Publications by authors named "Christopher W J Smith"

49 Publications

A drug-repositioning screen using splicing-sensitive fluorescent reporters identifies novel modulators of VEGF-A splicing with anti-angiogenic properties.

Oncogenesis 2021 May 3;10(5):36. Epub 2021 May 3.

Institute of Biomedical & Clinical Sciences, Medical School, College of Medicine and Health, University of Exeter, St Luke's Campus, Exeter, EX1 2LU, UK.

Alternative splicing of the vascular endothelial growth factor A (VEGF-A) terminal exon generates two protein families with differing functions. Pro-angiogenic VEGF-Aa isoforms are produced via selection of the proximal 3' splice site of the terminal exon. Use of an alternative distal splice site generates the anti-angiogenic VEGF-Ab proteins. A bichromatic splicing-sensitive reporter was designed to mimic VEGF-A alternative splicing and was used as a molecular tool to further investigate this alternative splicing event. Part of VEGF-A's terminal exon and preceding intron were inserted into a minigene construct followed by the coding sequences for two fluorescent proteins. A different fluorescent protein is expressed depending on which 3' splice site of the exon is used during splicing (dsRED denotes VEGF-Aa and EGFP denotes VEGF-Ab). The fluorescent output can be used to follow splicing decisions in vitro and in vivo. Following successful reporter validation in different cell lines and altering splicing using known modulators, a screen was performed using the LOPAC library of small molecules. Alterations to reporter splicing were measured using a fluorescent plate reader to detect dsRED and EGFP expression. Compounds of interest were further validated using flow cytometry and assessed for effects on endogenous VEGF-A alternative splicing at the mRNA and protein level. Ex vivo and in vitro angiogenesis assays were used to demonstrate the anti-angiogenic effect of the compounds. Furthermore, anti-angiogenic activity was investigated in a Matrigel in vivo model. To conclude, we have identified a set of compounds that have anti-angiogenic activity through modulation of VEGF-A terminal exon splicing.
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http://dx.doi.org/10.1038/s41389-021-00323-0DOI Listing
May 2021

A systems view of spliceosomal assembly and branchpoints with iCLIP.

Nat Struct Mol Biol 2019 10 30;26(10):930-940. Epub 2019 Sep 30.

MRC Laboratory of Molecular Biology, Cambridge, UK.

Studies of spliceosomal interactions are challenging due to their dynamic nature. Here we used spliceosome iCLIP, which immunoprecipitates SmB along with small nuclear ribonucleoprotein particles and auxiliary RNA binding proteins, to map spliceosome engagement with pre-messenger RNAs in human cell lines. This revealed seven peaks of spliceosomal crosslinking around branchpoints (BPs) and splice sites. We identified RNA binding proteins that crosslink to each peak, including known and candidate splicing factors. Moreover, we detected the use of over 40,000 BPs with strong sequence consensus and structural accessibility, which align well to nearby crosslinking peaks. We show how the position and strength of BPs affect the crosslinking patterns of spliceosomal factors, which bind more efficiently upstream of strong or proximally located BPs and downstream of weak or distally located BPs. These insights exemplify spliceosome iCLIP as a broadly applicable method for transcriptomic studies of splicing mechanisms.
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http://dx.doi.org/10.1038/s41594-019-0300-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6859068PMC
October 2019

Heteromeric RNP Assembly at LINEs Controls Lineage-Specific RNA Processing.

Cell 2018 08 2;174(5):1067-1081.e17. Epub 2018 Aug 2.

The Francis Crick Institute, Midland Road 1, Kings Cross, London NW1 1AT, UK; Department of Molecular Neuroscience, UCL Institute of Neurology, Queen Square, London WC1N 3BG, UK. Electronic address:

Long mammalian introns make it challenging for the RNA processing machinery to identify exons accurately. We find that LINE-derived sequences (LINEs) contribute to this selection by recruiting dozens of RNA-binding proteins (RBPs) to introns. This includes MATR3, which promotes binding of PTBP1 to multivalent binding sites within LINEs. Both RBPs repress splicing and 3' end processing within and around LINEs. Notably, repressive RBPs preferentially bind to evolutionarily young LINEs, which are located far from exons. These RBPs insulate the LINEs and the surrounding intronic regions from RNA processing. Upon evolutionary divergence, changes in RNA motifs within LINEs lead to gradual loss of their insulation. Hence, older LINEs are located closer to exons, are a common source of tissue-specific exons, and increasingly bind to RBPs that enhance RNA processing. Thus, LINEs are hubs for the assembly of repressive RBPs and also contribute to the evolution of new, lineage-specific transcripts in mammals. VIDEO ABSTRACT.
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http://dx.doi.org/10.1016/j.cell.2018.07.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6108849PMC
August 2018

Impact of spliceosome mutations on RNA splicing in myelodysplasia: dysregulated genes/pathways and clinical associations.

Blood 2018 09 21;132(12):1225-1240. Epub 2018 Jun 21.

Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, Oxford, United Kingdom.

, , and are the most frequently mutated splicing factor genes in the myelodysplastic syndromes (MDS). We have performed a comprehensive and systematic analysis to determine the effect of these commonly mutated splicing factors on pre-mRNA splicing in the bone marrow stem/progenitor cells and in the erythroid and myeloid precursors in splicing factor mutant MDS. Using RNA-seq, we determined the aberrantly spliced genes and dysregulated pathways in CD34 cells of 84 patients with MDS. Splicing factor mutations result in different alterations in splicing and largely affect different genes, but these converge in common dysregulated pathways and cellular processes, focused on RNA splicing, protein synthesis, and mitochondrial dysfunction, suggesting common mechanisms of action in MDS. Many of these dysregulated pathways and cellular processes can be linked to the known disease pathophysiology associated with splicing factor mutations in MDS, whereas several others have not been previously associated with MDS, such as sirtuin signaling. We identified aberrantly spliced events associated with clinical variables, and isoforms that independently predict survival in MDS and implicate dysregulation of focal adhesion and extracellular exosomes as drivers of poor survival. Aberrantly spliced genes and dysregulated pathways were identified in the MDS-affected lineages in splicing factor mutant MDS. Functional studies demonstrated that knockdown of the mitosis regulators and aberrantly spliced target genes of and mutations, respectively, led to impaired erythroid cell growth and differentiation. This study illuminates the effect of the common spliceosome mutations on the MDS phenotype and provides novel insights into disease pathophysiology.
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http://dx.doi.org/10.1182/blood-2018-04-843771DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6172604PMC
September 2018

SRSF3 maintains transcriptome integrity in oocytes by regulation of alternative splicing and transposable elements.

Cell Discov 2018 19;4:33. Epub 2018 Jun 19.

1Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QN UK.

The RNA-binding protein SRSF3 (also known as SRp20) has critical roles in the regulation of pre-mRNA splicing. Zygotic knockout of results in embryo arrest at the blastocyst stage. However, SRSF3 is also present in oocytes, suggesting that it might be critical as a maternally inherited factor. Here we identify SRSF3 as an essential regulator of alternative splicing and of transposable elements to maintain transcriptome integrity in mouse oocyte. Using 3D time-lapse confocal live imaging, we show that conditional deletion of in fully grown germinal vesicle oocytes substantially compromises the capacity of germinal vesicle breakdown (GVBD), and consequently entry into meiosis. By combining single cell RNA-seq, and oocyte micromanipulation with steric blocking antisense oligonucleotides and RNAse-H inducing gapmers, we found that the GVBD defect in mutant oocytes is due to both aberrant alternative splicing and derepression of B2 SINE transposable elements. Together, our study highlights how control of transcriptional identity of the maternal transcriptome by the RNA-binding protein SRSF3 is essential to the development of fertilized-competent oocytes.
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http://dx.doi.org/10.1038/s41421-018-0032-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6006335PMC
June 2018

The RNA-binding protein PTBP1 is necessary for B cell selection in germinal centers.

Nat Immunol 2018 03 22;19(3):267-278. Epub 2018 Jan 22.

Laboratory of Lymphocyte Signaling and Development, The Babraham Institute, Cambridge, UK.

Antibody affinity maturation occurs in germinal centers (GCs), where B cells cycle between the light zone (LZ) and the dark zone. In the LZ, GC B cells bearing immunoglobulins with the highest affinity for antigen receive positive selection signals from helper T cells, which promotes their rapid proliferation. Here we found that the RNA-binding protein PTBP1 was needed for the progression of GC B cells through late S phase of the cell cycle and for affinity maturation. PTBP1 was required for proper expression of the c-MYC-dependent gene program induced in GC B cells receiving T cell help and directly regulated the alternative splicing and abundance of transcripts that are increased during positive selection to promote proliferation.
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http://dx.doi.org/10.1038/s41590-017-0035-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5842895PMC
March 2018

Intron retention as a component of regulated gene expression programs.

Hum Genet 2017 09 8;136(9):1043-1057. Epub 2017 Apr 8.

Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QW, UK.

Intron retention has long been an exemplar of regulated splicing with case studies of individual events serving as models that provided key mechanistic insights into the process of splicing control. In organisms such as plants and budding yeast, intron retention is well understood as a major mechanism of gene expression regulation. In contrast, in mammalian systems, the extent and functional significance of intron retention have, until recently, remained greatly underappreciated. Technical challenges to the global detection and quantitation of transcripts with retained introns have often led to intron retention being overlooked or dismissed as "noise". Now, however, with the wealth of information available from high-throughput deep sequencing, combined with focused computational and statistical analyses, we are able to distinguish clear intron retention patterns in various physiological and pathological contexts. Several recent studies have demonstrated intron retention as a central component of gene expression programs during normal development as well as in response to stress and disease. Furthermore, these studies revealed various ways in which intron retention regulates protein isoform production, RNA stability and translation efficiency, and rapid induction of expression via post-transcriptional splicing of retained introns. In this review, we highlight critical findings from these transcriptomic studies and discuss commonalties in the patterns prevalent in intron retention networks at the functional and regulatory levels.
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http://dx.doi.org/10.1007/s00439-017-1791-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5602073PMC
September 2017

Functional interactions between polypyrimidine tract binding protein and PRI peptide ligand containing proteins.

Biochem Soc Trans 2016 08;44(4):1058-65

Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, U.K.

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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http://dx.doi.org/10.1042/BST20160080DOI Listing
August 2016

The alternative splicing program of differentiated smooth muscle cells involves concerted non-productive splicing of post-transcriptional regulators.

Nucleic Acids Res 2016 Oct 17;44(18):8933-8950. Epub 2016 Jun 17.

Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QW, UK

Alternative splicing (AS) is a key component of gene expression programs that drive cellular differentiation. Smooth muscle cells (SMCs) are important in the function of a number of physiological systems; however, investigation of SMC AS has been restricted to a handful of events. We profiled transcriptome changes in mouse de-differentiating SMCs and observed changes in hundreds of AS events. Exons included in differentiated cells were characterized by particularly weak splice sites and by upstream binding sites for Polypyrimidine Tract Binding protein (PTBP1). Consistent with this, knockdown experiments showed that that PTBP1 represses many smooth muscle specific exons. We also observed coordinated splicing changes predicted to downregulate the expression of core components of U1 and U2 snRNPs, splicing regulators and other post-transcriptional factors in differentiated cells. The levels of cognate proteins were lower or similar in differentiated compared to undifferentiated cells. However, levels of snRNAs did not follow the expression of splicing proteins, and in the case of U1 snRNP we saw reciprocal changes in the levels of U1 snRNA and U1 snRNP proteins. Our results suggest that the AS program in differentiated SMCs is orchestrated by the combined influence of auxiliary RNA binding proteins, such as PTBP1, along with altered activity and stoichiometry of the core splicing machinery.
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http://dx.doi.org/10.1093/nar/gkw560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5062968PMC
October 2016

Matrin3: connecting gene expression with the nuclear matrix.

Wiley Interdiscip Rev RNA 2016 05 26;7(3):303-15. Epub 2016 Jan 26.

Department of Biochemistry, University of Cambridge, Cambridge, UK.

As indicated by its name, Matrin3 was discovered as a component of the nuclear matrix, an insoluble fibrogranular network that structurally organizes the nucleus. Matrin3 possesses both DNA- and RNA-binding domains and, consistent with this, has been shown to function at a number of stages in the life cycle of messenger RNAs. These numerous activities indicate that Matrin3, and indeed the nuclear matrix, do not just provide a structural framework for nuclear activities but also play direct functional roles in these activities. Here, we review the structure, functions, and molecular interactions of Matrin3 and of Matrin3-related proteins, and the pathologies that can arise upon mutation of Matrin3. WIREs RNA 2016, 7:303-315. doi: 10.1002/wrna.1336 For further resources related to this article, please visit the WIREs website.
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http://dx.doi.org/10.1002/wrna.1336DOI Listing
May 2016

Generation of functionally distinct isoforms of PTBP3 by alternative splicing and translation initiation.

Nucleic Acids Res 2015 Jun 4;43(11):5586-600. Epub 2015 May 4.

Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK

Polypyrimidine tract binding protein (PTBP1) is a widely expressed RNA binding protein that acts as a regulator of alternative splicing and of cytoplasmic mRNA functions. Vertebrates contain two closely-related paralogs with >75% amino acid sequence identity. Early replacement of PTBP1 by PTBP2 during neuronal differentiation causes a concerted set of splicing changes. By comparison, very little is known about the molecular functions or physiological roles of PTBP3, although its expression and conservation throughout the vertebrates suggest a role in haematopoietic cells. To begin to understand its functions we have characterized the mRNA and protein isoform repertoire of PTBP3. Combinatorial alternative splicing events at the 5' end of the gene allow for the generation of eight mRNA and three major protein isoforms. Individual mRNAs generate up to three protein isoforms via alternative translation initiation by re-initiation and leaky scanning using downstream AUG codons. The N-terminally truncated PTBP3 isoforms lack nuclear localization signals and/or most of the RRM1 domain and vary in their RNA binding properties and nuclear/cytoplasmic distribution, suggesting that PTBP3 may have major post-transcriptional cytoplasmic roles. Our findings set the stage for understanding the non-redundant physiological roles of PTBP3.
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http://dx.doi.org/10.1093/nar/gkv429DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4477659PMC
June 2015

Nuclear matrix protein Matrin3 regulates alternative splicing and forms overlapping regulatory networks with PTB.

EMBO J 2015 Mar 19;34(5):653-68. Epub 2015 Jan 19.

Department of Biochemistry, University of Cambridge, Cambridge, UK

Matrin3 is an RNA- and DNA-binding nuclear matrix protein found to be associated with neural and muscular degenerative diseases. A number of possible functions of Matrin3 have been suggested, but no widespread role in RNA metabolism has yet been clearly demonstrated. We identified Matrin3 by its interaction with the second RRM domain of the splicing regulator PTB. Using a combination of RNAi knockdown, transcriptome profiling and iCLIP, we find that Matrin3 is a regulator of hundreds of alternative splicing events, principally acting as a splicing repressor with only a small proportion of targeted events being co-regulated by PTB. In contrast to other splicing regulators, Matrin3 binds to an extended region within repressed exons and flanking introns with no sharply defined peaks. The identification of this clear molecular function of Matrin3 should help to clarify the molecular pathology of ALS and other diseases caused by mutations of Matrin3.
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http://dx.doi.org/10.15252/embj.201489852DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4365034PMC
March 2015

The organization of RNA contacts by PTB for regulation of FAS splicing.

Nucleic Acids Res 2014 Jul 23;42(13):8605-20. Epub 2014 Jun 23.

Department of Biochemistry, University of Cambridge, Downing Site, Tennis Court Road, Cambridge, CB2 1QW, UK

Post-transcriptional steps of gene expression are regulated by RNA binding proteins. Major progress has been made in characterizing RNA-protein interactions, from high resolution structures to transcriptome-wide profiling. Due to the inherent technical challenges, less attention has been paid to the way in which proteins with multiple RNA binding domains engage with target RNAs. We have investigated how the four RNA recognition motif (RRM) domains of Polypyrimidine tract binding (PTB) protein, a major splicing regulator, interact with FAS pre-mRNA under conditions in which PTB represses FAS exon 6 splicing. A combination of tethered hydroxyl radical probing, targeted inactivation of individual RRMs and single molecule analyses revealed an unequal division of labour between the four RRMs of PTB. RNA binding by RRM4 is the most important for function despite the low intrinsic binding specificity and the complete lack of effect of disrupting individual RRM4 contact points on the RNA. The ordered RRM3-4 di-domain packing provides an extended binding surface for RNA interacting at RRM4, via basic residues in the preceding linker. Our results illustrate how multiple alternative low-specificity binding configurations of RRM4 are consistent with repressor function as long as the overall ribonucleoprotein architecture provided by appropriate di-domain packing is maintained.
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http://dx.doi.org/10.1093/nar/gku519DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4117754PMC
July 2014

Regulation of alternative pre-mRNA splicing.

Methods Mol Biol 2014 ;1126:55-82

Department of Biochemistry, University of Cambridge, Cambridge, UK.

Alternative splicing plays a prevalent role in generating functionally diversified proteomes from genomes with a more limited repertoire of protein-coding genes. Alternative splicing is frequently regulated with cell type or developmental specificity and in response to signaling pathways, and its mis-regulation can lead to disease. Co-regulated programs of alternative splicing involve interplay between a host of cis-acting transcript features and trans-acting RNA-binding proteins. Here, we review the current state of understanding of the logic and mechanism of regulated alternative splicing and indicate how this understanding can be exploited to manipulate splicing for therapeutic purposes.
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http://dx.doi.org/10.1007/978-1-62703-980-2_5DOI Listing
October 2014

Dissecting domains necessary for activation and repression of splicing by Muscleblind-like protein 1.

BMC Mol Biol 2013 Dec 27;14:29. Epub 2013 Dec 27.

Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK.

Background: Alternative splicing contributes to the diversity of the proteome, and provides the cell with an important additional layer of regulation of gene expression. Among the many RNA binding proteins that regulate alternative splicing pathways are the Muscleblind-like (MBNL) proteins. MBNL proteins bind YGCY motifs in RNA via four CCCH zinc fingers arranged in two tandem arrays, and play a crucial role in the transition from embryonic to adult muscle splicing patterns, deregulation of which leads to Myotonic Dystrophy. Like many other RNA binding proteins, MBNL proteins can act as both activators or repressors of different splicing events.

Results: We used targeted point mutations to interfere with the RNA binding of MBNL1 zinc fingers individually and in combination. The effects of the mutations were tested in assays for splicing repression and activation, including overexpression, complementation of siRNA-mediated knockdown, and artificial tethering using MS2 coat protein. Mutations were tested in the context of both full length MBNL1 as well as a series of truncation mutants. Individual mutations within full length MBNL1 had little effect, but mutations in ZF1 and 2 combined were more detrimental than those in ZF 3 and 4, upon splicing activation, repression and RNA binding. Activation and repression both required linker sequences between ZF2 and 3, but activation was more sensitive to loss of linker sequences.

Conclusions: Our results highlight the importance of RNA binding by MBNL ZF domains 1 and 2 for splicing regulatory activity, even when the protein is artificially recruited to its regulatory location on target RNAs. However, RNA binding is not sufficient for activity; additional regions between ZF 2 and 3 are also essential. Activation and repression show differential sensitivity to truncation of this linker region, suggesting interactions with different sets of cofactors for the two types of activity.
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http://dx.doi.org/10.1186/1471-2199-14-29DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3880588PMC
December 2013

MBNL1 and PTB cooperate to repress splicing of Tpm1 exon 3.

Nucleic Acids Res 2013 May 19;41(9):4765-82. Epub 2013 Mar 19.

Department of Biochemistry, University of Cambridge, CB2 1QW, UK.

Exon 3 of the rat α-tropomyosin (Tpm1) gene is repressed in smooth muscle cells, allowing inclusion of the mutually exclusive partner exon 2. Two key types of elements affect repression of exon 3 splicing: binding sites for polypyrimidine tract-binding protein (PTB) and additional negative regulatory elements consisting of clusters of UGC or CUG motifs. Here, we show that the UGC clusters are bound by muscleblind-like proteins (MBNL), which act as repressors of Tpm1 exon 3. We show that the N-terminal region of MBNL1, containing its four CCCH zinc-finger domains, is sufficient to mediate repression. The same region of MBNL1 can make a direct protein-to-protein interaction with PTB, and RNA binding by MBNL promotes this interaction, apparently by inducing a conformational change in MBNL. Moreover, single molecule analysis showed that MBNL-binding sites increase the binding of PTB to its own sites. Our data suggest that the smooth muscle splicing of Tpm1 is mediated by allosteric assembly of an RNA-protein complex minimally comprising PTB, MBNL and their cognate RNA-binding sites.
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http://dx.doi.org/10.1093/nar/gkt168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3643581PMC
May 2013

A pathway involving HDAC5, cFLIP and caspases regulates expression of the splicing regulator polypyrimidine tract binding protein in the heart.

J Cell Sci 2013 Apr 19;126(Pt 7):1682-91. Epub 2013 Feb 19.

Departament de Ciències Mèdiques Bàsiques, Universitat de Lleida - IRBLLEIDA, Av Rovira Roure, 80, Lleida, 25198 Spain.

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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http://dx.doi.org/10.1242/jcs.121384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3647441PMC
April 2013

Defining the roles and interactions of PTB.

Biochem Soc Trans 2012 Aug;40(4):815-20

Department of Biochemistry, University of Cambridge, Cambridge, UK.

PTB (polypyrimidine tract-binding protein) is an abundant and widely expressed RNA-binding protein with four RRM (RNA recognition motif) domains. PTB is involved in numerous post-transcriptional steps in gene expression in both the nucleus and cytoplasm, but has been best characterized as a regulatory repressor of some ASEs (alternative splicing events), and as an activator of translation driven by IRESs (internal ribosome entry segments). We have used a variety of approaches to characterize the activities of PTB and its molecular interactions with RNA substrates and protein partners. Using splice-sensitive microarrays we found that PTB acts not only as a splicing repressor but also as an activator, and that these two activities are determined by the location at which PTB binds relative to target exons. We have identified minimal splicing repressor and activator domains, and have determined high resolution structures of the second RRM domain of PTB binding to peptide motifs from the co-repressor protein Raver1. Using single-molecule techniques we have determined the stoichiometry of PTB binding to a regulated splicing substrate in whole nuclear extracts. Finally, we have used tethered hydroxyl radical probing to determine the locations on viral IRESs at which each of the four RRM domains bind. We are now combining tethered probing with single molecule analyses to gain a detailed understanding of how PTB interacts with pre-mRNA substrates to effect either repression or activation of splicing.
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http://dx.doi.org/10.1042/BST20120044DOI Listing
August 2012

Crystallographic analysis of polypyrimidine tract-binding protein-Raver1 interactions involved in regulation of alternative splicing.

Structure 2011 Dec;19(12):1816-25

Division of Cell and Molecular Biology, Imperial College, Exhibition Road, London SW7 2AZ, UK.

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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http://dx.doi.org/10.1016/j.str.2011.09.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3420021PMC
December 2011

Decoding muscle alternative splicing.

Curr Opin Genet Dev 2011 Aug 21;21(4):380-7. Epub 2011 Apr 21.

Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK.

Muscle was one of the first tissues in which alternative splicing was widely observed. Cloning and sequencing of muscle-derived cDNAs in the early 1980's revealed that many of the abundant contractile proteins arise by alternative splicing of genes that are more widely expressed. Consequently alternative splicing events in contractile protein genes have long been used as models to dissect the mechanisms of alternative splicing. Transcriptomic and computational analyses have complemented traditional molecular analyses of alternative splicing in muscle and other tissues, illuminating the general underlying principles of coregulated splicing programs. This has culminated in the first attempt to computationally predict tissue-specific changes in splicing. Investigations of myotonic dystrophy (DM), in which CUG expansion RNA leads to misregulated splicing in muscle, have enhanced our understanding of developmentally regulated splicing and led to the development of promising therapeutic strategies based on targeting the toxic RNA repeats.
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http://dx.doi.org/10.1016/j.gde.2011.03.006DOI Listing
August 2011

Genome-wide association between branch point properties and alternative splicing.

PLoS Comput Biol 2010 Nov 24;6(11):e1001016. Epub 2010 Nov 24.

Computational Genomics, Universitat Pompeu Fabra, Barcelona, Spain.

The branch point (BP) is one of the three obligatory signals required for pre-mRNA splicing. In mammals, the degeneracy of the motif combined with the lack of a large set of experimentally verified BPs complicates the task of modeling it in silico, and therefore of predicting the location of natural BPs. Consequently, BPs have been disregarded in a considerable fraction of the genome-wide studies on the regulation of splicing in mammals. We present a new computational approach for mammalian BP prediction. Using sequence conservation and positional bias we obtained a set of motifs with good agreement with U2 snRNA binding stability. Using a Support Vector Machine algorithm, we created a model complemented with polypyrimidine tract features, which considerably improves the prediction accuracy over previously published methods. Applying our algorithm to human introns, we show that BP position is highly dependent on the presence of AG dinucleotides in the 3' end of introns, with distance to the 3' splice site and BP strength strongly correlating with alternative splicing. Furthermore, experimental BP mapping for five exons preceded by long AG-dinucleotide exclusion zones revealed that, for a given intron, more than one BP can be chosen throughout the course of splicing. Finally, the comparison between exons of different evolutionary ages and pseudo exons suggests a key role of the BP in the pathway of exon creation in human. Our computational and experimental analyses suggest that BP recognition is more flexible than previously assumed, and it appears highly dependent on the presence of downstream polypyrimidine tracts. The reported association between BP features and the splicing outcome suggests that this, so far disregarded but yet crucial, element buries information that can complement current acceptor site models.
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http://dx.doi.org/10.1371/journal.pcbi.1001016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2991248PMC
November 2010

Expression proteomics of UPF1 knockdown in HeLa cells reveals autoregulation of hnRNP A2/B1 mediated by alternative splicing resulting in nonsense-mediated mRNA decay.

BMC Genomics 2010 Oct 14;11:565. Epub 2010 Oct 14.

Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QW, UK.

Background: In addition to acting as an RNA quality control pathway, nonsense-mediated mRNA decay (NMD) plays roles in regulating normal gene expression. In particular, the extent to which alternative splicing is coupled to NMD and the roles of NMD in regulating uORF containing transcripts have been a matter of debate.

Results: In order to achieve a greater understanding of NMD regulated gene expression we used 2D-DiGE proteomics technology to examine the changes in protein expression induced in HeLa cells by UPF1 knockdown. QPCR based validation of the corresponding mRNAs, in response to both UPF1 knockdown and cycloheximide treatment, identified 17 bona fide NMD targets. Most of these were associated with bioinformatically predicted NMD activating features, predominantly upstream open reading frames (uORFs). Strikingly, however, the majority of transcripts up-regulated by UPF1 knockdown were either insensitive to, or even down-regulated by, cycloheximide treatment. Furthermore, the mRNA abundance of several down-regulated proteins failed to change upon UPF1 knockdown, indicating that UPF1's role in regulating mRNA and protein abundance is more complex than previously appreciated. Among the bona fide NMD targets, we identified a highly conserved AS-NMD event within the 3' UTR of the HNRNPA2B1 gene. Overexpression of GFP tagged hnRNP A2 resulted in a decrease in endogenous hnRNP A2 and B1 mRNA with a concurrent increase in the NMD sensitive isoforms.

Conclusions: Despite the large number of changes in protein expression upon UPF1 knockdown, a relatively small fraction of them can be directly attributed to the action of NMD on the corresponding mRNA. From amongst these we have identified a conserved AS-NMD event within HNRNPA2B1 that appears to mediate autoregulation of HNRNPA2B1 expression levels.
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http://dx.doi.org/10.1186/1471-2164-11-565DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3091714PMC
October 2010

Position-dependent alternative splicing activity revealed by global profiling of alternative splicing events regulated by PTB.

Nat Struct Mol Biol 2010 Sep 15;17(9):1114-23. Epub 2010 Aug 15.

Department of Biochemistry, University of Cambridge, Cambridge, UK.

To gain global insights into the role of the well-known repressive splicing regulator PTB, we analyzed the consequences of PTB knockdown in HeLa cells using high-density oligonucleotide splice-sensitive microarrays. The major class of identified PTB-regulated splicing event was PTB-repressed cassette exons, but there was also a substantial number of PTB-activated splicing events. PTB-repressed and PTB-activated exons showed a distinct arrangement of motifs with pyrimidine-rich motif enrichment within and upstream of repressed exons but downstream of activated exons. The N-terminal half of PTB was sufficient to activate splicing when recruited downstream of a PTB-activated exon. Moreover, insertion of an upstream pyrimidine tract was sufficient to convert a PTB-activated exon to a PTB-repressed exon. Our results show that PTB, an archetypal splicing repressor, has variable splicing activity that predictably depends upon its binding location with respect to target exons.
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http://dx.doi.org/10.1038/nsmb.1881DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2933513PMC
September 2010

Stoichiometry of a regulatory splicing complex revealed by single-molecule analyses.

EMBO J 2010 Jul 25;29(13):2161-72. Epub 2010 May 25.

Department of Biochemistry, University of Leicester, Leicester, UK.

Splicing is regulated by complex interactions of numerous RNA-binding proteins. The molecular mechanisms involved remain elusive, in large part because of ignorance regarding the numbers of proteins in regulatory complexes. Polypyrimidine tract-binding protein (PTB), which regulates tissue-specific splicing, represses exon 3 of alpha-tropomyosin through distant pyrimidine-rich tracts in the flanking introns. Current models for repression involve either PTB-mediated looping or the propagation of complexes between tracts. To test these models, we used single-molecule approaches to count the number of bound PTB molecules both by counting the number of bleaching steps of GFP molecules linked to PTB within complexes and by analysing their total emissions. Both approaches showed that five or six PTB molecules assemble. Given the domain structures, this suggests that the molecules occupy primarily multiple overlapping potential sites in the polypyrimidine tracts, excluding propagation models. As an alternative to direct looping, we propose that repression involves a multistep process in which PTB binding forms small local loops, creating a platform for recruitment of other proteins that bring these loops into close proximity.
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http://dx.doi.org/10.1038/emboj.2010.103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2905242PMC
July 2010

Four exons of the serotonin receptor 4 gene are associated with multiple distant branch points.

RNA 2010 Apr 2;16(4):839-51. Epub 2010 Mar 2.

Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom.

Splicing of vertebrate introns involves recognition of three consensus elements at the 3' end. The branch point (BP) and polypyrimidine tract (PPT) are usually located within 40 nucleotides (nt) of the 3' splice site (3' ss), AG, but can be much more distant. A characteristic of the region between distant BPs (dBPs) and the 3' ss is the absence of intervening AG dinucleotides, leading to its designation as the "AG exclusion zone" (AGEZ). The human HTR4 gene, which encodes serotonin receptor 4 and has been associated with schizophrenia, bipolar disease, and gastrointestinal disorders, has four exons with extensive AGEZs. We have mapped the BPs for HTR4 exons 3, 4, 5, and g generated by in vitro splicing, and validated them by mutagenesis in exon-trapping vectors. All exons used dBPs up to 273 nt upstream of the exon. Strikingly, exons 4 and 5 used combinations of both distant and conventionally located BPs, suggesting that successful splicing of these exons can occur by distinct pathways. Our results emphasize the importance for single nucleotide polymorphism resequencing projects to take account of potential dBPs, as the extended AGEZs are vulnerable to mutations that could affect splicing itself or regulation of alternative splicing.
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http://dx.doi.org/10.1261/rna.2013110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2844630PMC
April 2010

Alternative splicing: global insights.

FEBS J 2010 Feb 15;277(4):856-66. Epub 2010 Jan 15.

Department of Biochemistry, University of Cambridge, UK.

Following the original reports of pre-mRNA splicing in 1977, it was quickly realized that splicing together of different combinations of splice sites--alternative splicing--allows individual genes to generate more than one mRNA isoform. The full extent of alternative splicing only began to be revealed once large-scale genome and transcriptome sequencing projects began, rapidly revealing that alternative splicing is the rule rather than the exception. Recent technical innovations have facilitated the investigation of alternative splicing at a global scale. Splice-sensitive microarray platforms and deep sequencing allow quantitative profiling of very large numbers of alternative splicing events, whereas global analysis of the targets of RNA binding proteins reveals the regulatory networks involved in post-transcriptional gene control. Combined with sophisticated computational analysis, these new approaches are beginning to reveal the so-called 'RNA code' that underlies tissue and developmentally regulated alternative splicing, and that can be disrupted by disease-causing mutations.
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http://dx.doi.org/10.1111/j.1742-4658.2009.07521.xDOI Listing
February 2010

Tropomyosin exons as models for alternative splicing.

Adv Exp Med Biol 2008 ;644:27-42

Department of Biochemistry, University of Cambridge, CB2 1GA, UK.

Three of the four mammalian tropomyosin (Tm) genes are alternatively spliced, most commonly by mutually exclusive selection from pairs of internal or 3' end exons. Alternative splicing events in the TPM1, 2 and 3 genes have been analysed experimentally in various levels ofdetail. In particular, mutually exclusive exon pairs in the betaTm (TPM2) and alphaTm (TPM1) genes are among the most intensively studied models for striated and smooth muscle specific alternative splicing, respectively. Analysis of these model systems has provided important insights into general mechanisms and strategies of splicing regulation.
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http://dx.doi.org/10.1007/978-0-387-85766-4_3DOI Listing
February 2009

A nonsense exon in the Tpm1 gene is silenced by hnRNP H and F.

RNA 2009 Jan 26;15(1):33-43. Epub 2008 Nov 26.

Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom.

As well as generating protein isoform diversity, in some cases alternative splicing generates RNAs that harbor premature termination codons and that are subject to nonsense-mediated decay (NMD). We previously identified an apparent pseudo-exon in the rat alpha-tropomyosin (Tpm1) gene as a probable genuine alternatively spliced exon that causes NMD when spliced into Tpm1 RNA. Here, we report the analysis of cis-acting splicing regulatory elements within this "nonsense exon." Guided by the data set of predicted splicing enhancer and silencer elements compiled by Zhang and Chasin, we made a series of mutations through the nonsense exon and found that like authentic exons it is densely packed with enhancer and silencer elements. Strikingly, 11 of 13 tested mutations behaved as predicted computationally. In particular, we found that a G-rich silencer at the 5' end, which is crucial for skipping of the nonsense exon, functions by binding hnRNP-H and F.
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http://dx.doi.org/10.1261/rna.1225209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2612778PMC
January 2009

Polypyrimidine tract binding protein regulates alternative splicing of an aberrant pseudoexon in NF1.

FEBS J 2008 Dec 1;275(24):6101-8. Epub 2008 Nov 1.

Human Genetics Division, University of Southampton, UK.

In disease-associated genes, understanding the functional significance of deep intronic nucleotide variants represents a difficult challenge. We previously reported that an NF1 intron 30 exonization event is triggered from a single correct nomenclature is 'c.293-279 A>G' mutation [Raponi M, Upadhyaya M & Baralle D (2006) Hum Mutat 27, 294-295]. In this paper, we investigate which characteristics play a role in regulating inclusion of the aberrant pseudoexon. Our investigation shows that pseudoexon inclusion levels are strongly downregulated by polypyrimidine tract binding protein and its homologue neuronal polypyrimidine tract binding protein. In particular, we provide evidence that the functional effect of polypyrimidine tract binding protein is proportional to its concentration, and map the cis-acting elements that are principally responsible for this negative regulation. These results highlight the importance of evaluating local sequence context for diagnostic purposes, and the utility of developing therapies to turn off activated pseudoexons.
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http://dx.doi.org/10.1111/j.1742-4658.2008.06734.xDOI Listing
December 2008