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Douglas L Black - One of the best experts on this subject based on the ideXlab platform.
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multiple determinants of splicing repression activity in the Polypyrimidine Tract binding proteins ptbp1 and ptbp2
RNA, 2016Co-Authors: Niroshika Keppetipola, Shalini Sharma, Kyuhyeon Yeom, Adrian L Hernandez, Tessa Bui, Douglas L BlackAbstract:Most human genes generate multiple protein isoforms through alternative pre-mRNA splicing, but the mechanisms controlling alternative splicing choices by RNA binding proteins are not well understood. These proteins can have multiple paralogs expressed in different cell types and exhibiting different splicing activities on target exons. We examined the paralogous Polypyrimidine Tract binding proteins PTBP1 and PTBP2 to understand how PTBP1 can exhibit greater splicing repression activity on certain exons. Using both an in vivo coexpression assay and an in vitro splicing assay, we show that PTBP1 is more repressive than PTBP2 per unit protein on a target exon. Constructing chimeras of PTBP1 and 2 to determine amino acid features that contribute to their differential activity, we find that multiple segments of PTBP1 increase the repressive activity of PTBP2. Notably, when either RRM1 of PTBP2 or the linker peptide separating RRM2 and RRM3 are replaced with the equivalent PTBP1 sequences, the resulting chimeras are highly active for splicing repression. These segments are distinct from the known region of interaction for the PTBP1 cofactors Raver1 and Matrin3 in RRM2. We find that RRM2 of PTBP1 also increases the repression activity of an otherwise PTBP2 sequence, and that this is potentially explained by stronger binding by Raver1. These results indicate that multiple features over the length of the two proteins affect their ability to repress an exon.
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neuronal regulation of pre mrna splicing by Polypyrimidine Tract binding proteins ptbp1 and ptbp2
Critical Reviews in Biochemistry and Molecular Biology, 2012Co-Authors: Niroshika Keppetipola, Shalini Sharma, Douglas L BlackAbstract:Alternative splicing patterns are regulated by RNA binding proteins that assemble onto each pre-mRNA to form a complex RNP structure. The Polypyrimidine Tract binding protein, PTB, has served as an informative model for understanding how RNA binding proteins affect spliceosome assembly and how changes in the expression of these proteins can control complex programs of splicing in tissues. In this review, we describe the mechanisms of splicing regulation by PTB and its function, along with its paralog PTBP2, in neuronal development.
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u1 snrna directly interacts with Polypyrimidine Tract binding protein during splicing repression
Molecular Cell, 2011Co-Authors: Shalini Sharma, Frederic H T Allain, Christophe Maris, Douglas L BlackAbstract:Summary Splicing of the c-src N1 exon is repressed by the Polypyrimidine Tract-binding protein (PTB or PTBP1). During exon repression, the U1 snRNP binds properly to the N1 exon 5′ splice site but is made inactive by the presence of PTB. Examining the patterns of nuclease protection at this 5′ splice site, we find that the interaction of U1 is altered by the adjacent PTB. Interestingly, UV crosslinking identifies a direct contact between the pre-mRNA-bound PTB and the U1 snRNA. EMSA, ITC, and NMR studies show that PTB RRMs 1 and 2 bind the pyrimidine-rich internal loop of U1 snRNA stem loop 4. The PTB/U1 interaction prevents further assembly of the U1 snRNP with spliceosomal components downstream. This precise interaction between a splicing regulator and an snRNA component of the spliceosome points to a range of different mechanisms for splicing regulation.
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A role for Polypyrimidine Tract binding protein in the establishment of focal adhesions.
Molecular and cellular biology, 2009Co-Authors: Ivan Babic, Shalini Sharma, Douglas L BlackAbstract:Polypyrimidine Tract binding protein (PTB) is a widely expressed RNA binding protein. In the nucleus PTB regulates the splicing of alternative exons, while in the cytoplasm it can affect mRNA stability, translation, and localization. Here we demonstrate that PTB transiently localizes to the cytoplasm and to protrusions in the cellular edge of mouse embryo fibroblasts during adhesion to fibronectin and the early stages of cell spreading. This cytoplasmic PTB is associated with transcripts encoding the focal adhesion scaffolding proteins vinculin and alpha-actinin 4. We demonstrate that vinculin mRNA colocalizes with PTB to cytoplasmic protrusions and that PTB depletion reduces vinculin mRNA at the cellular edge and limits the size of focal adhesions. The loss of PTB also alters cell morphology and limits the ability of cells to spread after adhesion. These data indicate that during the initial stages of cell adhesion, PTB shuttles from the nucleus to the cytoplasm and influences focal adhesion formation through coordinated control of scaffolding protein mRNAs.
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Polypyrimidine Tract binding protein controls the transition from exon definition to an intron defined spliceosome
Nature Structural & Molecular Biology, 2008Co-Authors: Shalini Sharma, Lori A Kohlstaedt, Andrey Damianov, Donald C Rio, Douglas L BlackAbstract:The Polypyrimidine Tract binding protein (PTB) binds pre-mRNAs to alter splice-site choice. We characterized a series of spliceosomal complexes that assemble on a pre-mRNA under conditions of either PTB-mediated splicing repression or its absence. In the absence of repression, exon definition complexes that were assembled downstream of the regulated exon could progress to pre-spliceosomal A complexes and functional spliceosomes. Under PTB-mediated repression, assembly was arrested at an A-like complex that was unable to transition to spliceosomal complexes. Trans-splicing experiments indicated that, even when the U1 and U2 small nuclear ribonucleoprotein particles (snRNPs) are properly bound to the upstream and downstream exons, the presence of PTB prevents the interaction of the two exon complexes. Proteomic analyses of these complexes provide a new description of exon definition complexes, and indicate that splicing regulators can act on the transition between the exon definition complex and an intron-defined spliceosome.
Christopher W.j. Smith - One of the best experts on this subject based on the ideXlab platform.
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essential requirement for Polypyrimidine Tract binding proteins 1 and 3 in the maturation and maintenance of mature b cells in mice
European Journal of Immunology, 2021Co-Authors: Christopher W.j. Smith, Elisa Monzoncasanova, Kirsty J Bates, Martin R TurnerAbstract:The maturation of immature B cells and the survival of mature B cells is stringently controlled to maintain a diverse repertoire of antibody specificities while avoiding self-reactivity. At the molecular level this is regulated by signalling from membrane immunoglobulin and the BAFF-receptor which sustain a pro-survival programme of gene expression. Whether and how posttranscriptional mechanisms contribute to B cell maturation and survival remains poorly understood. Here we show that the Polypyrimidine Tract binding proteins (PTBP) PTBP1 and PTBP3 bind to a large and overlapping set of transcripts in B cells. Both PTBP1 and PTBP3 bind to introns and exons where they are predicted to regulate alternative splicing. Moreover, they also show high-density of binding to 3' untranslated regions suggesting they influence the transcriptome in diverse ways. We show that PTBP1 and PTBP3 are required in B cells beyond the immature cell stage to sustain transitional B cells and the B1, marginal zone and follicular B cell lineages. Therefore, PTBP1 and PTBP3 promote the maturation of quiescent B cells by regulating gene expression at the post-transcriptional level. This article is protected by copyright. All rights reserved.
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Polypyrimidine Tract-binding proteins are essential for B cell development
'Organisation for Economic Co-Operation and Development (OECD)', 2020Co-Authors: Monzón-casanova Elisa, Christopher W.j. Smith, Matheson, Louise S, Tabbada Kristina, Zarnack Kathi, Turner MartinAbstract:Polypyrimidine Tract-binding protein 1 (PTBP1) is a RNA-binding protein (RBP) expressed throughout B cell development. Deletion of Ptbp1 in mouse pro-B cells results in upregulation of PTBP2 and normal B cell development. We show that PTBP2 compensates for PTBP1 in B cell ontogeny as deletion of both Ptbp1 and Ptbp2 results in a complete block at the pro-B cell stage and a lack of mature B cells. In pro-B cells PTBP1 ensures precise synchronisation of the activity of cyclin dependent kinases at distinct stages of the cell cycle, suppresses S-phase entry and promotes progression into mitosis. PTBP1 controls mRNA abundance and alternative splicing of important cell cycle regulators including CYCLIN-D2, c-MYC, p107 and CDC25B. Our results reveal a previously unrecognised mechanism mediated by a RBP that is essential for B cell ontogeny and integrates transcriptional and post-translational determinants of progression through the cell cycle
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Functional interactions between Polypyrimidine Tract binding protein and PRI peptide ligand containing proteins
Biochemical Society transactions, 2016Co-Authors: Miguel Coelho, David B. Ascher, Clare Gooding, Emma Lang, Hannah Maude, David A. Turner, Miriam Llorian, Douglas E. V. Pires, Jan Attig, Christopher W.j. SmithAbstract:Polypyrimidine Tract binding protein (PTBP1) is a heterogeneous nuclear ribonucleoprotein (hnRNP) that plays roles in most stages of the life-cycle of pre-mRNA and mRNAs in the nucleus and cytoplasm. PTBP1 has four RNA binding domains of the RNA recognition motif (RRM) family, each of which can bind to pyrimidine motifs. In addition, RRM2 can interact via its dorsal surface with proteins containing short peptide ligands known as PTB RRM2 interacting (PRI) motifs, originally found in the protein Raver1. Here we review our recent progress in understanding the interactions of PTB with RNA and with various proteins containing PRI ligands.
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a pathway involving hdac5 cflip and caspases regulates expression of the splicing regulator Polypyrimidine Tract binding protein in the heart
Journal of Cell Science, 2013Co-Authors: Miriam Llorian, Christopher W.j. Smith, Maria Cardona, Anthony Rongvaux, Rana S Moubarak, Joan X Comella, Rhonda Basselduby, Richard A Flavell, Eric N Olson, Daniel SanchisAbstract:Polypyrimidine Tract binding protein (PTB) regulates pre-mRNA splicing, having special relevance for determining gene expression in the differentiating muscle. We have previously shown that PTB protein abundance is progressively reduced during heart development without reduction of its own transcript. Simultaneous reduction of histone deacetylase (HDAC) expression prompted us to investigate the potential link between these events. HDAC5-deficient mice have reduced cardiac PTB protein abundance, and HDAC inhibition in myocytes causes a reduction in endogenous expression of cellular FLICE-like inhibitory protein (cFLIP) and caspase-dependent cleavage of PTB. In agreement with this, cardiac PTB expression is abnormally high in mice with cardiac-specific executioner caspase deficiency, and cFLIP overexpression prevents PTB cleavage in vitro. Caspase-dependent cleavage triggers further fragmentation of PTB, and these fragments accumulate in the presence of proteasome inhibitors. Experimental modification of the above processes in vivo and in vitro results in coherent changes in the alternative splicing of genes encoding tropomyosin-1 (TPM1), tropomyosin-2 (TPM2) and myocyte enhancer factor-2 (MEF2). Thus, we report a pathway connecting HDAC, cFLIP and caspases regulating the progressive disappearance of PTB, which enables the expression of the adult variants of proteins involved in the regulation of conTraction and transcription during cardiac muscle development.
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crystallographic analysis of Polypyrimidine Tract binding protein raver1 interactions involved in regulation of alternative splicing
Structure, 2011Co-Authors: Amar Joshi, Miguel Coelho, Christopher W.j. Smith, Stephen Matthews, Olga Kotikkogan, Peter J Simpson, Stephen CurryAbstract:The Polypyrimidine Tract-binding protein (PTB) is an important regulator of alternative splicing. PTB-regulated splicing of α-tropomyosin is enhanced by Raver1, a protein with four PTB-Raver1 interacting motifs (PRIs) that bind to the helical face of the second RNA recognition motif (RRM2) in PTB. We present the crystal structures of RRM2 in complex with PRI3 and PRI4 from Raver1, which—along with structure-based mutagenesis—reveal the molecular basis of their differential binding. High-affinity binding by Raver1 PRI3 involves shape-matched apolar contacts complemented by specific hydrogen bonds, a new variant of an established mode of peptide-RRM interaction. Our results refine the sequence of the PRI motif and place important structural constraints on functional models of PTB-Raver1 interactions. Our analysis indicates that the observed Raver1-PTB interaction is a general mode of binding that applies to Raver1 complexes with PTB paralogues such as nPTB and to complexes of Raver2 with PTB.
Stephen Curry - One of the best experts on this subject based on the ideXlab platform.
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solution and crystal structures of a c terminal fragment of the neuronal isoform of the Polypyrimidine Tract binding protein nptb
PeerJ, 2014Co-Authors: Amar Joshi, Frederic H T Allain, Vicent Esteve, Adrian N Buckroyd, Markus Blatter, Stephen CurryAbstract:The eukaryotic Polypyrimidine Tract binding protein (PTB) serves primarily as a regulator of alternative splicing of messenger RNA, but is also co-opted to other roles such as RNA localisation and translation initiation from internal ribosome entry sites. The neuronal paralogue of PTB (nPTB) is 75% identical in amino acid sequence with PTB. Although the two proteins have broadly similar RNA binding specificities and effects on RNA splicing, differential expression of PTB and nPTB can lead to the generation of alternatively spliced mRNAs. RNA binding by PTB and nPTB is mediated by four RNA recognition motifs (RRMs). We present here the crystal and solution structures of the C-terminal domain of nPTB (nPTB34) which contains RRMs 3 and 4. As expected the structures are similar to each other and to the solution structure of the equivalent fragment from PTB (PTB34). The result confirms that, as found for PTB, RRMs 3 and 4 of nPTB interact with one another to form a stable unit that presents the RNA-binding surfaces of the component RRMs on opposite sides that face away from each other. The major differences between PTB34 and nPTB34 arise from amino acid side chain substitutions on the exposed β-sheet surfaces and adjoining loops of each RRM, which are likely to modulate interactions with RNA.
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crystallographic analysis of Polypyrimidine Tract binding protein raver1 interactions involved in regulation of alternative splicing
Structure, 2011Co-Authors: Amar Joshi, Miguel Coelho, Christopher W.j. Smith, Stephen Matthews, Olga Kotikkogan, Peter J Simpson, Stephen CurryAbstract:The Polypyrimidine Tract-binding protein (PTB) is an important regulator of alternative splicing. PTB-regulated splicing of α-tropomyosin is enhanced by Raver1, a protein with four PTB-Raver1 interacting motifs (PRIs) that bind to the helical face of the second RNA recognition motif (RRM2) in PTB. We present the crystal structures of RRM2 in complex with PRI3 and PRI4 from Raver1, which—along with structure-based mutagenesis—reveal the molecular basis of their differential binding. High-affinity binding by Raver1 PRI3 involves shape-matched apolar contacts complemented by specific hydrogen bonds, a new variant of an established mode of peptide-RRM interaction. Our results refine the sequence of the PRI motif and place important structural constraints on functional models of PTB-Raver1 interactions. Our analysis indicates that the observed Raver1-PTB interaction is a general mode of binding that applies to Raver1 complexes with PTB paralogues such as nPTB and to complexes of Raver2 with PTB.
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Polypyrimidine Tract binding protein stabilizes the encephalomyocarditis virus ires structure via binding multiple sites in a unique orientation
Molecular Cell, 2009Co-Authors: Panagiota Kafasla, Stephen Curry, Carol V Robinson, Nina Morgner, Tuija Poyry, Richard J JacksonAbstract:Summary Polypyrimidine Tract binding (PTB) protein is a regulator of alternative pre-mRNA splicing, and also stimulates the initiation of translation dependent on many viral internal ribosome entry segments/sites (IRESs). It has four RNA-binding domains (RBDs), but although the contacts with many IRESs have been mapped, the orientation of binding (i.e., which RBD binds to which site in the IRES) is unknown. To answer this question, 16 derivatives of PTB1, each with a single cysteine flanking the RNA-binding surface in an RBD, were constructed and used in directed hydroxyl radical probing with the encephalomyocarditis virus IRES. The results, together with mass spectrometry data on the stoichiometry of PTB binding to different IRES derivatives, show that the minimal IRES binds a single PTB in a unique orientation, with RBD1 and RBD2 binding near the 3′ end, and RBD3 contacting the 5′ end, thereby constraining and stabilizing the three-dimensional structural fold of the IRES.
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conformation of Polypyrimidine Tract binding protein in solution
Structure, 2006Co-Authors: Maxim V Petoukhov, Frederic H T Allain, Tom P Monie, Stephen Matthews, Stephen Curry, Dmitri I SvergunAbstract:The Polypyrimidine Tract binding protein (PTB) is an RNA binding protein that normally functions as a regulator of alternative splicing but can also be recruited to stimulate translation initiation by certain picornaviruses. High-resolution structures of the four RNA recognition motifs (RRMs) that make up PTB have previously been determined by NMR. Here, we have used small-angle X-ray scattering to determine the low-resolution structure of the entire protein. Scattering patterns from full-length PTB and deletion mutants containing all possible sequential combinations of the RRMs were collected. All constructs were found to be monomeric in solution. Ab initio analysis and rigid-body modeling utilizing the high-resolution models of the RRMs yielded a consistent low-resolution model of the spatial organization of domains in PTB. Domains 3 and 4 were found to be in close contact, whereas domains 2 and especially 1 had loose contacts with the rest of the protein.
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the Polypyrimidine Tract binding protein is a monomer
RNA, 2005Co-Authors: Tom P Monie, Stephen Matthews, Helena Hernandez, Carol V Robinson, Peter T Simpson, Stephen CurryAbstract:The Polypyrimidine Tract binding (PTB) protein is a potent regulator of alternative mRNA splicing. It also participates in other essential cellular functions, including translation initiation and polyadenylation. Several published reports have suggested that the protein forms a dimer in solution, a feature that has been widely incorporated into mechanistic models of protein function. However, recent studies have provided indications that full-length PTB is a monomer. Here we present new biophysical and biochemical evidence supporting the monomeric status of the protein. By use of blue-native polyacrylamide gel electrophoresis and size-exclusion chromatography, PTB was observed as a single molecular species under native reducing environments, though in oxidizing conditions, a larger protein species was also detected. Further analyses of wild-type and mutant PTB molecules with SDS-PAGE and time-of-flight electrospray ionization mass spectroscopy confirmed these observations. They also identified the single reduced species as monomeric PTB and the higher-molecular-weight nonreduced species as disulphide-linked PTB dimer mediated by Cys23. Our results indicate that the use of oxidizing environments in previous studies is likely to have contributed to the misassignment of PTB as a dimer. Although purified PTB may form disulphide-linked dimers under these conditions, in the reducing intracellular environment the protein will be monomeric. These findings have implications for the construction of models of PTB function in regulating mRNA metabolism.
Frederic H T Allain - One of the best experts on this subject based on the ideXlab platform.
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a transient α helix in the n terminal rna recognition motif of Polypyrimidine Tract binding protein senses rna secondary structure
Nucleic Acids Research, 2020Co-Authors: Christophe Maris, Fred F Damberger, Sandrine Jayne, Irene Beusch, Georg Dorn, Sapna Ravindranathan, Frederic H T AllainAbstract:The Polypyrimidine Tract binding protein (PTB) is a multi-domain protein involved in alternative splicing, mRNA localization, stabilization, polyadenylation and translation initiation from internal ribosome entry sites (IRES). In this latter process, PTB promotes viral translation by interacting extensively with complex structured regions in the 5'-untranslated regions of viral RNAs at pyrimidine-rich targets located in single strand and hairpin regions. To better understand how PTB recognizes structured elements in RNA targets, we solved the solution structure of the N-terminal RNA recognition motif (RRM) in complex with an RNA hairpin embedding the loop sequence UCUUU, which is frequently found in IRESs of the picornovirus family. Surprisingly, a new three-turn α3 helix C-terminal to the RRM, folds upon binding the RNA hairpin. Although α3 does not mediate any contacts to the RNA, it acts as a sensor of RNA secondary structure, suggesting a role for RRM1 in detecting pyrimidine Tracts in the context of structured RNA. Moreover, the degree of helix formation depends on the RNA loop sequence. Finally, we show that the α3 helix region, which is highly conserved in vertebrates, is crucial for PTB function in enhancing Encephalomyocarditis virus IRES activity.
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solution and crystal structures of a c terminal fragment of the neuronal isoform of the Polypyrimidine Tract binding protein nptb
PeerJ, 2014Co-Authors: Amar Joshi, Frederic H T Allain, Vicent Esteve, Adrian N Buckroyd, Markus Blatter, Stephen CurryAbstract:The eukaryotic Polypyrimidine Tract binding protein (PTB) serves primarily as a regulator of alternative splicing of messenger RNA, but is also co-opted to other roles such as RNA localisation and translation initiation from internal ribosome entry sites. The neuronal paralogue of PTB (nPTB) is 75% identical in amino acid sequence with PTB. Although the two proteins have broadly similar RNA binding specificities and effects on RNA splicing, differential expression of PTB and nPTB can lead to the generation of alternatively spliced mRNAs. RNA binding by PTB and nPTB is mediated by four RNA recognition motifs (RRMs). We present here the crystal and solution structures of the C-terminal domain of nPTB (nPTB34) which contains RRMs 3 and 4. As expected the structures are similar to each other and to the solution structure of the equivalent fragment from PTB (PTB34). The result confirms that, as found for PTB, RRMs 3 and 4 of nPTB interact with one another to form a stable unit that presents the RNA-binding surfaces of the component RRMs on opposite sides that face away from each other. The major differences between PTB34 and nPTB34 arise from amino acid side chain substitutions on the exposed β-sheet surfaces and adjoining loops of each RRM, which are likely to modulate interactions with RNA.
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u1 snrna directly interacts with Polypyrimidine Tract binding protein during splicing repression
Molecular Cell, 2011Co-Authors: Shalini Sharma, Frederic H T Allain, Christophe Maris, Douglas L BlackAbstract:Summary Splicing of the c-src N1 exon is repressed by the Polypyrimidine Tract-binding protein (PTB or PTBP1). During exon repression, the U1 snRNP binds properly to the N1 exon 5′ splice site but is made inactive by the presence of PTB. Examining the patterns of nuclease protection at this 5′ splice site, we find that the interaction of U1 is altered by the adjacent PTB. Interestingly, UV crosslinking identifies a direct contact between the pre-mRNA-bound PTB and the U1 snRNA. EMSA, ITC, and NMR studies show that PTB RRMs 1 and 2 bind the pyrimidine-rich internal loop of U1 snRNA stem loop 4. The PTB/U1 interaction prevents further assembly of the U1 snRNP with spliceosomal components downstream. This precise interaction between a splicing regulator and an snRNA component of the spliceosome points to a range of different mechanisms for splicing regulation.
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Structure-function relationships of the Polypyrimidine Tract binding protein
Cellular and Molecular Life Sciences, 2007Co-Authors: S. D. Auweter, Frederic H T AllainAbstract:The Polypyrimidine Tract binding protein (PTB) is a 58-kDa RNA binding protein involved in multiple aspects of mRNA metabolism including splicing regulation, polyadenylation, 3′end formation, internal ribosomal entry site-mediated translation, RNA localization and stability. PTB contains four RNA recognition motifs (RRMs) separated by three linkers. In this review we summarize structural information on PTB in solution that has been gathered during the past 7 years using NMR spectroscopy and small-angle X-ray scattering. The structures of all RRMs of PTB in their free state and in complex with short pyrimidine Tracts, as well as a structural model of PTB RRM2 in complex with a peptide, revealed unusual structural features that provided new insights into the mechanisms of action of PTB in the different processes of RNA metabolism and in particular splicing regulation.
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conformation of Polypyrimidine Tract binding protein in solution
Structure, 2006Co-Authors: Maxim V Petoukhov, Frederic H T Allain, Tom P Monie, Stephen Matthews, Stephen Curry, Dmitri I SvergunAbstract:The Polypyrimidine Tract binding protein (PTB) is an RNA binding protein that normally functions as a regulator of alternative splicing but can also be recruited to stimulate translation initiation by certain picornaviruses. High-resolution structures of the four RNA recognition motifs (RRMs) that make up PTB have previously been determined by NMR. Here, we have used small-angle X-ray scattering to determine the low-resolution structure of the entire protein. Scattering patterns from full-length PTB and deletion mutants containing all possible sequential combinations of the RRMs were collected. All constructs were found to be monomeric in solution. Ab initio analysis and rigid-body modeling utilizing the high-resolution models of the RRMs yielded a consistent low-resolution model of the spatial organization of domains in PTB. Domains 3 and 4 were found to be in close contact, whereas domains 2 and especially 1 had loose contacts with the rest of the protein.
Shalini Sharma - One of the best experts on this subject based on the ideXlab platform.
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multiple determinants of splicing repression activity in the Polypyrimidine Tract binding proteins ptbp1 and ptbp2
RNA, 2016Co-Authors: Niroshika Keppetipola, Shalini Sharma, Kyuhyeon Yeom, Adrian L Hernandez, Tessa Bui, Douglas L BlackAbstract:Most human genes generate multiple protein isoforms through alternative pre-mRNA splicing, but the mechanisms controlling alternative splicing choices by RNA binding proteins are not well understood. These proteins can have multiple paralogs expressed in different cell types and exhibiting different splicing activities on target exons. We examined the paralogous Polypyrimidine Tract binding proteins PTBP1 and PTBP2 to understand how PTBP1 can exhibit greater splicing repression activity on certain exons. Using both an in vivo coexpression assay and an in vitro splicing assay, we show that PTBP1 is more repressive than PTBP2 per unit protein on a target exon. Constructing chimeras of PTBP1 and 2 to determine amino acid features that contribute to their differential activity, we find that multiple segments of PTBP1 increase the repressive activity of PTBP2. Notably, when either RRM1 of PTBP2 or the linker peptide separating RRM2 and RRM3 are replaced with the equivalent PTBP1 sequences, the resulting chimeras are highly active for splicing repression. These segments are distinct from the known region of interaction for the PTBP1 cofactors Raver1 and Matrin3 in RRM2. We find that RRM2 of PTBP1 also increases the repression activity of an otherwise PTBP2 sequence, and that this is potentially explained by stronger binding by Raver1. These results indicate that multiple features over the length of the two proteins affect their ability to repress an exon.
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neuronal regulation of pre mrna splicing by Polypyrimidine Tract binding proteins ptbp1 and ptbp2
Critical Reviews in Biochemistry and Molecular Biology, 2012Co-Authors: Niroshika Keppetipola, Shalini Sharma, Douglas L BlackAbstract:Alternative splicing patterns are regulated by RNA binding proteins that assemble onto each pre-mRNA to form a complex RNP structure. The Polypyrimidine Tract binding protein, PTB, has served as an informative model for understanding how RNA binding proteins affect spliceosome assembly and how changes in the expression of these proteins can control complex programs of splicing in tissues. In this review, we describe the mechanisms of splicing regulation by PTB and its function, along with its paralog PTBP2, in neuronal development.
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u1 snrna directly interacts with Polypyrimidine Tract binding protein during splicing repression
Molecular Cell, 2011Co-Authors: Shalini Sharma, Frederic H T Allain, Christophe Maris, Douglas L BlackAbstract:Summary Splicing of the c-src N1 exon is repressed by the Polypyrimidine Tract-binding protein (PTB or PTBP1). During exon repression, the U1 snRNP binds properly to the N1 exon 5′ splice site but is made inactive by the presence of PTB. Examining the patterns of nuclease protection at this 5′ splice site, we find that the interaction of U1 is altered by the adjacent PTB. Interestingly, UV crosslinking identifies a direct contact between the pre-mRNA-bound PTB and the U1 snRNA. EMSA, ITC, and NMR studies show that PTB RRMs 1 and 2 bind the pyrimidine-rich internal loop of U1 snRNA stem loop 4. The PTB/U1 interaction prevents further assembly of the U1 snRNP with spliceosomal components downstream. This precise interaction between a splicing regulator and an snRNA component of the spliceosome points to a range of different mechanisms for splicing regulation.
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A role for Polypyrimidine Tract binding protein in the establishment of focal adhesions.
Molecular and cellular biology, 2009Co-Authors: Ivan Babic, Shalini Sharma, Douglas L BlackAbstract:Polypyrimidine Tract binding protein (PTB) is a widely expressed RNA binding protein. In the nucleus PTB regulates the splicing of alternative exons, while in the cytoplasm it can affect mRNA stability, translation, and localization. Here we demonstrate that PTB transiently localizes to the cytoplasm and to protrusions in the cellular edge of mouse embryo fibroblasts during adhesion to fibronectin and the early stages of cell spreading. This cytoplasmic PTB is associated with transcripts encoding the focal adhesion scaffolding proteins vinculin and alpha-actinin 4. We demonstrate that vinculin mRNA colocalizes with PTB to cytoplasmic protrusions and that PTB depletion reduces vinculin mRNA at the cellular edge and limits the size of focal adhesions. The loss of PTB also alters cell morphology and limits the ability of cells to spread after adhesion. These data indicate that during the initial stages of cell adhesion, PTB shuttles from the nucleus to the cytoplasm and influences focal adhesion formation through coordinated control of scaffolding protein mRNAs.
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Polypyrimidine Tract binding protein controls the transition from exon definition to an intron defined spliceosome
Nature Structural & Molecular Biology, 2008Co-Authors: Shalini Sharma, Lori A Kohlstaedt, Andrey Damianov, Donald C Rio, Douglas L BlackAbstract:The Polypyrimidine Tract binding protein (PTB) binds pre-mRNAs to alter splice-site choice. We characterized a series of spliceosomal complexes that assemble on a pre-mRNA under conditions of either PTB-mediated splicing repression or its absence. In the absence of repression, exon definition complexes that were assembled downstream of the regulated exon could progress to pre-spliceosomal A complexes and functional spliceosomes. Under PTB-mediated repression, assembly was arrested at an A-like complex that was unable to transition to spliceosomal complexes. Trans-splicing experiments indicated that, even when the U1 and U2 small nuclear ribonucleoprotein particles (snRNPs) are properly bound to the upstream and downstream exons, the presence of PTB prevents the interaction of the two exon complexes. Proteomic analyses of these complexes provide a new description of exon definition complexes, and indicate that splicing regulators can act on the transition between the exon definition complex and an intron-defined spliceosome.