagiwara, Yuhei Koyama, Hiromi Ito, Tsuneyoshi HorigomeAbstract:Participation of multiple kinases in regulation of the binding of lamin B receptor (LBR) to chromatin was suggested previously (Takano, M., Takeuchi, M., Ito, H., Furukawa, K., Sugimoto, K., Omata, S., and Horigome, T. (2002) Eur. J. Biochem. 269, 943-953). To identify these kinases, regulation of the binding of the nucleoplasmic region (NK, amino acid residues 1-211) of LBR to sperm chromatin was studied using a cell cycle-dependent Xenopus egg extract in vitro. The binding was stimulated on specific phosphorylation of the NK fragment by an S-phase egg extract. Protein depletion with beads bearing SF2/ASF, which binds SR Protein kinases, abolished this stimulation, suggesting that an SR Protein kinase(s) is responsible for the activation of LBR. This was confirmed by direct phosphorylation and activation with recombinant SR Protein-specific kinase 1. The binding of the NK fragment to chromatin pretreated with an S-phase extract was suppressed by incubation with an M-phase extract. Enzyme inhibitor experiments revealed that multiple kinases participate in the suppression. One of these kinases was shown to be cdc2 kinase using a specific inhibitor, roscovitine, and Protein depletion with beads bearing p13, which specifically binds cdc2 kinase. Experiments involving a mutant NK fragment showed that the phosphorylation of serine 71 by cdc2 kinase is responsible for the suppression.
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spk 1 a c elegans SR Protein kinase homologue is essential for embryogenesis and required for germline development
Mechanisms of Development, 2000Co-Authors: Hidehito Kuroyanagi, Tomomi Kimura, Kazuhiro Wada, Naoki Hisamoto, Kunihiro Matsumoto, Masatoshi HagiwaraAbstract:SR-Protein kinases (SRPKs) and their substrates, serine/arginine-rich pre-mRNA splicing factors, are key components of splicing machinery and are well conserved across phyla. Despite extensive biochemical investigation, the physiological functions of SRPKs remain unclear. In the present study, cDNAs for SPK-1, a C. elegans SRPK homologue, and CeSF2, an SPK-1 substrate, were cloned. SPK-1 binds directly to and phosphorylates the RS domain of CeSF2 in vitro. Both spk-1 and CeSF2 are predominantly expressed in germlines. RNA interference (RNAi) experiments revealed that spk-1 and CeSF2 play an essential role at the embryonic stage of C. elegans. Furthermore, RNAi studies demonstrated that spk-1 is required for germline development in C. elegans. We provide evidence that RNAi, achieved by the soaking of L1 larvae, is beneficial in the study of gene function in post-embryonic germline development.
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the subcellular localization of sf2 asf is regulated by direct interaction with SR Protein kinases SRpks
Journal of Biological Chemistry, 1999Co-Authors: Jun Koizumi, Hiroshi Onogi, Adrian R Krainer, Akila Mayeda, Yoshichika Okamoto, Masatoshi HagiwaraAbstract:Abstract Serine/arginine-rich (SR) Proteins play an important role in constitutive and alternative pre-mRNA splicing. The C-terminal arginine-serine domain of these Proteins, such as SF2/ASF, mediates Protein-Protein interactions and is phosphorylatedin vivo. Using glutathione S-transferase (GST)-SF2/ASF-affinity chromatography, the SF2/ASF kinase activity was co-purified from HeLa cells with a 95-kDa Protein, which was recognized by an anti-SR Protein kinase (SRPK) 1 monoclonal antibody. Recombinant SRPK1 and SRPK2 bound to and phosphorylated GST-SF2/ASF in vitro. Phosphopeptide mapping showed that identical sites were phosphorylated in the pull-down kinase reaction with HeLa extracts and by recombinant SRPKs. Epitope-tagged SF2/ASF transiently expressed in COS7 cells co-immunoprecipitated with SRPKs. Deletion analysis mapped the phosphorylation sites to a region containing an (Arg-Ser)8 repeat beginning at residue 204, and far-Western analysis showed that the region is required for binding of SRPKs to SF2/ASF. Further binding studies showed that SRPKs bound unphosphorylated SF2/ASF but did not bind phosphorylated SF2/ASF. Expression of an SRPK2 kinase-inactive mutant caused accumulation of SF2/ASF in the cytoplasm. These results suggest that the formation of complexes between SF2/ASF and SRPKs, which is influenced by the phosphorylation state of SF2/ASF, may have regulatory roles in the assembly and localization of this splicing factor.
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Novel SR-Protein-specific kinase, SRPK2, disassembles nuclear speckles.
Biochemical and biophysical research communications, 1998Co-Authors: Noriyo Kuroyanagi, Hiroshi Onogi, Takashi Wakabayashi, Masatoshi HagiwaraAbstract:SR-Protein-specific kinase 1 (SRPK1) is first identified as a specific kinase for SR splicing factors. By RT-PCR of a conserved kinase domain, novel SR-Protein-specific kinase clones were isolated from mouse brain. The cloned cDNAs encode a 106 kDa Protein (648 amino acids, 92% identical to human SRPK1) and a 120 kDa Protein (681 amino acids, 58% identical to human SRPK1). Therefore, they were designated mSRPK1 and mSRPK2, respectively. Northern blotting revealed the ubiquitous expression of mSRPK1 in all tissues examined and the tissue-specific expression of mSRPK2 in testis, lung, and brain. Both kinases phosphorylated SF2/ASF, a member of SR Proteins in vitro and the phosphopeptide mappings were identical, indicating that these kinases phosphorylate the same site of SF2/ASF. Overexpression of mSRPK2 caused disassembly of cotransfected SF2/ASF and endogenous SC35. Our results indicate that SRPK family members may regulate the disassembly of the SR Proteins in a tissue-specific manner.
