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Sample records for alternative splicing regulatory

  1. Control of alternative splicing by signal-dependent degradation of splicing-regulatory proteins.

    PubMed

    Katzenberger, Rebeccah J; Marengo, Matthew S; Wassarman, David A

    2009-04-17

    Alternative pre-mRNA splicing is a major gene expression regulatory mechanism in metazoan organisms. Proteins that bind pre-mRNA elements and control assembly of splicing complexes regulate utilization of pre-mRNA alternative splice sites. To understand how signaling pathways impact this mechanism, an RNA interference screen in Drosophila S2 cells was used to identify proteins that regulate TAF1 (TBP-associated factor 1) alternative splicing in response to activation of the ATR (ATM-RAD3-related) signaling pathway by the chemotherapeutic drug camptothecin (CPT). The screen identified 15 proteins that, when knocked down, caused the same change in TAF1 alternative splicing as CPT treatment. However, combined RNA interference and CPT treatment experiments indicated that only a subset of the identified proteins are targets of the CPT-induced signal, suggesting that multiple independent pathways regulate TAF1 alternative splicing. To understand how signals modulate the function of splicing factors, we characterized one of the CPT targets, Tra2 (Transformer-2). CPT was found to down-regulate Tra2 protein levels. CPT-induced Tra2 down-regulation was ATR-dependent and temporally paralleled the change in TAF1 alternative splicing, supporting the conclusion that Tra2 directly regulates TAF1 alternative splicing. Additionally, CPT-induced Tra2 down-regulation occurred independently of new protein synthesis, suggesting a post-translational mechanism. The proteasome inhibitor MG132 reduced CPT-induced Tra2 degradation and TAF1 alternative splicing, and mutation of evolutionarily conserved Tra2 lysine 81, a potential ubiquitin conjugation site, to arginine inhibited CPT-induced Tra2 degradation, supporting a proteasome-dependent alternative splicing mechanism. We conclude that CPT-induced TAF1 alternative splicing occurs through ATR-signaled degradation of a subset of splicing-regulatory proteins.

  2. Multilayered Control of Alternative Splicing Regulatory Networks by Transcription Factors.

    PubMed

    Han, Hong; Braunschweig, Ulrich; Gonatopoulos-Pournatzis, Thomas; Weatheritt, Robert J; Hirsch, Calley L; Ha, Kevin C H; Radovani, Ernest; Nabeel-Shah, Syed; Sterne-Weiler, Tim; Wang, Juli; O'Hanlon, Dave; Pan, Qun; Ray, Debashish; Zheng, Hong; Vizeacoumar, Frederick; Datti, Alessandro; Magomedova, Lilia; Cummins, Carolyn L; Hughes, Timothy R; Greenblatt, Jack F; Wrana, Jeffrey L; Moffat, Jason; Blencowe, Benjamin J

    2017-02-02

    Networks of coordinated alternative splicing (AS) events play critical roles in development and disease. However, a comprehensive knowledge of the factors that regulate these networks is lacking. We describe a high-throughput system for systematically linking trans-acting factors to endogenous RNA regulatory events. Using this system, we identify hundreds of factors associated with diverse regulatory layers that positively or negatively control AS events linked to cell fate. Remarkably, more than one-third of the regulators are transcription factors. Further analyses of the zinc finger protein Zfp871 and BTB/POZ domain transcription factor Nacc1, which regulate neural and stem cell AS programs, respectively, reveal roles in controlling the expression of specific splicing regulators. Surprisingly, these proteins also appear to regulate target AS programs via binding RNA. Our results thus uncover a large "missing cache" of splicing regulators among annotated transcription factors, some of which dually regulate AS through direct and indirect mechanisms.

  3. Predominant contribution of cis-regulatory divergence in the evolution of mouse alternative splicing

    PubMed Central

    Gao, Qingsong; Sun, Wei; Ballegeer, Marlies; Libert, Claude; Chen, Wei

    2015-01-01

    Divergence of alternative splicing represents one of the major driving forces to shape phenotypic diversity during evolution. However, the extent to which these divergences could be explained by the evolving cis-regulatory versus trans-acting factors remains unresolved. To globally investigate the relative contributions of the two factors for the first time in mammals, we measured splicing difference between C57BL/6J and SPRET/EiJ mouse strains and allele-specific splicing pattern in their F1 hybrid. Out of 11,818 alternative splicing events expressed in the cultured fibroblast cells, we identified 796 with significant difference between the parental strains. After integrating allele-specific data from F1 hybrid, we demonstrated that these events could be predominately attributed to cis-regulatory variants, including those residing at and beyond canonical splicing sites. Contrary to previous observations in Drosophila, such predominant contribution was consistently observed across different types of alternative splicing. Further analysis of liver tissues from the same mouse strains and reanalysis of published datasets on other strains showed similar trends, implying in general the predominant contribution of cis-regulatory changes in the evolution of mouse alternative splicing. PMID:26134616

  4. New Insights into VEGF-A Alternative Splicing: Key Regulatory Switching in the Pathological Process.

    PubMed

    Dehghanian, Fariba; Hojati, Zohreh; Kay, Maryam

    2014-10-01

    Vascular endothelial growth factor (VEGF-A) is one of the most important regulatory factors in pathological and physiological angiogenesis. Alternative splicing is a complicated molecular process in VEGF-A gene expression which adds complexity to VEGF-A biology. Among all VEGF-A exons, alternative splicing of exon 8 is the key determinant of isoform switching from pro-angio-genic VEGF-xxx to anti-angiogenic VEGF-xxxb. This is known as a key molecular switching in many pathological situations. In fact, the balance between VEGF-xxx and VEGF-xxxb isoforms is a critical controlling switch in both conditions of health and disease. Here, the properties of VEGF-xxx and VEGF-xxxb isoforms were discussed and their regulatory mechanism and their roles in certain pathological processes were evaluated. In summary, it was suggested that C-terminal VEGF-A alternative splicing can provide a new treatment opportunity in angiogenic diseases.

  5. Regulatory Expansion in Mammals of Multivalent hnRNP Assemblies that Globally Control Alternative Splicing.

    PubMed

    Gueroussov, Serge; Weatheritt, Robert J; O'Hanlon, Dave; Lin, Zhen-Yuan; Narula, Ashrut; Gingras, Anne-Claude; Blencowe, Benjamin J

    2017-07-13

    Alternative splicing (AS) patterns have diverged rapidly during vertebrate evolution, yet the functions of most species- and lineage-specific splicing events are not known. We observe that mammalian-specific AS events are enriched in transcript sequences encoding intrinsically disordered regions (IDRs) of proteins, in particular those containing glycine/tyrosine repeats that mediate formation of higher-order protein assemblies implicated in gene regulation and human disease. These evolutionary changes impact nearly all members of the hnRNP A and D families of RNA binding proteins. Regulation of these events requires formation of unusual, long-range mammalian-specific RNA duplexes. Differential inclusion of the alternative exons controls the formation of tyrosine-dependent multivalent hnRNP assemblies that, in turn, function to globally regulate splicing. Together, our results demonstrate that AS control of IDR-mediated interactions between hnRNPs represents an important and recurring mechanism underlying splicing regulation. Furthermore, this mechanism has expanded the regulatory capacity of mammalian cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. HRP-2, the Caenorhabditis elegans homolog of mammalian heterogeneous nuclear ribonucleoproteins Q and R, is an alternative splicing factor that binds to UCUAUC splicing regulatory elements.

    PubMed

    Kabat, Jennifer L; Barberan-Soler, Sergio; Zahler, Alan M

    2009-10-16

    Alternative splicing is regulated by cis sequences in the pre-mRNA that serve as binding sites for trans-acting alternative splicing factors. In a previous study, we used bioinformatics and molecular biology to identify and confirm that the intronic hexamer sequence UCUAUC is a nematode alternative splicing regulatory element. In this study, we used RNA affinity chromatography to identify trans factors that bind to this sequence. HRP-2, the Caenorhabditis elegans homolog of human heterogeneous nuclear ribonucleoproteins Q and R, binds to UCUAUC in the context of unc-52 intronic regulatory sequences as well as to RNAs containing tandem repeats of this sequence. The three Us in the hexamer are the most important determinants of this binding specificity. We demonstrate, using RNA interference, that HRP-2 regulates the alternative splicing of two genes, unc-52 and lin-10, both of which have cassette exons flanked by an intronic UCUAUC motif. We propose that HRP-2 is a protein responsible for regulating alternative splicing through binding interactions with the UCUAUC sequence.

  7. SIRT1 Undergoes Alternative Splicing in a Novel Auto-Regulatory Loop with p53

    PubMed Central

    Lynch, Cian J.; Ahmed, Shafiq U.; Ford, Jack; Warnock, Lorna J.; Li, Han; Serrano, Manuel; Milner, Jo

    2010-01-01

    Background The NAD-dependent deacetylase SIRT1 is a nutrient-sensitive coordinator of stress-tolerance, multiple homeostatic processes and healthspan, while p53 is a stress-responsive transcription factor and our paramount tumour suppressor. Thus, SIRT1-mediated inhibition of p53 has been identified as a key node in the common biology of cancer, metabolism, development and ageing. However, precisely how SIRT1 integrates such diverse processes remains to be elucidated. Methodology/Principal Findings Here we report that SIRT1 is alternatively spliced in mammals, generating a novel SIRT1 isoform: SIRT1-ΔExon8. We show that SIRT1-ΔExon8 is expressed widely throughout normal human and mouse tissues, suggesting evolutionary conservation and critical function. Further studies demonstrate that the SIRT1-ΔExon8 isoform retains minimal deacetylase activity and exhibits distinct stress sensitivity, RNA/protein stability, and protein-protein interactions compared to classical SIRT1-Full-Length (SIRT1-FL). We also identify an auto-regulatory loop whereby SIRT1-ΔExon8 can regulate p53, while in reciprocal p53 can influence SIRT1 splice variation. Conclusions/Significance We characterize the first alternative isoform of SIRT1 and demonstrate its evolutionary conservation in mammalian tissues. The results also reveal a new level of inter-dependency between p53 and SIRT1, two master regulators of multiple phenomena. Thus, previously-attributed SIRT1 functions may in fact be distributed between SIRT1 isoforms, with important implications for SIRT1 functional studies and the current search for SIRT1-activating therapeutics to combat age-related decline. PMID:20975832

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

    PubMed Central

    Coelho, Miguel B; Attig, Jan; Bellora, Nicolás; König, Julian; Hallegger, Martina; Kayikci, Melis; Eyras, Eduardo; Ule, Jernej; Smith, Christopher WJ

    2015-01-01

    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. PMID:25599992

  9. Understanding alternative splicing: towards a cellular code.

    PubMed

    Matlin, Arianne J; Clark, Francis; Smith, Christopher W J

    2005-05-01

    In violation of the 'one gene, one polypeptide' rule, alternative splicing allows individual genes to produce multiple protein isoforms - thereby playing a central part in generating complex proteomes. Alternative splicing also has a largely hidden function in quantitative gene control, by targeting RNAs for nonsense-mediated decay. Traditional gene-by-gene investigations of alternative splicing mechanisms are now being complemented by global approaches. These promise to reveal details of the nature and operation of cellular codes that are constituted by combinations of regulatory elements in pre-mRNA substrates and by cellular complements of splicing regulators, which together determine regulated splicing pathways.

  10. Chromatin, DNA structure and alternative splicing.

    PubMed

    Nieto Moreno, Nicolás; Giono, Luciana E; Cambindo Botto, Adrián E; Muñoz, Manuel J; Kornblihtt, Alberto R

    2015-11-14

    Coupling of transcription and alternative splicing via regulation of the transcriptional elongation rate is a well-studied phenomenon. Template features that act as roadblocks for the progression of RNA polymerase II comprise histone modifications and variants, DNA-interacting proteins and chromatin compaction. These may affect alternative splicing decisions by inducing pauses or decreasing elongation rate that change the time-window for splicing regulatory sequences to be recognized. Herein we discuss the evidence supporting the influence of template structural modifications on transcription and splicing, and provide insights about possible roles of non-B DNA conformations on the regulation of alternative splicing.

  11. Investigating alternative RNA splicing in Xenopus.

    PubMed

    Mereau, Agnès; Hardy, Serge

    2012-01-01

    Alternative splicing, the process by which distinct mature mRNAs can be produced from a single primary transcript, is a key mechanism to increase the organism complexity. The generation of alternative splicing pattern is a means to expand the proteome diversity and also to control gene expression through the regulation of mRNA abundance. Alternative splicing is therefore particularly prevalent during development and accordingly numerous splicing events are regulated in a tissue or temporal manner. To study the roles of alternative splicing during developmental processes and decipher the molecular mechanisms that underlie temporal and spatial regulation, it is important to develop in vivo whole animal studies. In this chapter, we present the advantages of using the amphibian Xenopus as a fully in vivo model to study alternative splicing and we describe the experimental procedures that can be used with Xenopus laevis embryos and oocytes to define the cis-regulatory elements and identify the associated trans-acting factors.

  12. Regulation of alternative splicing within the supraspliceosome.

    PubMed

    Sebbag-Sznajder, Naama; Raitskin, Oleg; Angenitzki, Minna; Sato, Taka-Aki; Sperling, Joseph; Sperling, Ruth

    2012-01-01

    Alternative splicing is a fundamental feature in regulating the eukaryotic transcriptome, as ~95% of multi-exon human Pol II transcripts are subject to this process. Regulated splicing operates through the combinatorial interplay of positive and negative regulatory signals present in the pre-mRNA, which are recognized by trans-acting factors. All these RNA and protein components are assembled in a gigantic, 21 MDa, ribonucleoprotein splicing machine - the supraspliceosome. Because most alternatively spliced mRNA isoforms vary between different cell and tissue types, the ability to perform alternative splicing is expected to be an integral part of the supraspliceosome, which constitutes the splicing machine in vivo. Here we show that both the constitutively and alternatively spliced mRNAs of the endogenous human pol II transcripts: hnRNP A/B, survival of motor neuron (SMN) and ADAR2 are predominantly found in supraspliceosomes. This finding is consistent with our observations that the splicing regulators hnRNP G as well as all phosphorylated SR proteins are predominantly associated with supraspliceosomes. We further show that changes in alternative splicing of hnRNP A/B, affected by up regulation of SRSF5 (SRp40) or by treatment with C6-ceramide, occur within supraspliceosomes. These observations support the proposed role of the supraspliceosome in splicing regulation and alternative splicing. Copyright © 2011 Elsevier Inc. All rights reserved.

  13. Regulation of alternative splicing within the supraspliceosome

    PubMed Central

    Sebbag-Sznajder, Naama; Raitskin, Oleg; Angenitzki, Minna; Sato, Taka-Aki; Sperling, Joseph; Sperling, Ruth

    2012-01-01

    Alternative splicing is a fundamental feature in regulating the eukaryotic transcriptome, as ~95% of multi-exon human Pol II transcripts are subject to this process. Regulated splicing operates through the combinatorial interplay of positive and negative regulatory signals present in the pre-mRNA, which are recognized by trans-acting factors. All these RNA and protein components are assembled in a gigantic, 21 MDa, ribonucleoprotein splicing machine – the supraspliceosome. Because most alternatively spliced mRNA isoforms vary between different cell and tissue types, the ability to perform alternative splicing is expected to be an integral part of the supraspliceosome, which constitutes the splicing machine in vivo. Here we show that both the constitutively and alternatively spliced mRNAs of the endogenous human pol II transcripts: hnRNP A/B, survival of motor neuron (SMN) and ADAR2 are predominantly found in supraspliceosomes. This finding is consistent with our observations that the splicing regulators hnRNP G as well as all phosphorylated SR proteins are predominantly associated with supraspliceosomes. We further show that changes in alternative splicing of hnRNP A/B, affected by up regulation of SRSF5 (SRp40) or by treatment with C6-ceramide, occur within supraspliceosomes. These observations support the proposed role of the supraspliceosome in splicing regulation and alternative splicing. PMID:22100336

  14. Promoter usage and alternative splicing.

    PubMed

    Kornblihtt, Alberto R

    2005-06-01

    Recent findings justify a renewed interest in alternative splicing (AS): the process is more a rule than an exception as it affects the expression of 60% of human genes; it explains how a vast mammalian proteomic complexity is achieved with a limited number of genes; and mutations in AS regulatory sequences are a widespread source of human disease. AS regulation not only depends on the interaction of splicing factors with their target sequences in the pre-mRNA but is coupled to transcription. A clearer picture is emerging of the mechanisms by which transcription affects AS through promoter identity and occupation. These mechanisms involve the recruitment of factors with dual functions in transcription and splicing (i.e. that contain both functional domains and hence link the two processes) and the control of RNA polymerase II elongation.

  15. Coupling transcription and alternative splicing.

    PubMed

    Kornblihtt, Alberto R

    2007-01-01

    Alternative splicing regulation not only depends on the interaction of splicing factors with splicing enhancers and silencers in the pre-mRNA, but also on the coupling between transcription and splicing. This coupling is possible because splicing is often cotranscriptional and promoter identity and occupation may affect alternative splicing. We discuss here the different mechanisms by which transcription regulates alternative splicing. These include the recruitment of splicing factors to the transcribing polymerase and "kinetic coupling", which involves changes in the rate of transcriptional elongation that in turn affect the timing in which splice sites are presented to the splicing machinery. The recruitment mechanism may depend on the particular features of the carboxyl terminal domain of RNA polymerase II, whereas kinetic coupling seems to be linked to how changes in chromatin structure and other factors affect transcription elongation.

  16. Alternative splicing and nonsense-mediated decay modulate expression of important regulatory genes in Arabidopsis

    PubMed Central

    Kalyna, Maria; Simpson, Craig G.; Syed, Naeem H.; Lewandowska, Dominika; Marquez, Yamile; Kusenda, Branislav; Marshall, Jacqueline; Fuller, John; Cardle, Linda; McNicol, Jim; Dinh, Huy Q.; Barta, Andrea; Brown, John W. S.

    2012-01-01

    Alternative splicing (AS) coupled to nonsense-mediated decay (NMD) is a post-transcriptional mechanism for regulating gene expression. We have used a high-resolution AS RT–PCR panel to identify endogenous AS isoforms which increase in abundance when NMD is impaired in the Arabidopsis NMD factor mutants, upf1-5 and upf3-1. Of 270 AS genes (950 transcripts) on the panel, 102 transcripts from 97 genes (32%) were identified as NMD targets. Extrapolating from these data around 13% of intron-containing genes in the Arabidopsis genome are potentially regulated by AS/NMD. This cohort of naturally occurring NMD-sensitive AS transcripts also allowed the analysis of the signals for NMD in plants. We show the importance of AS in introns in 5′ or 3′UTRs in modulating NMD-sensitivity of mRNA transcripts. In particular, we identified upstream open reading frames overlapping the main start codon as a new trigger for NMD in plants and determined that NMD is induced if 3′-UTRs were >350 nt. Unexpectedly, although many intron retention transcripts possess NMD features, they are not sensitive to NMD. Finally, we have shown that AS/NMD regulates the abundance of transcripts of many genes important for plant development and adaptation including transcription factors, RNA processing factors and stress response genes. PMID:22127866

  17. Determination of a Comprehensive Alternative Splicing Regulatory Network and Combinatorial Regulation by Key Factors during the Epithelial-to-Mesenchymal Transition

    PubMed Central

    Yang, Yueqin; Park, Juw Won; Bebee, Thomas W.; Warzecha, Claude C.; Guo, Yang; Shang, Xuequn

    2016-01-01

    The epithelial-to-mesenchymal transition (EMT) is an essential biological process during embryonic development that is also implicated in cancer metastasis. While the transcriptional regulation of EMT has been well studied, the role of alternative splicing (AS) regulation in EMT remains relatively uncharacterized. We previously showed that the epithelial cell-type-specific proteins epithelial splicing regulatory proteins 1 (ESRP1) and ESRP2 are important for the regulation of many AS events that are altered during EMT. However, the contributions of the ESRPs and other splicing regulators to the AS regulatory network in EMT require further investigation. Here, we used a robust in vitro EMT model to comprehensively characterize splicing switches during EMT in a temporal manner. These investigations revealed that the ESRPs are the major regulators of some but not all AS events during EMT. We determined that the splicing factor RBM47 is downregulated during EMT and also regulates numerous transcripts that switch splicing during EMT. We also determined that Quaking (QKI) broadly promotes mesenchymal splicing patterns. Our study highlights the broad role of posttranscriptional regulation during the EMT and the important role of combinatorial regulation by different splicing factors to fine tune gene expression programs during these physiological and developmental transitions. PMID:27044866

  18. Rethinking gene regulatory networks in light of alternative splicing, intrinsically disordered protein domains, and post-translational modifications

    PubMed Central

    Niklas, Karl J.; Bondos, Sarah E.; Dunker, A. Keith; Newman, Stuart A.

    2015-01-01

    Models for genetic regulation and cell fate specification characteristically assume that gene regulatory networks (GRNs) are essentially deterministic and exhibit multiple stable states specifying alternative, but pre-figured cell fates. Mounting evidence shows, however, that most eukaryotic precursor RNAs undergo alternative splicing (AS) and that the majority of transcription factors contain intrinsically disordered protein (IDP) domains whose functionalities are context dependent as well as subject to post-translational modification (PTM). Consequently, many transcription factors do not have fixed cis-acting regulatory targets, and developmental determination by GRNs alone is untenable. Modeling these phenomena requires a multi-scale approach to explain how GRNs operationally interact with the intra- and intercellular environments. Evidence shows that AS, IDP, and PTM complicate gene expression and act synergistically to facilitate and promote time- and cell-specific protein modifications involved in cell signaling and cell fate specification and thereby disrupt a strict deterministic GRN-phenotype mapping. The combined effects of AS, IDP, and PTM give proteomes physiological plasticity, adaptive responsiveness, and developmental versatility without inefficiently expanding genome size. They also help us understand how protein functionalities can undergo major evolutionary changes by buffering mutational consequences. PMID:25767796

  19. Alternative RNA splicing and cancer

    PubMed Central

    Liu, Sali; Cheng, Chonghui

    2015-01-01

    Alternative splicing of pre-messenger RNA (mRNA) is a fundamental mechanism by which a gene can give rise to multiple distinct mRNA transcripts, yielding protein isoforms with different, even opposing, functions. With the recognition that alternative splicing occurs in nearly all human genes, its relationship with cancer-associated pathways has emerged as a rapidly growing field. In this review, we summarize recent findings that have implicated the critical role of alternative splicing in cancer and discuss current understandings of the mechanisms underlying dysregulated alternative splicing in cancer cells. PMID:23765697

  20. Alternative RNA splicing and cancer.

    PubMed

    Liu, Sali; Cheng, Chonghui

    2013-01-01

    Alternative splicing of pre-messenger RNA (mRNA) is a fundamental mechanism by which a gene can give rise to multiple distinct mRNA transcripts, yielding protein isoforms with different, even opposing, functions. With the recognition that alternative splicing occurs in nearly all human genes, its relationship with cancer-associated pathways has emerged as a rapidly growing field. In this review, we summarize recent findings that have implicated the critical role of alternative splicing in cancer and discuss current understandings of the mechanisms underlying dysregulated alternative splicing in cancer cells.

  1. Alternative splicing interference by xenobiotics.

    PubMed

    Zaharieva, Emanuela; Chipman, J Kevin; Soller, Matthias

    2012-06-14

    The protein coding sequence of most eukaryotic genes (exons) is interrupted by non-coding parts (introns), which are excised in a process termed splicing. To generate a mature messenger RNA (mRNA) hundreds of combinatorial protein-protein and RNA-protein interactions are required to splice out often very large introns with high fidelity and accuracy. Inherent to splicing is the use of alternative splice sites generating immense proteomic diversity from a limited number of genes. In humans, alternative splicing is a major mode of regulating gene expression, occurs in over 90% of genes and is particularly abundant in the brain. Only recently, it has been recognized that the complexity of the splicing process makes it susceptible to interference by various xenobiotics. These compounds include antineoplastic substances, commonly used drugs and food supplements and cause a spectrum of effects ranging from deleterious inhibition of general splicing to highly specific modifications of alternative splicing affecting only certain genes. Alterations in splicing have been implicated in numerous diseases such as cancer and neurodegeneration. Splicing regulation plays an important role in the execution of programmed cell death. The switch between anti- and pro-apoptotic isoforms by alternative splice site selection and misregulation of a number of splicing factors impacts on cell survival and disease. Here, our current knowledge is summarized on compounds interfering with general and alternative splicing and of the current methodology to study changes in these processes relevant to the field of toxicology and future risk assessments. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  2. The neurogenetics of alternative splicing

    PubMed Central

    Vuong, Celine K.; Black, Douglas L.; Zheng, Sika

    2016-01-01

    Alternative precursor-mRNA splicing is a key mechanism for regulating gene expression in mammals and is controlled by specialized RNA-binding proteins. The misregulation of splicing is implicated in multiple neurological disorders. We describe recent mouse genetic studies of alternative splicing that reveal its critical role in both neuronal development and the function of mature neurons. We discuss the challenges in understanding the extensive genetic programmes controlled by proteins that regulate splicing, both during development and in the adult brain. PMID:27094079

  3. Alternative splicing and muscular dystrophy

    PubMed Central

    Pistoni, Mariaelena; Ghigna, Claudia; Gabellini, Davide

    2013-01-01

    Alternative splicing of pre-mRNAs is a major contributor to proteomic diversity and to the control of gene expression in higher eukaryotic cells. For this reasons, alternative splicing is tightly regulated in different tissues and developmental stages and its disruption can lead to a wide range of human disorders. The aim of this review is to focus on the relevance of alternative splicing for muscle function and muscle disease. We begin by giving a brief overview of alternative splicing, muscle-specific gene expression and muscular dystrophy. Next, to illustrate these concepts we focus on two muscular dystrophy, myotonic muscular dystrophy and facioscapulohumeral muscular dystrophy, both associated to disruption of splicing regulation in muscle. PMID:20603608

  4. Alternative Splice in Alternative Lice

    PubMed Central

    Tovar-Corona, Jaime M.; Castillo-Morales, Atahualpa; Chen, Lu; Olds, Brett P.; Clark, John M.; Reynolds, Stuart E.; Pittendrigh, Barry R.; Feil, Edward J.; Urrutia, Araxi O.

    2015-01-01

    Genomic and transcriptomics analyses have revealed human head and body lice to be almost genetically identical; although con-specific, they nevertheless occupy distinct ecological niches and have differing feeding patterns. Most importantly, while head lice are not known to be vector competent, body lice can transmit three serious bacterial diseases; epidemictyphus, trench fever, and relapsing fever. In order to gain insights into the molecular bases for these differences, we analyzed alternative splicing (AS) using next-generation sequencing data for one strain of head lice and one strain of body lice. We identified a total of 3,598 AS events which were head or body lice specific. Exon skipping AS events were overrepresented among both head and body lice, whereas intron retention events were underrepresented in both. However, both the enrichment of exon skipping and the underrepresentation of intron retention are significantly stronger in body lice compared with head lice. Genes containing body louse-specific AS events were found to be significantly enriched for functions associated with development of the nervous system, salivary gland, trachea, and ovarian follicle cells, as well as regulation of transcription. In contrast, no functional categories were overrepresented among genes with head louse-specific AS events. Together, our results constitute the first evidence for transcript pool differences in head and body lice, providing insights into molecular adaptations that enabled human lice to adapt to clothing, and representing a powerful illustration of the pivotal role AS can play in functional adaptation. PMID:26169943

  5. Alternative Splice in Alternative Lice.

    PubMed

    Tovar-Corona, Jaime M; Castillo-Morales, Atahualpa; Chen, Lu; Olds, Brett P; Clark, John M; Reynolds, Stuart E; Pittendrigh, Barry R; Feil, Edward J; Urrutia, Araxi O

    2015-10-01

    Genomic and transcriptomics analyses have revealed human head and body lice to be almost genetically identical; although con-specific, they nevertheless occupy distinct ecological niches and have differing feeding patterns. Most importantly, while head lice are not known to be vector competent, body lice can transmit three serious bacterial diseases; epidemictyphus, trench fever, and relapsing fever. In order to gain insights into the molecular bases for these differences, we analyzed alternative splicing (AS) using next-generation sequencing data for one strain of head lice and one strain of body lice. We identified a total of 3,598 AS events which were head or body lice specific. Exon skipping AS events were overrepresented among both head and body lice, whereas intron retention events were underrepresented in both. However, both the enrichment of exon skipping and the underrepresentation of intron retention are significantly stronger in body lice compared with head lice. Genes containing body louse-specific AS events were found to be significantly enriched for functions associated with development of the nervous system, salivary gland, trachea, and ovarian follicle cells, as well as regulation of transcription. In contrast, no functional categories were overrepresented among genes with head louse-specific AS events. Together, our results constitute the first evidence for transcript pool differences in head and body lice, providing insights into molecular adaptations that enabled human lice to adapt to clothing, and representing a powerful illustration of the pivotal role AS can play in functional adaptation.

  6. HOLLYWOOD: a comparative relational database of alternative splicing.

    PubMed

    Holste, Dirk; Huo, George; Tung, Vivian; Burge, Christopher B

    2006-01-01

    RNA splicing is an essential step in gene expression, and is often variable, giving rise to multiple alternatively spliced mRNA and protein isoforms from a single gene locus. The design of effective databases to support experimental and computational investigations of alternative splicing (AS) is a significant challenge. In an effort to integrate accurate exon and splice site annotation with current knowledge about splicing regulatory elements and predicted AS events, and to link information about the splicing of orthologous genes in different species, we have developed the Hollywood system. This database was built upon genomic annotation of splicing patterns of known genes derived from spliced alignment of complementary DNAs (cDNAs) and expressed sequence tags, and links features such as splice site sequence and strength, exonic splicing enhancers and silencers, conserved and non-conserved patterns of splicing, and cDNA library information for inferred alternative exons. Hollywood was implemented as a relational database and currently contains comprehensive information for human and mouse. It is accompanied by a web query tool that allows searches for sets of exons with specific splicing characteristics or splicing regulatory element composition, or gives a graphical or sequence-level summary of splicing patterns for a specific gene. A streamlined graphical representation of gene splicing patterns is provided, and these patterns can alternatively be layered onto existing information in the UCSC Genome Browser. The database is accessible at http://hollywood.mit.edu.

  7. [Exon 5 alternative splicing of the cytochrome P450 aromatase could be a regulatory mechanism for estrogen production in humans].

    PubMed

    Pepe, Carolina M; Saraco, Nora I; Baquedano, María Sonia; Guercio, Gabriela; Vaiani, Elisa; Berensztein, Esperanza; Rivarola, Marco A; Belgorosky, Alicia

    2007-01-01

    P450 aromatase (P450Aro), involved in androgen to estrogen conversion, is encoded by the CYP19 gene. P450Aro c655G>A mutation described in heterozygous form in a girl and in homozygous form in an adult male with P450Aro deficiency results in an aberrant splicing due to disruption of a donor splice site. A truncated inactive protein would be expected if intron5 is retained. Surprisingly, the girl described with this mutation showed spontaneous breast development and pubertal estradiol (E2) levels suggesting residual P450Aro activity (AA). Formerly, we postulate the in frame E5 skipping as a consequence of this mutation generating a protein with some degree of activity. When P450Aro mRNA expression was analysed from patient's lymphocytes, an aberrant spliced mRNA lacking E5 (-E5mRNA) was detected, suggesting an association between E5 skipping and the presence of the mutation. Splicing assays in Y1 cells confirmed this association. -Ex5 cDNA expression in Y1 cells resulted in an inactive protein that could not explain patient's phenotype. Exon 5 might be predicted as a poorly defined exon suggesting a susceptibility to splicing mutations and physiological alternative splicing (AS) events. Therefore, -Ex5mRNA was assessed as a natural occurring alternative transcript in normal human steroidogenic tissues. As P450Aro -E5mRNA expression was detected in human term placenta, prepubertal testis and prepubertal adrenal, we might speculate that AS of P450Aro coding region would occur in humans and would be involved in the complex AA regulation. Furthermore, tissue specific regulation of AS might suggest low expression of +E5mRNA from the c655G>A allele explaining residual AA evidenced in the affected girl.

  8. Alternative splicing: a pivotal step between eukaryotic transcription and translation.

    PubMed

    Kornblihtt, Alberto R; Schor, Ignacio E; Alló, Mariano; Dujardin, Gwendal; Petrillo, Ezequiel; Muñoz, Manuel J

    2013-03-01

    Alternative splicing was discovered simultaneously with splicing over three decades ago. Since then, an enormous body of evidence has demonstrated the prevalence of alternative splicing in multicellular eukaryotes, its key roles in determining tissue- and species-specific differentiation patterns, the multiple post- and co-transcriptional regulatory mechanisms that control it, and its causal role in hereditary disease and cancer. The emerging evidence places alternative splicing in a central position in the flow of eukaryotic genetic information, between transcription and translation, in that it can respond not only to various signalling pathways that target the splicing machinery but also to transcription factors and chromatin structure.

  9. Methods for Characterization of Alternative RNA Splicing.

    PubMed

    Harvey, Samuel E; Cheng, Chonghui

    2016-01-01

    Quantification of alternative splicing to detect the abundance of differentially spliced isoforms of a gene in total RNA can be accomplished via RT-PCR using both quantitative real-time and semi-quantitative PCR methods. These methods require careful PCR primer design to ensure specific detection of particular splice isoforms. We also describe analysis of alternative splicing using a splicing "minigene" in mammalian cell tissue culture to facilitate investigation of the regulation of alternative splicing of a particular exon of interest.

  10. Alternative splicing modulates stem cell differentiation.

    PubMed

    Fu, Ru-Huei; Liu, Shih-Ping; Ou, Chen-Wei; Yu, Hsiu-Hui; Li, Kuo-Wei; Tsai, Chang-Hai; Shyu, Woei-Cherng; Lin, Shinn-Zong

    2009-01-01

    Stem cells have the surprising potential to develop into many different cell types. Therefore, major research efforts have focused on transplantation of stem cells and/or derived progenitors for restoring depleted diseased cells in degenerative disorders. Understanding the molecular controls, including alternative splicing, that arise during lineage differentiation of stem cells is crucial for developing stem cell therapeutic approaches in regeneration medicine. Alternative splicing to allow a single gene to encode multiple transcripts with different protein coding sequences and RNA regulatory elements increases genomic complexities. Utilizing differences in alternative splicing as a molecular marker may be more sensitive than simply gene expression in various degrees of stem cell differentiation. Moreover, alternative splicing maybe provide a new concept to acquire induced pluripotent stem cells or promote cell-cell transdifferentiation for restorative therapies and basic medicine researches. In this review, we highlight the recent advances of alternative splicing regulation in stem cells and their progenitors. It will hopefully provide much needed knowledge into realizing stem cell biology and related applications.

  11. Variation in alternative splicing across human tissues

    PubMed Central

    Yeo, Gene; Holste, Dirk; Kreiman, Gabriel; Burge, Christopher B

    2004-01-01

    Background Alternative pre-mRNA splicing (AS) is widely used by higher eukaryotes to generate different protein isoforms in specific cell or tissue types. To compare AS events across human tissues, we analyzed the splicing patterns of genomically aligned expressed sequence tags (ESTs) derived from libraries of cDNAs from different tissues. Results Controlling for differences in EST coverage among tissues, we found that the brain and testis had the highest levels of exon skipping. The most pronounced differences between tissues were seen for the frequencies of alternative 3' splice site and alternative 5' splice site usage, which were about 50 to 100% higher in the liver than in any other human tissue studied. Quantifying differences in splice junction usage, the brain, pancreas, liver and the peripheral nervous system had the most distinctive patterns of AS. Analysis of available microarray expression data showed that the liver had the most divergent pattern of expression of serine-arginine protein and heterogeneous ribonucleoprotein genes compared to the other human tissues studied, possibly contributing to the unusually high frequency of alternative splice site usage seen in liver. Sequence motifs enriched in alternative exons in genes expressed in the brain, testis and liver suggest specific splicing factors that may be important in AS regulation in these tissues. Conclusions This study distinguishes the human brain, testis and liver as having unusually high levels of AS, highlights differences in the types of AS occurring commonly in different tissues, and identifies candidate cis-regulatory elements and trans-acting factors likely to have important roles in tissue-specific AS in human cells. PMID:15461793

  12. Heritability of alternative splicing in the human genome

    PubMed Central

    Kwan, Tony; Benovoy, David; Dias, Christel; Gurd, Scott; Serre, David; Zuzan, Harry; Clark, Tyson A.; Schweitzer, Anthony; Staples, Michelle K.; Wang, Hui; Blume, John E.; Hudson, Thomas J.; Sladek, Rob; Majewski, Jacek

    2007-01-01

    Alternative pre-mRNA splicing increases proteomic diversity and provides a potential mechanism underlying both phenotypic diversity and susceptibility to genetic disorders in human populations. To investigate the variation in splicing among humans on a genome-wide scale, we use a comprehensive exon-targeted microarray to examine alternative splicing in lymphoblastoid cell lines (LCLs) derived from the CEPH HapMap population. We show the identification of transcripts containing sequence verified exon skipping, intron retention, and cryptic splice site usage that are specific between individuals. A number of novel alternative splicing events with no previous annotations in either the RefSeq and EST databases were identified, indicating that we are able to discover de novo splicing events. Using family-based linkage analysis, we demonstrate Mendelian inheritance and segregation of specific splice isoforms with regulatory haplotypes for three genes: OAS1, CAST, and CRTAP. Allelic association was further used to identify individual SNPs or regulatory haplotype blocks linked to the alternative splicing event, taking advantage of the high-resolution genotype information from the CEPH HapMap population. In one candidate, we identified a regulatory polymorphism that disrupts a 5′ splice site of an exon in the CAST gene, resulting in its exclusion in the mutant allele. This report illustrates that our approach can detect both annotated and novel alternatively spliced variants, and that such variation among individuals is heritable and genetically controlled. PMID:17671095

  13. Control of Human PLP1 Expression Through Transcriptional Regulatory Elements and Alternatively Spliced Exons in Intron 1

    PubMed Central

    Hamdan, Hamdan; Kockara, Neriman T.; Jolly, Lee Ann; Haun, Shirley

    2015-01-01

    *These authors contributed equally to this work.Although the myelin proteolipid protein gene (PLP1) encodes the most abundant protein in central nervous system (CNS) myelin, not much is known about the mechanisms that govern expression of the human gene (hPLP1). Much more is known about the processes that regulate Plp1 gene expression in rodents. From studies with Plp1-lacZ transgenic mice, it was determined that the first intron of mouse Plp1 (mPlp1) is required to attain high levels of expression in brain, concurrent with the active myelination period. Other studies have suggested that within mPlp1 intron 1 (>8 kb) lie several regions with enhancer-like activity. To test whether these sequences (and possibly others) in hPLP1 intron 1 are functional, deletion-transfection analysis was performed with hPLP1-lacZ constructs that contain various portions of the intron, or lack it altogether. Results presented here demonstrate the importance of hPLP1 intron 1 in achieving maximal levels of expression in the immortalized oligodendroglial cell line, Oli-neu. Deletion analysis indicates that the intron contains multiple positive regulatory elements which are active in Oli-neu cells. Some of these elements appear to be functionally conserved between human and mouse, while others are not. Furthermore, our studies demonstrate that multiple splice variants can be formed due to inclusion of extra (supplementary) exons from what is classically thought of as hPLP1 intron 1. Thus, splicing of these novel exons (which are not recognized as such in mPlp1 due to lack of conserved splice sites) must utilize factors common to both human and mouse since Oli-neu cells are of mouse origin. PMID:25694552

  14. Regulation of splicing factors by alternative splicing and NMD is conserved between kingdoms yet evolutionarily flexible.

    PubMed

    Lareau, Liana F; Brenner, Steven E

    2015-04-01

    Ultraconserved elements, unusually long regions of perfect sequence identity, are found in genes encoding numerous RNA-binding proteins including arginine-serine rich (SR) splicing factors. Expression of these genes is regulated via alternative splicing of the ultraconserved regions to yield mRNAs that are degraded by nonsense-mediated mRNA decay (NMD), a process termed unproductive splicing (Lareau et al. 2007; Ni et al. 2007). As all human SR genes are affected by alternative splicing and NMD, one might expect this regulation to have originated in an early SR gene and persisted as duplications expanded the SR family. But in fact, unproductive splicing of most human SR genes arose independently (Lareau et al. 2007). This paradox led us to investigate the origin and proliferation of unproductive splicing in SR genes. We demonstrate that unproductive splicing of the splicing factor SRSF5 (SRp40) is conserved among all animals and even observed in fungi; this is a rare example of alternative splicing conserved between kingdoms, yet its effect is to trigger mRNA degradation. As the gene duplicated, the ancient unproductive splicing was lost in paralogs, and distinct unproductive splicing evolved rapidly and repeatedly to take its place. SR genes have consistently employed unproductive splicing, and while it is exceptionally conserved in some of these genes, turnover in specific events among paralogs shows flexible means to the same regulatory end. © The Author 2015. Published by Oxford University Press on behalf of the society for Molecular Biology and Evolution.

  15. Regulation of Splicing Factors by Alternative Splicing and NMD Is Conserved between Kingdoms Yet Evolutionarily Flexible

    PubMed Central

    Lareau, Liana F.; Brenner, Steven E.

    2015-01-01

    Ultraconserved elements, unusually long regions of perfect sequence identity, are found in genes encoding numerous RNA-binding proteins including arginine-serine rich (SR) splicing factors. Expression of these genes is regulated via alternative splicing of the ultraconserved regions to yield mRNAs that are degraded by nonsense-mediated mRNA decay (NMD), a process termed unproductive splicing (Lareau et al. 2007; Ni et al. 2007). As all human SR genes are affected by alternative splicing and NMD, one might expect this regulation to have originated in an early SR gene and persisted as duplications expanded the SR family. But in fact, unproductive splicing of most human SR genes arose independently (Lareau et al. 2007). This paradox led us to investigate the origin and proliferation of unproductive splicing in SR genes. We demonstrate that unproductive splicing of the splicing factor SRSF5 (SRp40) is conserved among all animals and even observed in fungi; this is a rare example of alternative splicing conserved between kingdoms, yet its effect is to trigger mRNA degradation. As the gene duplicated, the ancient unproductive splicing was lost in paralogs, and distinct unproductive splicing evolved rapidly and repeatedly to take its place. SR genes have consistently employed unproductive splicing, and while it is exceptionally conserved in some of these genes, turnover in specific events among paralogs shows flexible means to the same regulatory end. PMID:25576366

  16. Alternative splicing regulation of telomerase: a new paradigm?

    PubMed

    Wong, Mandy S; Wright, Woodring E; Shay, Jerry W

    2014-10-01

    Alternative splicing affects approximately 95% of eukaryotic genes, greatly expanding the coding capacity of complex genomes. Although our understanding of alternative splicing has increased rapidly, current knowledge of splicing regulation has largely been derived from studies of highly expressed mRNAs. Telomerase is a key example of a protein that is alternatively spliced, but it is expressed at very low levels and although it is known that misregulation of telomerase splicing is a hallmark of nearly all cancers, the details of this process are unclear. Here we review work showing that hTERT expression is in part regulated by atypical alternative splicing, perhaps due to its exceptionally low expression level. We propose that these differential regulatory mechanisms may be widely applicable to other genes and may provide new opportunities for the development of cancer therapeutics. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. COMMUNICATION: Alternative splicing and genomic stability

    NASA Astrophysics Data System (ADS)

    Cahill, Kevin

    2004-06-01

    Alternative splicing allows an organism to make different proteins in different cells at different times, all from the same gene. In a cell that uses alternative splicing, the total length of all the exons is much shorter than in a cell that encodes the same set of proteins without alternative splicing. This economical use of exons makes genes more stable during reproduction and development because a genome with a shorter exon length is more resistant to harmful mutations. Genomic stability may be the reason why higher vertebrates splice alternatively. For a broad class of alternatively spliced genes, a formula is given for the increase in their stability.

  18. Expression of Alternatively Spliced Human T-Cell Leukemia Virus Type 1 mRNAs Is Influenced by Mitosis and by a Novel cis-Acting Regulatory Sequence

    PubMed Central

    Cavallari, Ilaria; Rende, Francesca; Bona, Marion K.; Sztuba-Solinska, Joanna; Silic-Benussi, Micol; Tognon, Martina; Franchini, Genoveffa; D'Agostino, Donna M.

    2015-01-01

    expression. The findings reported in this study revealed a novel cis-acting regulatory element and indicated that mitosis partially bypasses the requirement for Rex to export Rex-dependent HTLV-1 transcripts. Our results add a layer of complexity to the mechanisms controlling the expression of alternatively spliced HTLV-1 mRNAs and suggest a link between the cycling properties of the host cell and the temporal pattern of viral expression/latency that might influence the ability of the virus to spread and evade the immune system. PMID:26581997

  19. Expression of Alternatively Spliced Human T-Cell Leukemia Virus Type 1 mRNAs Is Influenced by Mitosis and by a Novel cis-Acting Regulatory Sequence.

    PubMed

    Cavallari, Ilaria; Rende, Francesca; Bona, Marion K; Sztuba-Solinska, Joanna; Silic-Benussi, Micol; Tognon, Martina; LeGrice, Stuart F J; Franchini, Genoveffa; D'Agostino, Donna M; Ciminale, Vincenzo

    2015-11-18

    in this study revealed a novel cis-acting regulatory element and indicated that mitosis partially bypasses the requirement for Rex to export Rex-dependent HTLV-1 transcripts. Our results add a layer of complexity to the mechanisms controlling the expression of alternatively spliced HTLV-1 mRNAs and suggest a link between the cycling properties of the host cell and the temporal pattern of viral expression/latency that might influence the ability of the virus to spread and evade the immune system. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  20. Evolutionary conservation of alternative splicing in chicken

    PubMed Central

    Katyal, S.; Gao, Z.; Liu, R.-Z.; Godbout, R.

    2013-01-01

    Alternative splicing represents a source of great diversity for regulating protein expression and function. It has been estimated that one-third to two-thirds of mammalian genes are alternatively spliced. With the sequencing of the chicken genome and analysis of transcripts expressed in chicken tissues, we are now in a position to address evolutionary conservation of alternative splicing events in chicken and mammals. Here, we compare chicken and mammalian transcript sequences of 41 alternatively-spliced genes and 50 frequently accessed genes. Our results support a high frequency of splicing events in chicken, similar to that observed in mammals. PMID:17675855

  1. Alcoholism and alternative splicing of candidate genes.

    PubMed

    Sasabe, Toshikazu; Ishiura, Shoichi

    2010-04-01

    Gene expression studies have shown that expression patterns of several genes have changed during the development of alcoholism. Gene expression is regulated not only at the level of transcription but also through alternative splicing of pre-mRNA. In this review, we discuss some of the evidence suggesting that alternative splicing of candidate genes such as DRD2 (encoding dopamine D2 receptor) may form the basis of the mechanisms underlying the pathophysiology of alcoholism. These reports suggest that aberrant expression of splice variants affects alcohol sensitivities, and alcohol consumption also regulates alternative splicing. Thus, investigations of alternative splicing are essential for understanding the molecular events underlying the development of alcoholism.

  2. Anti-Müllerian hormone (AMH/AMH) in the European sea bass: its gene structure, regulatory elements, and the expression of alternatively-spliced isoforms.

    PubMed

    Halm, S; Rocha, A; Miura, T; Prat, F; Zanuy, S

    2007-02-15

    In mammals, a multitude of studies have shown that anti-Müllerian hormone (AMH/AMH), apart from inducing Müllerian duct regression during male sexual differentiation, exerts inhibitory effects on male and female gonadal steroidogenesis and differentiation. However, in lower vertebrates like teleost fish, the function of AMH/AMH has been far less explored. As a first step to unravel its potential role in reproduction in teleost fish, we isolated and characterised the AMH gene in the European sea bass (sb), Dicentrachus labrax, determined putative regulatory elements of its 5'-flanking region, and analysed its gene expression and those of alternatively-spliced transcripts. The characterisation of sb-AMH revealed distinct features that distinguishes it from mammalian and bird AMH, suggesting a high rate of diversification of AMH during vertebrate evolution. It contained 7 exons that were divided by 6 introns, of which the last intron (intron vi) was localised only a few nucleotides upstream of the putative peptide cleavage site. The guanine and cytosine content of the open reading frame (ORF) was 52.7% and thus notably lower than that of bird and mammalian AMH. Sb-AMH cDNA was 2045 base pairs (bp) long, containing an ORF of 1599 bp encoding 533 amino acids. Deduced amino acid similarities of the conserved, carboxyterminal domain were highest with AMH in Japanese flounder (84.2%) and lowest with chicken AMH (45.5%). In the proximal promoter sequence of sb-AMH, a steroidogenic factor-1 (SF-1) binding site was present; however other regulatory sequences essential for transcriptional activation of AMH in mammals were absent. Likewise, there was no sequence homology to an SF3A2 sequence within the first 3200 bp upstream of the sb-AMH translation start site. Gene expression of sb-AMH and of alternatively-spliced sb-AMH transcripts were analysed in male and female juvenile and adult gonads as well as in somatic tissues of juvenile males. sb-AMH expression was highest in

  3. Conserved RNA secondary structures promote alternative splicing.

    PubMed

    Shepard, Peter J; Hertel, Klemens J

    2008-08-01

    Pre-mRNA splicing is carried out by the spliceosome, which identifies exons and removes intervening introns. Alternative splicing in higher eukaryotes results in the generation of multiple protein isoforms from gene transcripts. The extensive alternative splicing observed implies a flexibility of the spliceosome to identify exons within a given pre-mRNA. To reach this flexibility, splice-site selection in higher eukaryotes has evolved to depend on multiple parameters such as splice-site strength, splicing regulators, the exon/intron architecture, and the process of pre-mRNA synthesis itself. RNA secondary structures have also been proposed to influence alternative splicing as stable RNA secondary structures that mask splice sites are expected to interfere with splice-site recognition. Using structural and functional conservation, we identified RNA structure elements within the human genome that associate with alternative splice-site selection. Their frequent involvement with alternative splicing demonstrates that RNA structure formation is an important mechanism regulating gene expression and disease.

  4. Pharmacology of Modulators of Alternative Splicing

    PubMed Central

    Morris, Jonathan C.; Oltean, Sebastian; Donaldson, Lucy F.

    2017-01-01

    More than 95% of genes in the human genome are alternatively spliced to form multiple transcripts, often encoding proteins with differing or opposing function. The control of alternative splicing is now being elucidated, and with this comes the opportunity to develop modulators of alternative splicing that can control cellular function. A number of approaches have been taken to develop compounds that can experimentally, and sometimes clinically, affect splicing control, resulting in potential novel therapeutics. Here we develop the concepts that targeting alternative splicing can result in relatively specific pathway inhibitors/activators that result in dampening down of physiologic or pathologic processes, from changes in muscle physiology to altering angiogenesis or pain. The targets and pharmacology of some of the current inhibitors/activators of alternative splicing are demonstrated and future directions discussed. PMID:28034912

  5. Pharmacology of Modulators of Alternative Splicing.

    PubMed

    Bates, David O; Morris, Jonathan C; Oltean, Sebastian; Donaldson, Lucy F

    2017-01-01

    More than 95% of genes in the human genome are alternatively spliced to form multiple transcripts, often encoding proteins with differing or opposing function. The control of alternative splicing is now being elucidated, and with this comes the opportunity to develop modulators of alternative splicing that can control cellular function. A number of approaches have been taken to develop compounds that can experimentally, and sometimes clinically, affect splicing control, resulting in potential novel therapeutics. Here we develop the concepts that targeting alternative splicing can result in relatively specific pathway inhibitors/activators that result in dampening down of physiologic or pathologic processes, from changes in muscle physiology to altering angiogenesis or pain. The targets and pharmacology of some of the current inhibitors/activators of alternative splicing are demonstrated and future directions discussed. Copyright © 2016 by The Author(s).

  6. Peptidic tools applied to redirect alternative splicing events.

    PubMed

    Nancy, Martínez-Montiel; Nora, Rosas-Murrieta; Rebeca, Martínez-Contreras

    2015-05-01

    Peptides are versatile and attractive biomolecules that can be applied to modulate genetic mechanisms like alternative splicing. In this process, a single transcript yields different mature RNAs leading to the production of protein isoforms with diverse or even antagonistic functions. During splicing events, errors can be caused either by mutations present in the genome or by defects or imbalances in regulatory protein factors. In any case, defects in alternative splicing have been related to several genetic diseases including muscular dystrophy, Alzheimer's disease and cancer from almost every origin. One of the most effective approaches to redirect alternative splicing events has been to attach cell-penetrating peptides to oligonucleotides that can modulate a single splicing event and restore correct gene expression. Here, we summarize how natural existing and bioengineered peptides have been applied over the last few years to regulate alternative splicing and genetic expression. Under different genetic and cellular backgrounds, peptides have been shown to function as potent vehicles for splice correction, and their therapeutic benefits have reached clinical trials and patenting stages, emphasizing the use of regulatory peptides as an exciting therapeutic tool for the treatment of different genetic diseases.

  7. Comparative analysis identifies exonic splicing regulatory sequences--The complex definition of enhancers and silencers.

    PubMed

    Goren, Amir; Ram, Oren; Amit, Maayan; Keren, Hadas; Lev-Maor, Galit; Vig, Ida; Pupko, Tal; Ast, Gil

    2006-06-23

    Exonic splicing regulatory sequences (ESRs) are cis-acting factor binding sites that regulate constitutive and alternative splicing. A computational method based on the conservation level of wobble positions and the overabundance of sequence motifs between 46,103 human and mouse orthologous exons was developed, identifying 285 putative ESRs. Alternatively spliced exons that are either short in length or contain weak splice sites show the highest conservation level of those ESRs, especially toward the edges of exons. ESRs that are abundant in those subgroups show a different distribution between constitutively and alternatively spliced exons. Representatives of these ESRs and two SR protein binding sites were shown, experimentally, to display variable regulatory effects on alternative splicing, depending on their relative locations in the exon. This finding signifies the delicate positional effect of ESRs on alternative splicing regulation.

  8. Mechanisms and Regulation of Alternative Pre-mRNA Splicing

    PubMed Central

    Lee, Yeon

    2015-01-01

    Precursor messenger RNA (pre-mRNA) splicing is a critical step in the posttranscriptional regulation of gene expression, providing significant expansion of the functional proteome of eukaryotic organisms with limited gene numbers. Split eukaryotic genes contain intervening sequences or introns disrupting protein-coding exons, and intron removal occurs by repeated assembly of a large and highly dynamic ribonucleoprotein complex termed the spliceosome, which is composed of five small nuclear ribonucleoprotein particles, U1, U2, U4/U6, and U5. Biochemical studies over the past 10 years have allowed the isolation as well as compositional, functional, and structural analysis of splicing complexes at distinct stages along the spliceosome cycle. The average human gene contains eight exons and seven introns, producing an average of three or more alternatively spliced mRNA isoforms. Recent high-throughput sequencing studies indicate that 100% of human genes produce at least two alternative mRNA isoforms. Mechanisms of alternative splicing include RNA–protein interactions of splicing factors with regulatory sites termed silencers or enhancers, RNA–RNA base-pairing interactions, or chromatin-based effects that can change or determine splicing patterns. Disease-causing mutations can often occur in splice sites near intron borders or in exonic or intronic RNA regulatory silencer or enhancer elements, as well as in genes that encode splicing factors. Together, these studies provide mechanistic insights into how spliceosome assembly, dynamics, and catalysis occur; how alternative splicing is regulated and evolves; and how splicing can be disrupted by cis- and trans-acting mutations leading to disease states. These findings make the spliceosome an attractive new target for small-molecule, antisense, and genome-editing therapeutic interventions. PMID:25784052

  9. Hallmarks of alternative splicing in cancer.

    PubMed

    Oltean, S; Bates, D O

    2014-11-13

    The immense majority of genes are alternatively spliced and there are many isoforms specifically associated with cancer progression and metastasis. The splicing pattern of specific isoforms of numerous genes is altered as cells move through the oncogenic process of gaining proliferative capacity, acquiring angiogenic, invasive, antiapoptotic and survival properties, becoming free from growth factor dependence and growth suppression, altering their metabolism to cope with hypoxia, enabling them to acquire mechanisms of immune escape, and as they move through the epithelial-mesenchymal and mesenchymal-epithelial transitions and metastasis. Each of the 'hallmarks of cancer' is associated with a switch in splicing, towards a more aggressive invasive cancer phenotype. The choice of isoforms is regulated by several factors (signaling molecules, kinases, splicing factors) currently being identified systematically by a number of high-throughput, independent and unbiased methodologies. Splicing factors are de-regulated in cancer, and in some cases are themselves oncogenes or pseudo-oncogenes and can contribute to positive feedback loops driving cancer progression. Tumour progression may therefore be associated with a coordinated splicing control, meaning that there is the potential for a relatively small number of splice factors or their regulators to drive multiple oncogenic processes. The understanding of how splicing contributes to the various phenotypic traits acquired by tumours as they progress and metastasise, and in particular how alternative splicing is coordinated, can and is leading to the development of a new class of anticancer therapeutics-the alternative-splicing inhibitors.

  10. Aberrant Alternative Splicing Is Another Hallmark of Cancer

    PubMed Central

    Ladomery, Michael

    2013-01-01

    The vast majority of human genes are alternatively spliced. Not surprisingly, aberrant alternative splicing is increasingly linked to cancer. Splice isoforms often encode proteins that have distinct and even antagonistic properties. The abnormal expression of splice factors and splice factor kinases in cancer changes the alternative splicing of critically important pre-mRNAs. Aberrant alternative splicing should be added to the growing list of cancer hallmarks. PMID:24101931

  11. Abnormalities in Alternative Splicing of Apoptotic Genes and Cardiovascular Diseases

    PubMed Central

    Dlamini, Zodwa; Tshidino, Shonisani C.; Hull, Rodney

    2015-01-01

    Apoptosis is required for normal heart development in the embryo, but has also been shown to be an important factor in the occurrence of heart disease. Alternative splicing of apoptotic genes is currently emerging as a diagnostic and therapeutic target for heart disease. This review addresses the involvement of abnormalities in alternative splicing of apoptotic genes in cardiac disorders including cardiomyopathy, myocardial ischemia and heart failure. Many pro-apoptotic members of the Bcl-2 family have alternatively spliced isoforms that lack important active domains. These isoforms can play a negative regulatory role by binding to and inhibiting the pro-apoptotic forms. Alternative splicing is observed to be increased in various cardiovascular diseases with the level of alternate transcripts increasing elevated in diseased hearts compared to healthy subjects. In many cases these isoforms appear to be the underlying cause of the disease, while in others they may be induced in response to cardiovascular pathologies. Regardless of this, the detection of alternate splicing events in the heart can serve as useful diagnostic or prognostic tools, while those splicing events that seem to play a causative role in cardiovascular disease make attractive future drug targets. PMID:26580598

  12. A cis-acting regulatory element that affects the alternative splicing of a muscle-specific exon in the mouse NCAM gene.

    PubMed

    Kawahigashi, H; Harada, Y; Asano, A; Nakamura, M

    1998-05-11

    The pre-mRNA encoding the neural cell adhesion molecule (NCAM) is spliced to generate NCAM isoforms containing the muscle-specific domain (MSD) during myogenesis. Utilizing chimeric NCAM minigenes, we searched for cis-acting elements that contribute to the alternative selection of exon MSDb, one of the four exons encoding MSD, and identified an intronic cis-element located downstream of exon MSDb. The cis-element acted as a negative regulator for the selection of exon MSDb in nonmuscle fibroblasts but not in myoblasts, that are already destined to differentiate into muscle cells. The suppressive effect of this cis-element on the selection of exon MSDb was released in the process of myogenesis. When MyoD was co-expressed with a minigene containing this element in fibroblasts, the suppressive effect of the cis-element was released as the cells underwent differentiation. We propose that this cis-element contributes at least as one of the regulatory elements in the differentiation state-dependent selection of MSD exons in vivo.

  13. The BRCA1 alternative splicing variant Δ14-15 with an in-frame deletion of part of the regulatory serine-containing domain (SCD) impairs the DNA repair capacity in MCF-7 cells.

    PubMed

    Sevcik, Jan; Falk, Martin; Kleiblova, Petra; Lhota, Filip; Stefancikova, Lenka; Janatova, Marketa; Weiterova, Lenka; Lukasova, Emilie; Kozubek, Stanislav; Pohlreich, Petr; Kleibl, Zdenek

    2012-05-01

    The BRCA1 gene codes for a protein involved in the DNA double strand break (DDSB) repair. Alongside the dominant full-length splicing form of BRCA1, numerous endogenously expressed alternative splicing variants of unknown significance have been described in various tissues. Some of them retain the original BRCA1 reading frame but lack several critical BRCA1 structural domains, suggesting an altered function of the resulting protein in the BRCA1-regulated processes. To characterize the effect of the BRCA1Δ14-15 splicing variant (with an in-frame deletion affecting the regulatory serine-containing domain) on the DDSB repair, we constructed the MCF-7 clones stably expressing the analyzed variant with/without a shRNA-mediated downregulation of the endogenous full-length wild-type BRCA1 expression. Our results show that the expression of the BRCA1Δ14-15 variant delays the γ-radiation-induced DDSB repair, alters the kinetics of irradiation-induced foci formation/decomposition and reduces the non-homologous end-joining capacity in MCF-7 cells. Therefore, the BRCA1Δ14-15 is not able to functionally replace the full-length wt BRCA1 in the DDSB repair. Our findings indicate that the endogenously expressed BRCA1 alternative splicing variants may negatively influence genome stability and support the growing evidence of the pathological potential of the sequence variants generated by an altered or misregulated alternative splicing in the process of mammary malignant transformation.

  14. Alternative splicing: new insights from global analyses.

    PubMed

    Blencowe, Benjamin J

    2006-07-14

    Recent analyses of sequence and microarray data have suggested that alternative splicing plays a major role in the generation of proteomic and functional diversity in metazoan organisms. Efforts are now being directed at establishing the full repertoire of functionally relevant transcript variants generated by alternative splicing, the specific roles of such variants in normal and disease physiology, and how alternative splicing is coordinated on a global level to achieve cell- and tissue-specific functions. Recent progress in these areas is summarized in this review.

  15. Intron cleavage affects processing of alternatively spliced transcripts

    PubMed Central

    Pastor, Tibor; Dal Mas, Andrea; Talotti, Gabriele; Bussani, Erica; Pagani, Franco

    2011-01-01

    We previously showed that the insertion of a hammerhead ribozyme (Rz) in a critical intronic position between the EDA exon and a downstream regulatory element affects alternative splicing. Here we evaluate the effect of other intronic cotranscriptional cleavage events on alternative pre-mRNA processing using different ribozymes (Rz) and Microprocessor target sequences (MTSs). In the context of the fibronectin EDA minigene, intronic MTSs were cleaved very inefficiently and did not affect alternative splicing or the level of mature transcripts. On the contrary, all hammerhead Rz derivatives and hepatitis δ Rz were completely cleaved before a splicing decision and able to affect alternative splicing. Despite the very efficient Rz-mediated cleavage, the levels of mature mRNA were only reduced to ∼40%. We show that this effect on mature transcripts occurs regardless of the type and intronic position of Rzs, or changes in alternative splicing and exon definition. Thus, we suggest that intron integrity is not strictly required for splicing but is necessary for efficient pre-mRNA biosynthesis. PMID:21673105

  16. Global genome splicing analysis reveals an increased number of alternatively spliced genes with aging.

    PubMed

    Rodríguez, Sofía A; Grochová, Diana; McKenna, Tomás; Borate, Bhavesh; Trivedi, Niraj S; Erdos, Michael R; Eriksson, Maria

    2016-04-01

    Alternative splicing (AS) is a key regulatory mechanism for the development of different tissues; however, not much is known about changes to alternative splicing during aging. Splicing events may become more frequent and widespread genome-wide as tissues age and the splicing machinery stringency decreases. Using skin, skeletal muscle, bone, thymus, and white adipose tissue from wild-type C57BL6/J male mice (4 and 18 months old), we examined the effect of age on splicing by AS analysis of the differential exon usage of the genome. The results identified a considerable number of AS genes in skeletal muscle, thymus, bone, and white adipose tissue between the different age groups (ranging from 27 to 246 AS genes corresponding to 0.3-3.2% of the total number of genes analyzed). For skin, skeletal muscle, and bone, we included a later age group (28 months old) that showed that the number of alternatively spliced genes increased with age in all three tissues (P < 0.01). Analysis of alternatively spliced genes across all tissues by gene ontology and pathway analysis identified 158 genes involved in RNA processing. Additional analysis of AS in a mouse model for the premature aging disease Hutchinson-Gilford progeria syndrome was performed. The results show that expression of the mutant protein, progerin, is associated with an impaired developmental splicing. As progerin accumulates, the number of genes with AS increases compared to in wild-type skin. Our results indicate the existence of a mechanism for increased AS during aging in several tissues, emphasizing that AS has a more important role in the aging process than previously known.

  17. Finding signals that regulate alternative splicing in the post-genomic era

    PubMed Central

    Ladd, Andrea N; Cooper, Thomas A

    2002-01-01

    Alternative splicing of pre-mRNAs is central to the generation of diversity from the relatively small number of genes in metazoan genomes. Auxiliary cis elements and trans-acting factors are required for the recognition of constitutive and alternatively spliced exons and their inclusion in pre-mRNA. Here, we discuss the regulatory elements that direct alternative splicing and how genome-wide analyses can aid in their identification. PMID:12429065

  18. Regulation of chemoresistance via alternative messenger RNA splicing.

    PubMed

    Eblen, Scott T

    2012-04-15

    The acquisition of resistance to chemotherapy is a significant problem in the treatment of cancer, greatly increasing patient morbidity and mortality. Tumors are often sensitive to chemotherapy upon initial treatment, but repeated treatments can select for those cells that were able to survive initial therapy and have acquired cellular mechanisms to enhance their resistance to subsequent chemotherapy treatment. Many cellular mechanisms of drug resistance have been identified, most of which result from changes in gene and protein expression. While changes at the transcriptional level have been duly noted, it is primarily the post-transcriptional processing of pre-mRNA into mature mRNA that regulates the composition of the proteome and it is the proteome that actually regulates the cell's response to chemotherapeutic insult, inducing cell survival or death. During pre-mRNA processing, intronic non-protein-coding sequences are removed and protein-coding exons are spliced to form a continuous template for protein translation. Alternative splicing involves the differential inclusion or exclusion of exonic sequences into the mature transcript, generating different mRNA templates for protein production. This regulatory mechanism enables the potential to produce many different protein isoforms from the same gene. In this review I will explain the mechanism of alternative pre-mRNA splicing and look at some specific examples of how splicing factors, splicing factor kinases and alternative splicing of specific pre-mRNAs from genes have been shown to contribute to acquisition of the drug resistant phenotype.

  19. Integrative Analysis of Many RNA-Seq Datasets to Study Alternative Splicing

    PubMed Central

    Li, Wenyuan; Dai, Chao; Kang, Shuli; Zhou, Xianghong Jasmine

    2014-01-01

    Alternative splicing is an important gene regulatory mechanism that dramatically increases the complexity of the proteome. However, how alternative splicing is regulated and how transcription and splicing are coordinated are still poorly understood, and functions of transcript isoforms have been studied only in a few limited cases. Nowadays, RNA-seq technology provides an exceptional opportunity to study alternative splicing on genome-wide scales and in an unbiased manner. With the rapid accumulation of data in public repositories, new challenges arise from the urgent need to effectively integrate many different RNA-seq datasets for study alterative splicing. This paper discusses a set of advanced computational methods that can integrate and analyze many RNA-seq datasets to systematically identify splicing modules, unravel the coupling of transcription and splicing, and predict the functions of splicing isoforms on a genome-wide scale. PMID:24583115

  20. Integrative analysis of many RNA-seq datasets to study alternative splicing.

    PubMed

    Li, Wenyuan; Dai, Chao; Kang, Shuli; Zhou, Xianghong Jasmine

    2014-06-01

    Alternative splicing is an important gene regulatory mechanism that dramatically increases the complexity of the proteome. However, how alternative splicing is regulated and how transcription and splicing are coordinated are still poorly understood, and functions of transcript isoforms have been studied only in a few limited cases. Nowadays, RNA-seq technology provides an exceptional opportunity to study alternative splicing on genome-wide scales and in an unbiased manner. With the rapid accumulation of data in public repositories, new challenges arise from the urgent need to effectively integrate many different RNA-seq datasets for study alterative splicing. This paper discusses a set of advanced computational methods that can integrate and analyze many RNA-seq datasets to systematically identify splicing modules, unravel the coupling of transcription and splicing, and predict the functions of splicing isoforms on a genome-wide scale.

  1. Pressure-overload cardiac hypertrophy is associated with distinct alternative splicing due to altered expression of splicing factors.

    PubMed

    Kim, Taeyong; Kim, Jin Ock; Oh, Jae Gyun; Hong, Seong-Eui; Kim, Do Han

    2014-01-01

    Chronic pressure-overload cardiac hypertrophy is associated with an increased risk of morbidity/mortality, largely due to maladaptive remodeling and dilatation that progresses to dilated cardiomyopathy. Alternative splicing is an important biological mechanism that generates proteomic complexity and diversity. The recent development of next-generation RNA sequencing has improved our understanding of the qualitative signatures associated with alternative splicing in various biological conditions. However, the role of alternative splicing in cardiac hypertrophy is yet unknown. The present study employed RNA-Seq and a bioinformatic approach to detect the RNA splicing regulatory elements involved in alternative splicing during pressure-overload cardiac hypertrophy. We found GC-rich exonic motifs that regulate intron retention in 5' UTRs and AT-rich exonic motifs that are involved in exclusion of the AT-rich elements that cause mRNA instability in 3' UTRs. We also identified motifs in the intronic regions involved in exon exclusion and inclusion, which predicted splicing factors that bind to these motifs. We found, through Western blotting, that the expression levels of three splicing factors, ESRP1, PTB and SF2/ASF, were significantly altered during cardiac hypertrophy. Collectively, the present results suggest that chronic pressure-overload hypertrophy is closely associated with distinct alternative splicing due to altered expression of splicing factors.

  2. Context-dependent control of alternative splicing by RNA-binding proteins

    PubMed Central

    Fu, Xiang-Dong; Ares, Manuel

    2015-01-01

    Sequence-specific RNA-binding proteins (RBPs) bind to pre-mRNA to control alternative splicing, but it is not yet possible to read the ‘splicing code’ that dictates splicing regulation on the basis of genome sequence. Each alternative splicing event is controlled by multiple RBPs, the combined action of which creates a distribution of alternatively spliced products in a given cell type. As each cell type expresses a distinct array of RBPs, the interpretation of regulatory information on a given RNA target is exceedingly dependent on the cell type. RBPs also control each other’s functions at many levels, including by mutual modulation of their binding activities on specific regulatory RNA elements. In this Review, we describe some of the emerging rules that govern the highly context-dependent and combinatorial nature of alternative splicing regulation. PMID:25112293

  3. Gene and alternative splicing annotation with AIR

    PubMed Central

    Florea, Liliana; Di Francesco, Valentina; Miller, Jason; Turner, Russell; Yao, Alison; Harris, Michael; Walenz, Brian; Mobarry, Clark; Merkulov, Gennady V.; Charlab, Rosane; Dew, Ian; Deng, Zuoming; Istrail, Sorin; Li, Peter; Sutton, Granger

    2005-01-01

    Designing effective and accurate tools for identifying the functional and structural elements in a genome remains at the frontier of genome annotation owing to incompleteness and inaccuracy of the data, limitations in the computational models, and shifting paradigms in genomics, such as alternative splicing. We present a methodology for the automated annotation of genes and their alternatively spliced mRNA transcripts based on existing cDNA and protein sequence evidence from the same species or projected from a related species using syntenic mapping information. At the core of the method is the splice graph, a compact representation of a gene, its exons, introns, and alternatively spliced isoforms. The putative transcripts are enumerated from the graph and assigned confidence scores based on the strength of sequence evidence, and a subset of the high-scoring candidates are selected and promoted into the annotation. The method is highly selective, eliminating the unlikely candidates while retaining 98% of the high-quality mRNA evidence in well-formed transcripts, and produces annotation that is measurably more accurate than some evidence-based gene sets. The process is fast, accurate, and fully automated, and combines the traditionally distinct gene annotation and alternative splicing detection processes in a comprehensive and systematic way, thus considerably aiding in the ensuing manual curation efforts. PMID:15632090

  4. Gene and alternative splicing annotation with AIR.

    PubMed

    Florea, Liliana; Di Francesco, Valentina; Miller, Jason; Turner, Russell; Yao, Alison; Harris, Michael; Walenz, Brian; Mobarry, Clark; Merkulov, Gennady V; Charlab, Rosane; Dew, Ian; Deng, Zuoming; Istrail, Sorin; Li, Peter; Sutton, Granger

    2005-01-01

    Designing effective and accurate tools for identifying the functional and structural elements in a genome remains at the frontier of genome annotation owing to incompleteness and inaccuracy of the data, limitations in the computational models, and shifting paradigms in genomics, such as alternative splicing. We present a methodology for the automated annotation of genes and their alternatively spliced mRNA transcripts based on existing cDNA and protein sequence evidence from the same species or projected from a related species using syntenic mapping information. At the core of the method is the splice graph, a compact representation of a gene, its exons, introns, and alternatively spliced isoforms. The putative transcripts are enumerated from the graph and assigned confidence scores based on the strength of sequence evidence, and a subset of the high-scoring candidates are selected and promoted into the annotation. The method is highly selective, eliminating the unlikely candidates while retaining 98% of the high-quality mRNA evidence in well-formed transcripts, and produces annotation that is measurably more accurate than some evidence-based gene sets. The process is fast, accurate, and fully automated, and combines the traditionally distinct gene annotation and alternative splicing detection processes in a comprehensive and systematic way, thus considerably aiding in the ensuing manual curation efforts.

  5. Sec16 alternative splicing dynamically controls COPII transport efficiency

    PubMed Central

    Wilhelmi, Ilka; Kanski, Regina; Neumann, Alexander; Herdt, Olga; Hoff, Florian; Jacob, Ralf; Preußner, Marco; Heyd, Florian

    2016-01-01

    The transport of secretory proteins from the endoplasmic reticulum (ER) to the Golgi depends on COPII-coated vesicles. While the basic principles of the COPII machinery have been identified, it remains largely unknown how COPII transport is regulated to accommodate tissue- or activation-specific differences in cargo load and identity. Here we show that activation-induced alternative splicing of Sec16 controls adaptation of COPII transport to increased secretory cargo upon T-cell activation. Using splice-site blocking morpholinos and CRISPR/Cas9-mediated genome engineering, we show that the number of ER exit sites, COPII dynamics and transport efficiency depend on Sec16 alternative splicing. As the mechanistic basis, we suggest the C-terminal Sec16 domain to be a splicing-controlled protein interaction platform, with individual isoforms showing differential abilities to recruit COPII components. Our work connects the COPII pathway with alternative splicing, adding a new regulatory layer to protein secretion and its adaptation to changing cellular environments. PMID:27492621

  6. Regulation of alternative splicing in Drosophila by 56 RNA binding proteins

    SciTech Connect

    Brooks, Angela N.; Duff, Michael O.; May, Gemma; Yang, Li; Bolisetty, Mohan; Landolin, Jane; Wan, Ken; Sandler, Jeremy; Booth, Benjamin W.; Celniker, Susan E.; Graveley, Brenton R.; Brenner, Steven E.

    2015-08-20

    Alternative splicing is regulated by RNA binding proteins (RBPs) that recognize pre-mRNA sequence elements and activate or repress adjacent exons. Here, we used RNA interference and RNA-seq to identify splicing events regulated by 56 Drosophila proteins, some previously unknown to regulate splicing. Nearly all proteins affected alternative first exons, suggesting that RBPs play important roles in first exon choice. Half of the splicing events were regulated by multiple proteins, demonstrating extensive combinatorial regulation. We observed that SR and hnRNP proteins tend to act coordinately with each other, not antagonistically. We also identified a cross-regulatory network where splicing regulators affected the splicing of pre-mRNAs encoding other splicing regulators. In conclusion, this large-scale study substantially enhances our understanding of recent models of splicing regulation and provides a resource of thousands of exons that are regulated by 56 diverse RBPs.

  7. Regulation of alternative splicing in Drosophila by 56 RNA binding proteins

    PubMed Central

    Brooks, Angela N.; Duff, Michael O.; May, Gemma; Yang, Li; Bolisetty, Mohan; Landolin, Jane; Wan, Ken; Sandler, Jeremy; Booth, Benjamin W.; Celniker, Susan E.; Graveley, Brenton R.; Brenner, Steven E.

    2015-01-01

    Alternative splicing is regulated by RNA binding proteins (RBPs) that recognize pre-mRNA sequence elements and activate or repress adjacent exons. Here, we used RNA interference and RNA-seq to identify splicing events regulated by 56 Drosophila proteins, some previously unknown to regulate splicing. Nearly all proteins affected alternative first exons, suggesting that RBPs play important roles in first exon choice. Half of the splicing events were regulated by multiple proteins, demonstrating extensive combinatorial regulation. We observed that SR and hnRNP proteins tend to act coordinately with each other, not antagonistically. We also identified a cross-regulatory network where splicing regulators affected the splicing of pre-mRNAs encoding other splicing regulators. This large-scale study substantially enhances our understanding of recent models of splicing regulation and provides a resource of thousands of exons that are regulated by 56 diverse RBPs. PMID:26294686

  8. Regulation of alternative splicing in Drosophila by 56 RNA binding proteins

    DOE PAGES

    Brooks, Angela N.; Duff, Michael O.; May, Gemma; ...

    2015-08-20

    Alternative splicing is regulated by RNA binding proteins (RBPs) that recognize pre-mRNA sequence elements and activate or repress adjacent exons. Here, we used RNA interference and RNA-seq to identify splicing events regulated by 56 Drosophila proteins, some previously unknown to regulate splicing. Nearly all proteins affected alternative first exons, suggesting that RBPs play important roles in first exon choice. Half of the splicing events were regulated by multiple proteins, demonstrating extensive combinatorial regulation. We observed that SR and hnRNP proteins tend to act coordinately with each other, not antagonistically. We also identified a cross-regulatory network where splicing regulators affected themore » splicing of pre-mRNAs encoding other splicing regulators. In conclusion, this large-scale study substantially enhances our understanding of recent models of splicing regulation and provides a resource of thousands of exons that are regulated by 56 diverse RBPs.« less

  9. Tumor microenvironment–associated modifications of alternative splicing

    PubMed Central

    Brosseau, Jean-Philippe; Lucier, Jean-François; Nwilati, Hanad; Thibault, Philippe; Garneau, Daniel; Gendron, Daniel; Durand, Mathieu; Couture, Sonia; Lapointe, Elvy; Prinos, Panagiotis; Klinck, Roscoe; Perreault, Jean-Pierre; Chabot, Benoit; Abou-Elela, Sherif

    2014-01-01

    Pre-mRNA alternative splicing is modified in cancer, but the origin and specificity of these changes remain unclear. Here, we probed ovarian tumors to identify cancer-associated splicing isoforms and define the mechanism by which splicing is modified in cancer cells. Using high-throughput quantitative PCR, we monitored the expression of splice variants in laser-dissected tissues from ovarian tumors. Surprisingly, changes in alternative splicing were not limited to the tumor tissues but were also found in the tumor microenvironment. Changes in the tumor-associated splicing events were found to be regulated by splicing factors that are differentially expressed in cancer tissues. Overall, ∼20% of the alternative splicing events affected by the down-regulation of the splicing factors QKI and RBFOX2 were altered in the microenvironment of ovarian tumors. Together, our results indicate that the tumor microenvironment undergoes specific changes in alternative splicing orchestrated by a limited number of splicing factors. PMID:24335142

  10. Tumor microenvironment-associated modifications of alternative splicing.

    PubMed

    Brosseau, Jean-Philippe; Lucier, Jean-François; Nwilati, Hanad; Thibault, Philippe; Garneau, Daniel; Gendron, Daniel; Durand, Mathieu; Couture, Sonia; Lapointe, Elvy; Prinos, Panagiotis; Klinck, Roscoe; Perreault, Jean-Pierre; Chabot, Benoit; Abou-Elela, Sherif

    2014-02-01

    Pre-mRNA alternative splicing is modified in cancer, but the origin and specificity of these changes remain unclear. Here, we probed ovarian tumors to identify cancer-associated splicing isoforms and define the mechanism by which splicing is modified in cancer cells. Using high-throughput quantitative PCR, we monitored the expression of splice variants in laser-dissected tissues from ovarian tumors. Surprisingly, changes in alternative splicing were not limited to the tumor tissues but were also found in the tumor microenvironment. Changes in the tumor-associated splicing events were found to be regulated by splicing factors that are differentially expressed in cancer tissues. Overall, ∼20% of the alternative splicing events affected by the down-regulation of the splicing factors QKI and RBFOX2 were altered in the microenvironment of ovarian tumors. Together, our results indicate that the tumor microenvironment undergoes specific changes in alternative splicing orchestrated by a limited number of splicing factors.

  11. Ultraconserved elements are associated with homeostatic control of splicing regulators by alternative splicing and nonsense-mediated decay

    PubMed Central

    Ni, Julie Z.; Grate, Leslie; Donohue, John Paul; Preston, Christine; Nobida, Naomi; O’Brien, Georgeann; Shiue, Lily; Clark, Tyson A.; Blume, John E.; Ares, Manuel

    2007-01-01

    Many alternative splicing events create RNAs with premature stop codons, suggesting that alternative splicing coupled with nonsense-mediated decay (AS-NMD) may regulate gene expression post-transcriptionally. We tested this idea in mice by blocking NMD and measuring changes in isoform representation using splicing-sensitive microarrays. We found a striking class of highly conserved stop codon-containing exons whose inclusion renders the transcript sensitive to NMD. A genomic search for additional examples identified >50 such exons in genes with a variety of functions. These exons are unusually frequent in genes that encode splicing activators and are unexpectedly enriched in the so-called “ultraconserved” elements in the mammalian lineage. Further analysis show that NMD of mRNAs for splicing activators such as SR proteins is triggered by splicing activation events, whereas NMD of the mRNAs for negatively acting hnRNP proteins is triggered by splicing repression, a polarity consistent with widespread homeostatic control of splicing regulator gene expression. We suggest that the extreme genomic conservation surrounding these regulatory splicing events within splicing factor genes demonstrates the evolutionary importance of maintaining tightly tuned homeostasis of RNA-binding protein levels in the vertebrate cell. PMID:17369403

  12. Alternative Splicing for Diseases, Cancers, Drugs, and Databases

    PubMed Central

    Lee, Jin-Ching; Hou, Ming-Feng; Wang, Chun-Lin; Chen, Chien-Chi; Huang, Hurng-Wern

    2013-01-01

    Alternative splicing is a major diversification mechanism in the human transcriptome and proteome. Several diseases, including cancers, have been associated with dysregulation of alternative splicing. Thus, correcting alternative splicing may restore normal cell physiology in patients with these diseases. This paper summarizes several alternative splicing-related diseases, including cancers and their target genes. Since new cancer drugs often target spliceosomes, several clinical drugs and natural products or their synthesized derivatives were analyzed to determine their effects on alternative splicing. Other agents known to have modulating effects on alternative splicing during therapeutic treatment of cancer are also discussed. Several commonly used bioinformatics resources are also summarized. PMID:23766705

  13. Origin of Spliceosomal Introns and Alternative Splicing

    PubMed Central

    Irimia, Manuel; Roy, Scott William

    2014-01-01

    In this work we review the current knowledge on the prehistory, origins, and evolution of spliceosomal introns. First, we briefly outline the major features of the different types of introns, with particular emphasis on the nonspliceosomal self-splicing group II introns, which are widely thought to be the ancestors of spliceosomal introns. Next, we discuss the main scenarios proposed for the origin and proliferation of spliceosomal introns, an event intimately linked to eukaryogenesis. We then summarize the evidence that suggests that the last eukaryotic common ancestor (LECA) had remarkably high intron densities and many associated characteristics resembling modern intron-rich genomes. From this intron-rich LECA, the different eukaryotic lineages have taken very distinct evolutionary paths leading to profoundly diverged modern genome structures. Finally, we discuss the origins of alternative splicing and the qualitative differences in alternative splicing forms and functions across lineages. PMID:24890509

  14. Alternative splicing in cancer: implications for biology and therapy.

    PubMed

    Chen, J; Weiss, W A

    2015-01-02

    Alternative splicing has critical roles in normal development and can promote growth and survival in cancer. Aberrant splicing, the production of noncanonical and cancer-specific mRNA transcripts, can lead to loss-of-function in tumor suppressors or activation of oncogenes and cancer pathways. Emerging data suggest that aberrant splicing products and loss of canonically spliced variants correlate with stage and progression in malignancy. Here, we review the splicing landscape of TP53, BARD1 and AR to illuminate roles for alternative splicing in cancer. We also examine the intersection between alternative splicing pathways and novel therapeutic approaches.

  15. Functional splicing network reveals extensive regulatory potential of the core spliceosomal machinery.

    PubMed

    Papasaikas, Panagiotis; Tejedor, J Ramón; Vigevani, Luisa; Valcárcel, Juan

    2015-01-08

    Pre-mRNA splicing relies on the poorly understood dynamic interplay between >150 protein components of the spliceosome. The steps at which splicing can be regulated remain largely unknown. We systematically analyzed the effect of knocking down the components of the splicing machinery on alternative splicing events relevant for cell proliferation and apoptosis and used this information to reconstruct a network of functional interactions. The network accurately captures known physical and functional associations and identifies new ones, revealing remarkable regulatory potential of core spliceosomal components, related to the order and duration of their recruitment during spliceosome assembly. In contrast with standard models of regulation at early steps of splice site recognition, factors involved in catalytic activation of the spliceosome display regulatory properties. The network also sheds light on the antagonism between hnRNP C and U2AF, and on targets of antitumor drugs, and can be widely used to identify mechanisms of splicing regulation.

  16. Schizophyllum commune has an extensive and functional alternative splicing repertoire

    SciTech Connect

    Gehrmann, Thies; Pelkmans, Jordi F.; Lugones, Luis G.; Wösten, Han A. B.; Abeel, Thomas; Reinders, Marcel J. T.

    2016-09-23

    Recent genome-wide studies have demonstrated that fungi possess the machinery to alternatively splice pre-mRNA. However, there has not been a systematic categorization of the functional impact of alternative splicing in a fungus. We investigate alternative splicing and its functional consequences in the model mushroom forming fungus Schizophyllum commune. Alternative splicing was demonstrated for 2,285 out of 12,988 expressed genes, resulting in 20% additional transcripts. Intron retentions were the most common alternative splicing events, accounting for 33% of all splicing events, and 43% of the events in coding regions. On the other hand, exon skipping events were rare in coding regions (1%) but enriched in UTRs where they accounted for 57% of the events. Specific functional groups, including transcription factors, contained alternatively spliced genes. Alternatively spliced transcripts were regulated differently throughout development in 19% of the 2,285 alternatively spliced genes. Notably, 69% of alternatively spliced genes have predicted alternative functionality by loss or gain of functional domains, or by acquiring alternative subcellular locations. S. commune exhibits more alternative splicing than any other studied fungus. Finally, taken together, alternative splicing increases the complexity of the S. commune proteome considerably and provides it with a rich repertoire of alternative functionality that is exploited dynamically.

  17. Schizophyllum commune has an extensive and functional alternative splicing repertoire.

    PubMed

    Gehrmann, Thies; Pelkmans, Jordi F; Lugones, Luis G; Wösten, Han A B; Abeel, Thomas; Reinders, Marcel J T

    2016-09-23

    Recent genome-wide studies have demonstrated that fungi possess the machinery to alternatively splice pre-mRNA. However, there has not been a systematic categorization of the functional impact of alternative splicing in a fungus. We investigate alternative splicing and its functional consequences in the model mushroom forming fungus Schizophyllum commune. Alternative splicing was demonstrated for 2,285 out of 12,988 expressed genes, resulting in 20% additional transcripts. Intron retentions were the most common alternative splicing events, accounting for 33% of all splicing events, and 43% of the events in coding regions. On the other hand, exon skipping events were rare in coding regions (1%) but enriched in UTRs where they accounted for 57% of the events. Specific functional groups, including transcription factors, contained alternatively spliced genes. Alternatively spliced transcripts were regulated differently throughout development in 19% of the 2,285 alternatively spliced genes. Notably, 69% of alternatively spliced genes have predicted alternative functionality by loss or gain of functional domains, or by acquiring alternative subcellular locations. S. commune exhibits more alternative splicing than any other studied fungus. Taken together, alternative splicing increases the complexity of the S. commune proteome considerably and provides it with a rich repertoire of alternative functionality that is exploited dynamically.

  18. Schizophyllum commune has an extensive and functional alternative splicing repertoire

    PubMed Central

    Gehrmann, Thies; Pelkmans, Jordi F.; Lugones, Luis G.; Wösten, Han A. B.; Abeel, Thomas; Reinders, Marcel J. T.

    2016-01-01

    Recent genome-wide studies have demonstrated that fungi possess the machinery to alternatively splice pre-mRNA. However, there has not been a systematic categorization of the functional impact of alternative splicing in a fungus. We investigate alternative splicing and its functional consequences in the model mushroom forming fungus Schizophyllum commune. Alternative splicing was demonstrated for 2,285 out of 12,988 expressed genes, resulting in 20% additional transcripts. Intron retentions were the most common alternative splicing events, accounting for 33% of all splicing events, and 43% of the events in coding regions. On the other hand, exon skipping events were rare in coding regions (1%) but enriched in UTRs where they accounted for 57% of the events. Specific functional groups, including transcription factors, contained alternatively spliced genes. Alternatively spliced transcripts were regulated differently throughout development in 19% of the 2,285 alternatively spliced genes. Notably, 69% of alternatively spliced genes have predicted alternative functionality by loss or gain of functional domains, or by acquiring alternative subcellular locations. S. commune exhibits more alternative splicing than any other studied fungus. Taken together, alternative splicing increases the complexity of the S. commune proteome considerably and provides it with a rich repertoire of alternative functionality that is exploited dynamically. PMID:27659065

  19. Schizophyllum commune has an extensive and functional alternative splicing repertoire

    DOE PAGES

    Gehrmann, Thies; Pelkmans, Jordi F.; Lugones, Luis G.; ...

    2016-09-23

    Recent genome-wide studies have demonstrated that fungi possess the machinery to alternatively splice pre-mRNA. However, there has not been a systematic categorization of the functional impact of alternative splicing in a fungus. We investigate alternative splicing and its functional consequences in the model mushroom forming fungus Schizophyllum commune. Alternative splicing was demonstrated for 2,285 out of 12,988 expressed genes, resulting in 20% additional transcripts. Intron retentions were the most common alternative splicing events, accounting for 33% of all splicing events, and 43% of the events in coding regions. On the other hand, exon skipping events were rare in coding regionsmore » (1%) but enriched in UTRs where they accounted for 57% of the events. Specific functional groups, including transcription factors, contained alternatively spliced genes. Alternatively spliced transcripts were regulated differently throughout development in 19% of the 2,285 alternatively spliced genes. Notably, 69% of alternatively spliced genes have predicted alternative functionality by loss or gain of functional domains, or by acquiring alternative subcellular locations. S. commune exhibits more alternative splicing than any other studied fungus. Finally, taken together, alternative splicing increases the complexity of the S. commune proteome considerably and provides it with a rich repertoire of alternative functionality that is exploited dynamically.« less

  20. [EDAS, databases of alternatively spliced human genes].

    PubMed

    Nurtdinov, R N; Neverov, A D; Mal'ko, D B; Kosmodem'ianskiĭ, I A; Ermakova, E O; Ramenskiĭ, V E; Mironov, A A; Gel'fand, M S

    2006-01-01

    EDAS, a database of alternatively spliced human genes, contains data on the alignment of proteins, mRNAs, and EST. It contains information on all exons and introns observed, as well as elementary alternatives formed from them. The database makes it possible to filter the output data by changing the cut-off threshold by the significance level. The database is accessible at http://www.gene-bee.msu.ru/edas/.

  1. Genomic architecture and functional relationships of intronless, constitutively- and alternatively-spliced genes in Brachypodium distachyon

    PubMed Central

    Mandadi, Kranthi K; Scholthof, Karen-Beth G

    2015-01-01

    Splicing and alternative splicing (AS) are widespread co- and post-transcriptional regulatory processes in plants. Recently, we characterized genome-wide AS landscapes and virus-induced AS patterns in Brachypodium distachyon (Brachypodium), a C3 model grass. Brachypodium plants infected with Panicum mosaic virus (PMV) alone or in mixed infections with its satellite virus (SPMV) were used for high-throughput, paired-end RNA sequencing. Here, using gene attributes of ∼5,655 intronless genes, ∼13,302 constitutively spliced, and ∼7,564 alternatively spliced genes, we analyzed the influence of genomic features on splicing incidence and AS frequency. In Brachypodium, gene length, coding sequence length, and exon and intron number were positively correlated to splicing incidence and AS frequency. In contrast, exon length and the percentage composition of GC (%GC) content were inversely correlated with splicing incidence and AS frequency. Although gene expression status had little correlation with splicing occurrence per se, it negatively correlated to AS frequency: i.e., genes with ≥5 alternatively spliced transcripts were significantly less expressed compared to genes encoding <5 alternative transcripts. Further gene set enrichment analysis uncovered unique functional relationships among nonspliced, constitutively spliced and alternatively spliced genes. PMID:26156297

  2. Genomic architecture and functional relationships of intronless, constitutively- and alternatively-spliced genes in Brachypodium distachyon.

    PubMed

    Mandadi, Kranthi K; Scholthof, Karen-Beth G

    2015-01-01

    Splicing and alternative splicing (AS) are widespread co- and post-transcriptional regulatory processes in plants. Recently, we characterized genome-wide AS landscapes and virus-induced AS patterns in Brachypodium distachyon (Brachypodium), a C3 model grass. Brachypodium plants infected with Panicum mosaic virus (PMV) alone or in mixed infections with its satellite virus (SPMV) were used for high-throughput, paired-end RNA sequencing. Here, using gene attributes of ∼5,655 intronless genes, ∼13,302 constitutively spliced, and ∼7,564 alternatively spliced genes, we analyzed the influence of genomic features on splicing incidence and AS frequency. In Brachypodium, gene length, coding sequence length, and exon and intron number were positively correlated to splicing incidence and AS frequency. In contrast, exon length and the percentage composition of GC (%GC) content were inversely correlated with splicing incidence and AS frequency. Although gene expression status had little correlation with splicing occurrence per se, it negatively correlated to AS frequency: i.e., genes with ≥5 alternatively spliced transcripts were significantly less expressed compared to genes encoding <5 alternative transcripts. Further gene set enrichment analysis uncovered unique functional relationships among nonspliced, constitutively spliced and alternatively spliced genes.

  3. Vitamin D and alternative splicing of RNA

    PubMed Central

    Zhou, Rui; Chun, Rene F.; Lisse, Thomas S.; Garcia, Alejandro J.; Xu, Jianzhong; Adams, John S.; Hewison, Martin

    2014-01-01

    The active form of vitamin D (1α,25-dihydroxyvitamin D, 1,25(OH)2D) exerts its genomic effects via binding to a nuclear high-affinity vitamin D receptor (VDR). Recent deep sequencing analysis of VDR binding locations across the complete genome has significantly expanded our understanding of the actions of vitamin D and VDR on gene transcription. However, these studies have also promoted appreciation of the extra-transcriptional impact of vitamin D on gene expression. It is now clear that vitamin D interacts with the epigenome via effects on DNA methylation, histone acetylation, and microRNA generation to maintain normal biological functions. There is also increasing evidence that vitamin D can influence pre-mRNA constitutive splicing and alternative splicing, although the mechanism for this remains unclear. Pre-mRNA splicing has long been thought to be a post-transcription RNA processing event, but current data indicate that this occurs co-transcriptionally. Several steroid hormones have been recognized to coordinately control gene transcription and pre-mRNA splicing through the recruitment of nuclear receptor co-regulators that can both control gene transcription and splicing. The current review will discuss this concept with specific reference to vitamin D, and the potential role of heterogeneous nuclear ribonucleoprotein C (hnRNPC), a nuclear factor with an established function in RNA splicing. hnRNPC, has been shown to be involved in the VDR transcriptional complex as a vitamin D-response element-binding protein (VDRE-BP), and may act as a coupling factor linking VDR-directed gene transcription with RNA splicing. In this way hnRNPC may provide an additional mechanism for the fine-tuning of vitamin D-regulated target gene expression. PMID:25447737

  4. Vitamin D and alternative splicing of RNA.

    PubMed

    Zhou, Rui; Chun, Rene F; Lisse, Thomas S; Garcia, Alejandro J; Xu, Jianzhong; Adams, John S; Hewison, Martin

    2015-04-01

    The active form of vitamin D (1α,25-dihydroxyvitamin D, 1,25(OH)2D) exerts its genomic effects via binding to a nuclear high-affinity vitamin D receptor (VDR). Recent deep sequencing analysis of VDR binding locations across the complete genome has significantly expanded our understanding of the actions of vitamin D and VDR on gene transcription. However, these studies have also promoted appreciation of the extra-transcriptional impact of vitamin D on gene expression. It is now clear that vitamin D interacts with the epigenome via effects on DNA methylation, histone acetylation, and microRNA generation to maintain normal biological functions. There is also increasing evidence that vitamin D can influence pre-mRNA constitutive splicing and alternative splicing, although the mechanism for this remains unclear. Pre-mRNA splicing has long been thought to be a post-transcription RNA processing event, but current data indicate that this occurs co-transcriptionally. Several steroid hormones have been recognized to coordinately control gene transcription and pre-mRNA splicing through the recruitment of nuclear receptor co-regulators that can both control gene transcription and splicing. The current review will discuss this concept with specific reference to vitamin D, and the potential role of heterogeneous nuclear ribonucleoprotein C (hnRNPC), a nuclear factor with an established function in RNA splicing. hnRNPC, has been shown to be involved in the VDR transcriptional complex as a vitamin D-response element-binding protein (VDRE-BP), and may act as a coupling factor linking VDR-directed gene transcription with RNA splicing. In this way hnRNPC may provide an additional mechanism for the fine-tuning of vitamin D-regulated target gene expression. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.

  5. Systematic Profiling of Poly(A)+ Transcripts Modulated by Core 3’ End Processing and Splicing Factors Reveals Regulatory Rules of Alternative Cleavage and Polyadenylation

    PubMed Central

    Li, Wencheng; You, Bei; Hoque, Mainul; Zheng, Dinghai; Luo, Wenting; Ji, Zhe; Park, Ji Yeon; Gunderson, Samuel I.; Kalsotra, Auinash; Manley, James L.; Tian, Bin

    2015-01-01

    Alternative cleavage and polyadenylation (APA) results in mRNA isoforms containing different 3’ untranslated regions (3’UTRs) and/or coding sequences. How core cleavage/polyadenylation (C/P) factors regulate APA is not well understood. Using siRNA knockdown coupled with deep sequencing, we found that several C/P factors can play significant roles in 3’UTR-APA. Whereas Pcf11 and Fip1 enhance usage of proximal poly(A) sites (pAs), CFI-25/68, PABPN1 and PABPC1 promote usage of distal pAs. Strong cis element biases were found for pAs regulated by CFI-25/68 or Fip1, and the distance between pAs plays an important role in APA regulation. In addition, intronic pAs are substantially regulated by splicing factors, with U1 mostly inhibiting C/P events in introns near the 5’ end of gene and U2 suppressing those in introns with features for efficient splicing. Furthermore, PABPN1 inhibits expression of transcripts with pAs near the transcription start site (TSS), a property possibly related to its role in RNA degradation. Finally, we found that groups of APA events regulated by C/P factors are also modulated in cell differentiation and development with distinct trends. Together, our results support an APA code where an APA event in a given cellular context is regulated by a number of parameters, including relative location to the TSS, splicing context, distance between competing pAs, surrounding cis elements and concentrations of core C/P factors. PMID:25906188

  6. Regulation of Chemoresistance Via Alternative Messenger RNA Splicing

    PubMed Central

    Eblen, Scott T.

    2012-01-01

    The acquisition of drug resistance to chemotherapy is a significant problem in the treatment of cancer, greatly increasing patient morbidity and mortality. Tumors are often sensitive to chemotherapy upon initial treatment, but repeated treatments can select for those cells that have were able to survive initial therapy and have acquired cellular mechanisms to enhance their resistance to subsequent chemotherapy treatment. Many cellular mechanisms of drug resistance have been identified, most of which result from changes in gene and protein expression. While changes at the transcriptional level have been duly noted, it is primarily the post-transcriptional processing of pre-mRNA into mature mRNA that regulates the composition of the proteome and it is the proteome that actually regulates the cell’s response to chemotherapeutic insult, inducing cell survival or death. During pre-mRNA processing, intronic non-protein-coding sequences are removed and protein-coding exons are spliced to form a continuous template for protein translation. Alternative splicing involves the differential inclusion or exclusion of exonic sequences into the mature transcript, generating different mRNA templates for protein production. This regulatory mechanism enables the potential to produce many different protein isoforms from the same gene. In this review I will explain the mechanism of alternative pre-mRNA splicing and look at some specific examples of how splicing factors, splicing factor kinases and alternative splicing of specific pre-mRNAs from genes have been shown to contribute to acquisition of the drug resistant phenotype. PMID:22248731

  7. Integrating alternative splicing detection into gene prediction

    PubMed Central

    Foissac, Sylvain; Schiex, Thomas

    2005-01-01

    Background Alternative splicing (AS) is now considered as a major actor in transcriptome/proteome diversity and it cannot be neglected in the annotation process of a new genome. Despite considerable progresses in term of accuracy in computational gene prediction, the ability to reliably predict AS variants when there is local experimental evidence of it remains an open challenge for gene finders. Results We have used a new integrative approach that allows to incorporate AS detection into ab initio gene prediction. This method relies on the analysis of genomically aligned transcript sequences (ESTs and/or cDNAs), and has been implemented in the dynamic programming algorithm of the graph-based gene finder EuGÈNE. Given a genomic sequence and a set of aligned transcripts, this new version identifies the set of transcripts carrying evidence of alternative splicing events, and provides, in addition to the classical optimal gene prediction, alternative optimal predictions (among those which are consistent with the AS events detected). This allows for multiple annotations of a single gene in a way such that each predicted variant is supported by a transcript evidence (but not necessarily with a full-length coverage). Conclusions This automatic combination of experimental data analysis and ab initio gene finding offers an ideal integration of alternatively spliced gene prediction inside a single annotation pipeline. PMID:15705189

  8. SplicingTypesAnno: annotating and quantifying alternative splicing events for RNA-Seq data.

    PubMed

    Sun, Xiaoyong; Zuo, Fenghua; Ru, Yuanbin; Guo, Jiqiang; Yan, Xiaoyan; Sablok, Gaurav

    2015-04-01

    Alternative splicing plays a key role in the regulation of the central dogma. Four major types of alternative splicing have been classified as intron retention, exon skipping, alternative 5 splice sites or alternative donor sites, and alternative 3 splice sites or alternative acceptor sites. A few algorithms have been developed to detect splice junctions from RNA-Seq reads. However, there are few tools targeting at the major alternative splicing types at the exon/intron level. This type of analysis may reveal subtle, yet important events of alternative splicing, and thus help gain deeper understanding of the mechanism of alternative splicing. This paper describes a user-friendly R package, extracting, annotating and analyzing alternative splicing types for sequence alignment files from RNA-Seq. SplicingTypesAnno can: (1) provide annotation for major alternative splicing at exon/intron level. By comparing the annotation from GTF/GFF file, it identifies the novel alternative splicing sites; (2) offer a convenient two-level analysis: genome-scale annotation for users with high performance computing environment, and gene-scale annotation for users with personal computers; (3) generate a user-friendly web report and additional BED files for IGV visualization. SplicingTypesAnno is a user-friendly R package for extracting, annotating and analyzing alternative splicing types at exon/intron level for sequence alignment files from RNA-Seq. It is publically available at https://sourceforge.net/projects/splicingtypes/files/ or http://genome.sdau.edu.cn/research/software/SplicingTypesAnno.html.

  9. Calcium-mediated histone modifications regulate alternative splicing in cardiomyocytes.

    PubMed

    Sharma, Alok; Nguyen, Hieu; Geng, Cuiyu; Hinman, Melissa N; Luo, Guangbin; Lou, Hua

    2014-11-18

    In cardiomyocytes, calcium is known to control gene expression at the level of transcription, whereas its role in regulating alternative splicing has not been explored. Here we report that, in mouse primary or embryonic stem cell-derived cardiomyocytes, increased calcium levels induce robust and reversible skipping of several alternative exons from endogenously expressed genes. Interestingly, we demonstrate a calcium-mediated splicing regulatory mechanism that depends on changes of histone modifications. Specifically, the regulation occurs through changes in calcium-responsive kinase activities that lead to alterations in histone modifications and subsequent changes in the transcriptional elongation rate and exon skipping. We demonstrate that increased intracellular calcium levels lead to histone hyperacetylation along the body of the genes containing calcium-responsive alternative exons by disrupting the histone deacetylase-to-histone acetyltransferase balance in the nucleus. Consequently, the RNA polymerase II elongation rate increases significantly on those genes, resulting in skipping of the alternative exons. These studies reveal a mechanism by which calcium-level changes in cardiomyocytes impact on the output of gene expression through altering alternative pre-mRNA splicing patterns.

  10. Neurogenesis: Regulation by Alternative Splicing and Related Posttranscriptional Processes.

    PubMed

    Lara-Pezzi, Enrique; Desco, Manuel; Gatto, Alberto; Gómez-Gaviro, María Victoria

    2016-11-10

    The complexity of the mammalian brain requires highly specialized protein function and diversity. As neurons differentiate and the neuronal circuitry is established, several mRNAs undergo alternative splicing and other posttranscriptional changes that expand the variety of protein isoforms produced. Recent advances are beginning to shed light on the molecular mechanisms that regulate isoform switching during neurogenesis and the role played by specific RNA binding proteins in this process. Neurogenesis and neuronal wiring were recently shown to also be regulated by RNA degradation through nonsense-mediated decay. An additional layer of regulatory complexity in these biological processes is the interplay between alternative splicing and long noncoding RNAs. Dysregulation of posttranscriptional regulation results in defective neuronal differentiation and/or synaptic connections that lead to neurodevelopmental and psychiatric disorders.

  11. Genome-wide analysis of SRSF10-regulated alternative splicing by deep sequencing of chicken transcriptome.

    PubMed

    Zhou, Xuexia; Wu, Wenwu; Wei, Ning; Cheng, Yuanming; Xie, Zhiqin; Feng, Ying

    2014-12-01

    Splicing factor SRSF10 is known to function as a sequence-specific splicing activator that is capable of regulating alternative splicing both in vitro and in vivo. We recently used an RNA-seq approach coupled with bioinformatics analysis to identify the extensive splicing network regulated by SRSF10 in chicken cells. We found that SRSF10 promoted both exon inclusion and exclusion. Functionally, many of the SRSF10-verified alternative exons are linked to pathways of response to external stimulus. Here we describe in detail the experimental design, bioinformatics analysis and GO/pathway enrichment analysis of SRSF10-regulated genes to correspond with our data in the Gene Expression Omnibus with accession number GSE53354. Our data thus provide a resource for studying regulation of alternative splicing in vivo that underlines biological functions of splicing regulatory proteins in cells.

  12. Dynamic integration of splicing within gene regulatory pathways

    PubMed Central

    Braunschweig, Ulrich; Gueroussov, Serge; Plocik, Alex; Graveley, Brenton R.; Blencowe, Benjamin J.

    2013-01-01

    Precursor mRNA splicing is one of the most highly regulated processes in metazoan species. In addition to generating vast repertoires of RNAs and proteins, splicing has a profound impact on other gene regulatory layers, including mRNA transcription, turnover, transport and translation. Conversely, factors regulating chromatin and transcription complexes impact the splicing process. This extensive cross-talk between gene regulatory layers takes advantage of dynamic spatial, physical and temporal organizational properties of the cell nucleus, and further emphasizes the importance of developing a multidimensional understanding of splicing control. PMID:23498935

  13. Alternative splicing in cancers: From aberrant regulation to new therapeutics.

    PubMed

    Song, Xiaowei; Zeng, Zhenyu; Wei, Huanhuan; Wang, Zefeng

    2017-09-14

    Alternative splicing is one of the most common mechanisms for gene regulation in humans, and plays a vital role to increase the complexity of functional proteins. In this article, we seek to provide a general review on the relationships between alternative splicing and tumorigenesis. We briefly introduce the basic rules for regulation of alternative splicing, and discuss recent advances on dynamic regulation of alternative splicing in cancers by highlighting the roles of a variety of RNA splicing factors in tumorigenesis. We further discuss several important questions regarding the splicing of long noncoding RNAs and back-splicing of circular RNAs in cancers. Finally, we discuss the current technologies that can be used to manipulate alternative splicing and serve as potential cancer treatment. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Vials: Visualizing Alternative Splicing of Genes

    PubMed Central

    Strobelt, Hendrik; Alsallakh, Bilal; Botros, Joseph; Peterson, Brant; Borowsky, Mark; Pfister, Hanspeter; Lex, Alexander

    2016-01-01

    Alternative splicing is a process by which the same DNA sequence is used to assemble different proteins, called protein isoforms. Alternative splicing works by selectively omitting some of the coding regions (exons) typically associated with a gene. Detection of alternative splicing is difficult and uses a combination of advanced data acquisition methods and statistical inference. Knowledge about the abundance of isoforms is important for understanding both normal processes and diseases and to eventually improve treatment through targeted therapies. The data, however, is complex and current visualizations for isoforms are neither perceptually efficient nor scalable. To remedy this, we developed Vials, a novel visual analysis tool that enables analysts to explore the various datasets that scientists use to make judgments about isoforms: the abundance of reads associated with the coding regions of the gene, evidence for junctions, i.e., edges connecting the coding regions, and predictions of isoform frequencies. Vials is scalable as it allows for the simultaneous analysis of many samples in multiple groups. Our tool thus enables experts to (a) identify patterns of isoform abundance in groups of samples and (b) evaluate the quality of the data. We demonstrate the value of our tool in case studies using publicly available datasets. PMID:26529712

  15. Alternative-splicing-mediated gene expression

    NASA Astrophysics Data System (ADS)

    Wang, Qianliang; Zhou, Tianshou

    2014-01-01

    Alternative splicing (AS) is a fundamental process during gene expression and has been found to be ubiquitous in eukaryotes. However, how AS impacts gene expression levels both quantitatively and qualitatively remains to be fully explored. Here, we analyze two common models of gene expression, each incorporating a simple splice mechanism that a pre-mRNA is spliced into two mature mRNA isoforms in a probabilistic manner. In the constitutive expression case, we show that the steady-state molecular numbers of two mature mRNA isoforms follow mutually independent Poisson distributions. In the bursting expression case, we demonstrate that the tail decay of the steady-state distribution for both mature mRNA isoforms that in general are not mutually independent can be characterized by the product of mean burst size and splicing probability. In both cases, we find that AS can efficiently modulate both the variability (measured by variance) and the noise level of the total mature mRNA, and in particular, the latter is always lower than the noise level of the pre-mRNA, implying that AS always reduces the noise. These results altogether reveal that AS is a mechanism of efficiently controlling the gene expression noise.

  16. Emerging roles of BRCA1 alternative splicing.

    PubMed

    Orban, T I; Olah, E

    2003-08-01

    Germline mutations of the BRCA1 gene predispose individuals mainly to the development of breast and/or ovarian cancer. However, the exact function of the gene is still unclear, although the encoded proteins are involved in various cellular processes, including transcriptional regulation and DNA repair pathways. Several BRCA1 splice variants are found in different tissues, but in spite of intense investigations, their regulation and possible functions are poorly understood at the moment. This review summarises current knowledge on the roles of these splice variants and the mechanisms responsible for their formation. Because alternative splicing is now widely accepted as an important source of genetic diversity, elucidating the functions of the BRCA1 splice variants would help in the understanding of the exact role(s) of this tumour suppressor. This should help to resolve the current paradox that, despite its seemingly vital cellular functions, mutations of this gene are associated with tissue specific tumour formation predominantly in the breast and the ovary.

  17. Alternative splicing of DNA damage response genes and gastrointestinal cancers.

    PubMed

    Rahmutulla, Bahityar; Matsushita, Kazuyuki; Nomura, Fumio

    2014-12-14

    Alternative splicing, which is a common phenomenon in mammalian genomes, is a fundamental process of gene regulation and contributes to great protein diversity. Alternative splicing events not only occur in the normal gene regulation process but are also closely related to certain diseases including cancer. In this review, we briefly demonstrate the concept of alternative splicing and DNA damage and describe the association of alternative splicing and cancer pathogenesis, focusing on the potential relationship of alternative splicing, DNA damage, and gastrointestinal cancers. We will also discuss whether alternative splicing leads to genetic instability, which is considered to be a driving force for tumorigenesis. Better understanding of the role and mechanism of alternative splicing in tumorigenesis may provide new directions for future cancer studies.

  18. Splicing-related genes are alternatively spliced upon changes in ambient temperatures in plants.

    PubMed

    Verhage, Leonie; Severing, Edouard I; Bucher, Johan; Lammers, Michiel; Busscher-Lange, Jacqueline; Bonnema, Guusje; Rodenburg, Nicole; Proveniers, Marcel C G; Angenent, Gerco C; Immink, Richard G H

    2017-01-01

    Plants adjust their development and architecture to small variations in ambient temperature. In a time in which temperatures are rising world-wide, the mechanism by which plants are able to sense temperature fluctuations and adapt to it, is becoming of special interest. By performing RNA-sequencing on two Arabidopsis accession and one Brassica species exposed to temperature alterations, we showed that alternative splicing is an important mechanism in ambient temperature sensing and adaptation. We found that amongst the differentially alternatively spliced genes, splicing related genes are enriched, suggesting that the splicing machinery itself is targeted for alternative splicing when temperature changes. Moreover, we showed that many different components of the splicing machinery are targeted for ambient temperature regulated alternative splicing. Mutant analysis of a splicing related gene that was differentially spliced in two of the genotypes showed an altered flowering time response to different temperatures. We propose a two-step mechanism where temperature directly influences alternative splicing of the splicing machinery genes, followed by a second step where the altered splicing machinery affects splicing of downstream genes involved in the adaptation to altered temperatures.

  19. Splicing-related genes are alternatively spliced upon changes in ambient temperatures in plants

    PubMed Central

    Bucher, Johan; Lammers, Michiel; Busscher-Lange, Jacqueline; Bonnema, Guusje; Rodenburg, Nicole; Proveniers, Marcel C. G.; Angenent, Gerco C.

    2017-01-01

    Plants adjust their development and architecture to small variations in ambient temperature. In a time in which temperatures are rising world-wide, the mechanism by which plants are able to sense temperature fluctuations and adapt to it, is becoming of special interest. By performing RNA-sequencing on two Arabidopsis accession and one Brassica species exposed to temperature alterations, we showed that alternative splicing is an important mechanism in ambient temperature sensing and adaptation. We found that amongst the differentially alternatively spliced genes, splicing related genes are enriched, suggesting that the splicing machinery itself is targeted for alternative splicing when temperature changes. Moreover, we showed that many different components of the splicing machinery are targeted for ambient temperature regulated alternative splicing. Mutant analysis of a splicing related gene that was differentially spliced in two of the genotypes showed an altered flowering time response to different temperatures. We propose a two-step mechanism where temperature directly influences alternative splicing of the splicing machinery genes, followed by a second step where the altered splicing machinery affects splicing of downstream genes involved in the adaptation to altered temperatures. PMID:28257507

  20. [Alternative splicing: a novel pharmacological target with wide therapeutic potential].

    PubMed

    Jeanteur, Philippe; Tazi, Jamal

    2005-05-01

    Alternative splicing is a process by which a single stretch of genomic DNA yields several mRNAs encoding different proteins. Once believed to be a marginal phenomenon, alternative splicing now appears to be widespread among higher organisms and to be behind a large repertoire of human diseases. It involves a flexible mechanism for selecting splice sites, based on regulatory sequences recognized by cognate trans-acting protein factors (stimulatory SR proteins, or their antagonists). This RNA-protein interaction provides two types of targets for therapeutic manipulation. Masking regulatory RNA sequences with an antisense strategy is the most obvious, and encouraging results are beginning to accrue. Our lab is currently developing an entirely new approach in which activating proteins are targeted by small chemical molecules. A large screening program has been conducted with the chemical library from the Curie Institute. Several molecules (all indole derivatives) were found to counter the stimulatory effects of individual activating proteins, and have been selected for further development.

  1. Common CYP2D6 polymorphisms affecting alternative splicing and transcription: long-range haplotypes with two regulatory variants modulate CYP2D6 activity.

    PubMed

    Wang, Danxin; Poi, Ming J; Sun, Xiaochun; Gaedigk, Andrea; Leeder, J Steven; Sadee, Wolfgang

    2014-01-01

    Cytochrome P450 2D6 (CYP2D6) is involved in the metabolism of 25% of clinically used drugs. Genetic polymorphisms cause substantial variation in CYP2D6 activity and serve as biomarkers guiding drug therapy. However, genotype-phenotype relationships remain ambiguous except for poor metabolizers carrying null alleles, suggesting the presence of yet unknown genetic variants. Searching for regulatory CYP2D6 polymorphisms, we find that a SNP defining the CYP2D6*2 allele, rs16947 [R296C, 17-60% minor allele frequency (MAF)], previously thought to convey normal activity, alters exon 6 splicing, thereby reducing CYP2D6 expression at least 2-fold. In addition, two completely linked SNPs (rs5758550/rs133333, MAF 13-42%) increase CYP2D6 transcription more than 2-fold, located in a distant downstream enhancer region (>100 kb) that interacts with the CYP2D6 promoter. In high linkage disequilibrium (LD) with each other, rs16947 and the enhancer SNPs form haplotypes that affect CYP2D6 enzyme activity in vivo. In a pediatric cohort of 164 individuals, rs16947 alone (minor haplotype frequency 28%) was associated with reduced CYP2D6 metabolic activity (measured as dextromethorphan/metabolite ratios), whereas rs5758550/rs133333 alone (frequency 3%) resulted in increased CYP2D6 activity, while haplotypes containing both rs16947 and rs5758550/rs133333 were similar to the wild-type. Other alleles used in biomarker panels carrying these variants such as CYP2D6*41 require re-evaluation of independent effects on CYP2D6 activity. The occurrence of two regulatory variants of high frequency and in high LD, residing on a long haplotype, highlights the importance of gene architecture, likely shaped by evolutionary selection pressures, in determining activity of encoded proteins.

  2. Alternative splicing of human cyclin E.

    PubMed

    Sewing, A; Rönicke, V; Bürger, C; Funk, M; Müller, R

    1994-02-01

    Cyclin E is a regulatory subunit of the cdc2-related protein kinase cdk2, which is activated shortly before S-phase entry, thus defining it as a G1 cyclin. We report here the existence of a 43 kDa splice variant of human cyclin E, termed cyclin Es, which lacks 49 amino acids within the cyclin box compared to the known 48 kDa cyclin E. Cyclin Es is expressed at approximately 1/10 of the level of full-length cyclin E in several cell lines analysed. The two cyclin E forms differ functionally in that cyclin E, but not cyclin Es, is able to complex with cdk2, to activate the histone H1, pRb and p107 in vitro kinase activity of cdk2 and to rescue a triple CLN mutation in S. cerevisiae. Cyclin Es is the first splice variant of a cell cycle regulatory protein to be described. Our findings also indicate that the cyclin box in cyclin E mediates the interaction with cdk2.

  3. Global regulation of alternative RNA splicing by the SR-rich protein RBM39.

    PubMed

    Mai, Sanyue; Qu, Xiuhua; Li, Ping; Ma, Qingjun; Cao, Cheng; Liu, Xuan

    2016-08-01

    RBM39 is a serine/arginine-rich RNA-binding protein that is highly homologous to the splicing factor U2AF65. However, the role of RBM39 in alternative splicing is poorly understood. In this study, RBM39-mediated global alternative splicing was investigated using RNA-Seq and genome-wide RBM39-RNA interactions were mapped via cross-linking and immunoprecipitation coupled with deep sequencing (CLIP-Seq) in wild-type and RBM39-knockdown MCF-7 cells. RBM39 was involved in the up- or down-regulation of the transcript levels of various genes. Hundreds of alternative splicing events regulated by endogenous RBM39 were identified. The majority of these events were cassette exons. Genes containing RBM39-regulated alternative exons were found to be linked to G2/M transition, cellular response to DNA damage, adherens junctions and endocytosis. CLIP-Seq analysis showed that the binding site of RBM39 was mainly in proximity to 5' and 3' splicing sites. Considerable RBM39 binding to mRNAs encoding proteins involved in translation was observed. Of particular importance, ~20% of the alternative splicing events that were significantly regulated by RBM39 were similarly regulated by U2AF65. RBM39 is extensively involved in alternative splicing of RNA and helps regulate transcript levels. RBM39 may modulate alternative splicing similarly to U2AF65 by either directly binding to RNA or recruiting other splicing factors, such as U2AF65. The current study offers a genome-wide view of RBM39's regulatory function in alternative splicing. RBM39 may play important roles in multiple cellular processes by regulating both alternative splicing of RNA molecules and transcript levels. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. The adaptive significance of unproductive alternative splicing in primates.

    PubMed

    Skandalis, Adonis; Frampton, Mark; Seger, Jon; Richards, Miriam H

    2010-10-01

    Alternative gene splicing is pervasive in metazoa, particularly in humans, where the majority of genes generate splice variant transcripts. Characterizing the biological significance of alternative transcripts is methodologically difficult since it is impractical to assess thousands of splice variants as to whether they actually encode proteins, whether these proteins are functional, or whether transcripts have a function independent of protein synthesis. Consequently, to elucidate the functional significance of splice variants and to investigate mechanisms underlying the fidelity of mRNA splicing, we used an indirect approach based on analyzing the evolutionary conservation of splice variants among species. Using DNA polymerase β as an indicator locus, we cloned and characterized the types and frequencies of transcripts generated in primary cell lines of five primate species. Overall, we found that in addition to the canonical DNA polymerase β transcript, there were 25 alternative transcripts generated, most containing premature terminating codons. We used a statistical method borrowed from community ecology to show that there is significant diversity and little conservation in alternative splicing patterns among species, despite high sequence similarity in the underlying genomic (exonic) sequences. However, the frequency of alternative splicing at this locus correlates well with life history parameters such as the maximal longevity of each species, indicating that the alternative splicing of unproductive splice variants may have adaptive significance, even if the specific RNA transcripts themselves have no function. These results demonstrate the validity of the phylogenetic conservation approach in elucidating the biological significance of alternative splicing.

  5. Widespread alternative and aberrant splicing revealed by lariat sequencing

    PubMed Central

    Stepankiw, Nicholas; Raghavan, Madhura; Fogarty, Elizabeth A.; Grimson, Andrew; Pleiss, Jeffrey A.

    2015-01-01

    Alternative splicing is an important and ancient feature of eukaryotic gene structure, the existence of which has likely facilitated eukaryotic proteome expansions. Here, we have used intron lariat sequencing to generate a comprehensive profile of splicing events in Schizosaccharomyces pombe, amongst the simplest organisms that possess mammalian-like splice site degeneracy. We reveal an unprecedented level of alternative splicing, including alternative splice site selection for over half of all annotated introns, hundreds of novel exon-skipping events, and thousands of novel introns. Moreover, the frequency of these events is far higher than previous estimates, with alternative splice sites on average activated at ∼3% the rate of canonical sites. Although a subset of alternative sites are conserved in related species, implying functional potential, the majority are not detectably conserved. Interestingly, the rate of aberrant splicing is inversely related to expression level, with lowly expressed genes more prone to erroneous splicing. Although we validate many events with RNAseq, the proportion of alternative splicing discovered with lariat sequencing is far greater, a difference we attribute to preferential decay of aberrantly spliced transcripts. Together, these data suggest the spliceosome possesses far lower fidelity than previously appreciated, highlighting the potential contributions of alternative splicing in generating novel gene structures. PMID:26261211

  6. Global regulation of alternative splicing by adenosine deaminase acting on RNA (ADAR)

    PubMed Central

    Solomon, Oz; Oren, Shirley; Safran, Michal; Deshet-Unger, Naamit; Akiva, Pinchas; Jacob-Hirsch, Jasmine; Cesarkas, Karen; Kabesa, Reut; Amariglio, Ninette; Unger, Ron; Rechavi, Gideon; Eyal, Eran

    2013-01-01

    Alternative mRNA splicing is a major mechanism for gene regulation and transcriptome diversity. Despite the extent of the phenomenon, the regulation and specificity of the splicing machinery are only partially understood. Adenosine-to-inosine (A-to-I) RNA editing of pre-mRNA by ADAR enzymes has been linked to splicing regulation in several cases. Here we used bioinformatics approaches, RNA-seq and exon-specific microarray of ADAR knockdown cells to globally examine how ADAR and its A-to-I RNA editing activity influence alternative mRNA splicing. Although A-to-I RNA editing only rarely targets canonical splicing acceptor, donor, and branch sites, it was found to affect splicing regulatory elements (SREs) within exons. Cassette exons were found to be significantly enriched with A-to-I RNA editing sites compared with constitutive exons. RNA-seq and exon-specific microarray revealed that ADAR knockdown in hepatocarcinoma and myelogenous leukemia cell lines leads to global changes in gene expression, with hundreds of genes changing their splicing patterns in both cell lines. This global change in splicing pattern cannot be explained by putative editing sites alone. Genes showing significant changes in their splicing pattern are frequently involved in RNA processing and splicing activity. Analysis of recently published RNA-seq data from glioblastoma cell lines showed similar results. Our global analysis reveals that ADAR plays a major role in splicing regulation. Although direct editing of the splicing motifs does occur, we suggest it is not likely to be the primary mechanism for ADAR-mediated regulation of alternative splicing. Rather, this regulation is achieved by modulating trans-acting factors involved in the splicing machinery. PMID:23474544

  7. Global regulation of alternative splicing by adenosine deaminase acting on RNA (ADAR).

    PubMed

    Solomon, Oz; Oren, Shirley; Safran, Michal; Deshet-Unger, Naamit; Akiva, Pinchas; Jacob-Hirsch, Jasmine; Cesarkas, Karen; Kabesa, Reut; Amariglio, Ninette; Unger, Ron; Rechavi, Gideon; Eyal, Eran

    2013-05-01

    Alternative mRNA splicing is a major mechanism for gene regulation and transcriptome diversity. Despite the extent of the phenomenon, the regulation and specificity of the splicing machinery are only partially understood. Adenosine-to-inosine (A-to-I) RNA editing of pre-mRNA by ADAR enzymes has been linked to splicing regulation in several cases. Here we used bioinformatics approaches, RNA-seq and exon-specific microarray of ADAR knockdown cells to globally examine how ADAR and its A-to-I RNA editing activity influence alternative mRNA splicing. Although A-to-I RNA editing only rarely targets canonical splicing acceptor, donor, and branch sites, it was found to affect splicing regulatory elements (SREs) within exons. Cassette exons were found to be significantly enriched with A-to-I RNA editing sites compared with constitutive exons. RNA-seq and exon-specific microarray revealed that ADAR knockdown in hepatocarcinoma and myelogenous leukemia cell lines leads to global changes in gene expression, with hundreds of genes changing their splicing patterns in both cell lines. This global change in splicing pattern cannot be explained by putative editing sites alone. Genes showing significant changes in their splicing pattern are frequently involved in RNA processing and splicing activity. Analysis of recently published RNA-seq data from glioblastoma cell lines showed similar results. Our global analysis reveals that ADAR plays a major role in splicing regulation. Although direct editing of the splicing motifs does occur, we suggest it is not likely to be the primary mechanism for ADAR-mediated regulation of alternative splicing. Rather, this regulation is achieved by modulating trans-acting factors involved in the splicing machinery.

  8. Tissue-specific alternative splicing of Tak1 is conserved in deuterostomes.

    PubMed

    Venables, Julian P; Vignal, Emmanuel; Baghdiguian, Stephen; Fort, Philippe; Tazi, Jamal

    2012-01-01

    Alternative splicing allows organisms to rapidly modulate protein functions to physiological changes and therefore represents a highly versatile adaptive process. We investigated the conservation of the evolutionary history of the "Fox" family of RNA-binding splicing factors (RBFOX) as well as the conservation of regulated alternative splicing of the genes they control. We found that the RBFOX proteins are conserved in all metazoans examined. In humans, Fox proteins control muscle-specific alternative splicing of many genes but despite the conservation of splicing factors, conservation of regulation of alternative splicing has never been demonstrated between man and nonvertebrate species. Therefore, we studied 40 known Fox-regulated human exons and found that 22 had a tissue-specific splicing pattern in muscle and heart. Of these, 11 were spliced in the same tissue-specific manner in mouse tissues and 4 were tissue-specifically spliced in muscle and heart of the frog Xenopus laevis. The inclusion of two of these alternative exons was also downregulated during tadpole development. Of the 40 in the starting set, the most conserved alternative splicing event was in the transforming growth factor (TGF) beta-activated kinase Tak1 (MAP3K7) as this was also muscle specific in urochordates and in Ambulacraria, the most ancient deuterostome clade. We found exclusion of the muscle-specific exon of Tak1 was itself under control of TGF beta in cell culture and consistently that TGF beta caused an upregulation of Fox2 (RBFOX2) expression. The alternative exon, which codes for an in-frame 27 amino acids between the kinase and known regulatory domain of TAK1, contains conserved features in all organisms including potential phosphorylation sites and likely has an important conserved function in TGF beta signaling and development. This study establishes that deuterostomes share a remarkable conserved physiological process that involves a splicing factor and expression of tissue

  9. MAASE: An alternative splicing database designed for supporting splicing microarray applications

    PubMed Central

    ZHENG, CHRISTINA L.; KWON, YOUNG-SOO; LI, HAI-RI; ZHANG, KUI; COUTINHO-MANSFIELD, GABRIELA; YANG, CANZHU; NAIR, T. MURLIDHARAN; GRIBSKOV, MICHAEL; FU, XIANG-DONG

    2005-01-01

    Alternative splicing is a prominent feature of higher eukaryotes. Understanding of the function of mRNA isoforms and the regulation of alternative splicing is a major challenge in the post-genomic era. The development of mRNA isoform sensitive microarrays, which requires precise splice-junction sequence information, is a promising approach. Despite the availability of a large number of mRNAs and ESTs in various databases and the efforts made to align transcript sequences to genomic sequences, existing alternative splicing databases do not offer adequate information in an appropriate format to aid in splicing array design. Here we describe our effort in constructing the Manually Annotated Alternatively Spliced Events (MAASE) database system, which is specifically designed to support splicing microarray applications. MAASE comprises two components: (1) a manual/computational annotation tool for the efficient extraction of critical sequence and functional information for alternative splicing events and (2) a user-friendly database of annotated events that allows convenient export of information to aid in microarray design and data analysis. We provide a detailed introduction and a step-by-step user guide to the MAASE database system to facilitate future large-scale annotation efforts, integration with other alternative splicing databases, and splicing array fabrication. PMID:16251387

  10. Impacts of Alternative Splicing Events on the Differentiation of Adipocytes

    PubMed Central

    Lin, Jung-Chun

    2015-01-01

    Alternative splicing was found to be a common phenomenon after the advent of whole transcriptome analyses or next generation sequencing. Over 90% of human genes were demonstrated to undergo at least one alternative splicing event. Alternative splicing is an effective mechanism to spatiotemporally expand protein diversity, which influences the cell fate and tissue development. The first focus of this review is to highlight recent studies, which demonstrated effects of alternative splicing on the differentiation of adipocytes. Moreover, use of evolving high-throughput approaches, such as transcriptome analyses (RNA sequencing), to profile adipogenic transcriptomes, is also addressed. PMID:26389882

  11. The influence of Argonaute proteins on alternative RNA splicing.

    PubMed

    Batsché, Eric; Ameyar-Zazoua, Maya

    2015-01-01

    Alternative splicing of precursor RNAs is an important process in multicellular species because it impacts several aspects of gene expression: from the increase of protein repertoire to the level of expression. A large body of evidences demonstrates that factors regulating chromatin and transcription impact the outcomes of alternative splicing. Argonaute (AGO) proteins were known to play key roles in the regulation of gene expression at the post-transcriptional level. More recently, their role in the nucleus of human somatic cells has emerged. Here, we will discuss some of the nuclear functions of AGO, with special emphasis on alternative splicing. The AGO-mediated modulation of alternative splicing is based on several properties of these proteins: their binding to transcripts on chromatin and their interactions with many proteins, especially histone tail-modifying enzymes, HP1γ and splicing factors. AGO proteins may favor a decrease in the RNA-polymerase II kinetics at actively transcribed genes leading to the modulation of alternative splicing decisions. They could also influence alternative splicing through their interaction with core components of the splicing machinery and several splicing factors. We will discuss the modes of AGO recruitment on chromatin at active genes. We suggest that long intragenic antisense transcripts (lincRNA) might be an important feature of genes containing splicing events regulated by AGO.

  12. Alternative splicing modulated by genetic variants demonstrates accelerated evolution regulated by highly conserved proteins

    PubMed Central

    Hsiao, Yun-Hua Esther; Bahn, Jae Hoon; Lin, Xianzhi; Chan, Tak-Ming; Wang, Rena; Xiao, Xinshu

    2016-01-01

    Identification of functional genetic variants and elucidation of their regulatory mechanisms represent significant challenges of the post-genomic era. A poorly understood topic is the involvement of genetic variants in mediating post-transcriptional RNA processing, including alternative splicing. Thus far, little is known about the genomic, evolutionary, and regulatory features of genetically modulated alternative splicing (GMAS). Here, we systematically identified intronic tag variants for genetic modulation of alternative splicing using RNA-seq data specific to cellular compartments. Combined with our previous method that identifies exonic tags for GMAS, this study yielded 622 GMAS exons. We observed that GMAS events are highly cell type independent, indicating that splicing-altering genetic variants could have widespread function across cell types. Interestingly, GMAS genes, exons, and single-nucleotide variants (SNVs) all demonstrated positive selection or accelerated evolution in primates. We predicted that GMAS SNVs often alter binding of splicing factors, with SRSF1 affecting the most GMAS events and demonstrating global allelic binding bias. However, in contrast to their GMAS targets, the predicted splicing factors are more conserved than expected, suggesting that cis-regulatory variation is the major driving force of splicing evolution. Moreover, GMAS-related splicing factors had stronger consensus motifs than expected, consistent with their susceptibility to SNV disruption. Intriguingly, GMAS SNVs in general do not alter the strongest consensus position of the splicing factor motif, except the more than 100 GMAS SNVs in linkage disequilibrium with polymorphisms reported by genome-wide association studies. Our study reports many GMAS events and enables a better understanding of the evolutionary and regulatory features of this phenomenon. PMID:26888265

  13. Genetic variations and alternative splicing: the Glioma associated oncogene 1, GLI1.

    PubMed

    Zaphiropoulos, Peter G

    2012-01-01

    Alternative splicing is a post-transcriptional regulatory process that is attaining stronger recognition as a modulator of gene expression. Alternative splicing occurs when the primary RNA transcript is differentially processed into more than one mature RNAs. This is the result of a variable definition/inclusion of the exons, the sequences that are excised from the primary RNA to form the mature RNAs. Consequently, RNA expression can generate a collection of differentially spliced RNAs, which may distinctly influence subsequent biological events, such as protein synthesis or other biomolecular interactions. Still the mechanisms that control exon definition and exon inclusion are not fully clarified. This mini-review highlights advances in this field as well as the impact of single nucleotide polymorphisms in affecting splicing decisions. The Glioma-associated oncogene 1, GLI1, is taken as an example in addressing the role of nucleotide substitutions for splicing regulation.

  14. Genetic variations and alternative splicing: the Glioma associated oncogene 1, GLI1

    PubMed Central

    Zaphiropoulos, Peter G.

    2012-01-01

    Alternative splicing is a post-transcriptional regulatory process that is attaining stronger recognition as a modulator of gene expression. Alternative splicing occurs when the primary RNA transcript is differentially processed into more than one mature RNAs. This is the result of a variable definition/inclusion of the exons, the sequences that are excised from the primary RNA to form the mature RNAs. Consequently, RNA expression can generate a collection of differentially spliced RNAs, which may distinctly influence subsequent biological events, such as protein synthesis or other biomolecular interactions. Still the mechanisms that control exon definition and exon inclusion are not fully clarified. This mini-review highlights advances in this field as well as the impact of single nucleotide polymorphisms in affecting splicing decisions. The Glioma-associated oncogene 1, GLI1, is taken as an example in addressing the role of nucleotide substitutions for splicing regulation. PMID:22833753

  15. The Arabidopsis splicing factor SR1 is regulated by alternative splicing.

    PubMed

    Lazar, G; Goodman, H M

    2000-03-01

    The serine-arginine (SR)-rich splicing factors play essential roles in general splicing and regulate alternative splice site utilization in a concentration-dependent manner. SR1 is a plant homologue of the human general/alternative splicing factor SF2/ASF. We report here that alternative splicing regulates SR1 itself. Of the five detected SR1 transcripts only one encodes the full-length protein, while the other four are different variants of the essential arginine-serine-rich domain. The data suggest that SR1 pre-mRNA could be committed to two alternate splicing pathways. One, dependent on the alternative utilization of competing 3' splice sites in intron 9, generates SR1, SR1B and SR1C. The other, regulated by suppression of intron 9 5' splice site utilization, generates SR1D and SR1E. The splicing pattern and molecular structure of SR1D indicates an evolutionary conservation of splicing-based regulation between plants and vertebrates and suggests that the various isoforms perform important functions. Results from transient gene expression assays indicate that alternative splicing is not an autoregulatory mechanism used to control the transcript level of the full-length protein. The ratio of SR1/SR1B transcripts, which are generated by alternative 3' splice site utilization in intron 9, is under temperature control. The temperature-dependent increase in SR1B/SR1 ratio suggests a role of SR1B in the adaptation to high-temperature environments. In addition, based on the regulated co-expression of SR1 transcripts, it is possible that some SR1 functions could be determined by the combinatorial action of the various isoforms.

  16. Functional association between promoter structure and transcript alternative splicing.

    PubMed

    Cramer, P; Pesce, C G; Baralle, F E; Kornblihtt, A R

    1997-10-14

    It has been assumed that constitutive and regulated splicing of RNA polymerase II transcripts depends exclusively on signals present in the RNA molecule. Here we show that changes in promoter structure strongly affect splice site selection. We investigated the splicing of the ED I exon, which encodes a facultative type III repeat of fibronectin, whose inclusion is regulated during development and in proliferative processes. We used an alternative splicing assay combined with promoter swapping to demonstrate that the extent of ED I splicing is dependent on the promoter structure from which the transcript originated and that this regulation is independent of the promoter strength. Thus, these results provide the first evidence for coupling between alternative splicing and promoter-specific transcription, which agrees with recent cytological and biochemical evidence of coordination between splicing and transcription.

  17. It's a bit over, is that ok? The subtle surplus from tandem alternative splicing.

    PubMed

    Szafranski, Karol; Kramer, Marcel

    2015-01-01

    Tandem alternative splice sites (TASS) form a defined class of alternative splicing and give rise to mRNA insertion/deletion variants with only small size differences. Previous work has confirmed evolutionary conservation of TASS elements while many cases show only low tissue specificity of isoform ratios. We pinpoint stochasticity and noise as important methodological issues for the dissection of TASS isoform patterns. Resolving such uncertainties, a recent report showed regulation in a cell culture system, with shifts of alternative splicing isoform ratios dependent on cell density. This novel type of regulation affects not only multiple TASS isoforms, but also other alternative splicing classes, in a concerted manner. Here, we discuss how specific regulatory network architectures may be realized through the novel regulation type and highlight the role of differential isoform functions as a key step in order to better understand the functional role of TASS.

  18. Regulation of Alternative Splicing in Vivo by Overexpression of Antagonistic Splicing Factors

    NASA Astrophysics Data System (ADS)

    Caceres, Javier F.; Stamm, Stefan; Helfman, David M.; Krainer, Adrian R.

    1994-09-01

    The opposing effects of SF2/ASF and heterogeneous nuclear ribonucleoprotein (hnRNP) A1 influence alternative splicing in vitro. SF2/ASF or hnRNP A1 complementary DNAs were transiently overexpressed in HeLa cells, and the effect on alternative splicing of several cotransfected reporter genes was measured. Increased expression of SF2/ASF activated proximal 5' splice sites, promoted inclusion of a neuron-specific exon, and prevented abnormal exon skipping. Increased expression of hnRNP A1 activated distal 5' splice sites. Therefore, variations in the intracellular levels of antagonistic splicing factors influence different modes of alternative splicing in vivo and may be a natural mechanism for tissue-specific or developmental regulation of gene expression.

  19. Unmasking alternative splicing inside protein-coding exons defines exitrons and their role in proteome plasticity

    PubMed Central

    Marquez, Yamile; Höpfler, Markus; Ayatollahi, Zahra; Barta, Andrea; Kalyna, Maria

    2015-01-01

    Alternative splicing (AS) diversifies transcriptomes and proteomes and is widely recognized as a key mechanism for regulating gene expression. Previously, in an analysis of intron retention events in Arabidopsis, we found unusual AS events inside annotated protein-coding exons. Here, we also identify such AS events in human and use these two sets to analyse their features, regulation, functional impact, and evolutionary origin. As these events involve introns with features of both introns and protein-coding exons, we name them exitrons (exonic introns). Though exitrons were detected as a subset of retained introns, they are clearly distinguishable, and their splicing results in transcripts with different fates. About half of the 1002 Arabidopsis and 923 human exitrons have sizes of multiples of 3 nucleotides (nt). Splicing of these exitrons results in internally deleted proteins and affects protein domains, disordered regions, and various post-translational modification sites, thus broadly impacting protein function. Exitron splicing is regulated across tissues, in response to stress and in carcinogenesis. Intriguingly, annotated intronless genes can be also alternatively spliced via exitron usage. We demonstrate that at least some exitrons originate from ancestral coding exons. Based on our findings, we propose a “splicing memory” hypothesis whereby upon intron loss imprints of former exon borders defined by vestigial splicing regulatory elements could drive the evolution of exitron splicing. Altogether, our studies show that exitron splicing is a conserved strategy for increasing proteome plasticity in plants and animals, complementing the repertoire of AS events. PMID:25934563

  20. Stochastic principles governing alternative splicing of RNA.

    PubMed

    Hu, Jianfei; Boritz, Eli; Wylie, William; Douek, Daniel C

    2017-09-01

    The dominance of the major transcript isoform relative to other isoforms from the same gene generated by alternative splicing (AS) is essential to the maintenance of normal cellular physiology. However, the underlying principles that determine such dominance remain unknown. Here, we analyzed the physical AS process and found that it can be modeled by a stochastic minimization process, which causes the scaled expression levels of all transcript isoforms to follow the same Weibull extreme value distribution. Surprisingly, we also found a simple equation to describe the median frequency of transcript isoforms of different dominance. This two-parameter Weibull model provides the statistical distribution of all isoforms of all transcribed genes, and reveals that previously unexplained observations concerning relative isoform expression derive from these principles.

  1. Cauliflower mosaic virus Transcriptome Reveals a Complex Alternative Splicing Pattern

    PubMed Central

    Bouton, Clément; Geldreich, Angèle; Ramel, Laëtitia; Ryabova, Lyubov A.; Dimitrova, Maria; Keller, Mario

    2015-01-01

    The plant pararetrovirus Cauliflower mosaic virus (CaMV) uses alternative splic-ing to generate several isoforms from its polycistronic pregenomic 35S RNA. This pro-cess has been shown to be essential for infectivity. Previous works have identified four splice donor sites and a single splice acceptor site in the 35S RNA 5’ region and sug-gested that the main role of CaMV splicing is to downregulate expression of open read-ing frames (ORFs) I and II. In this study, we show that alternative splicing is a conserved process among CaMV isolates. In Cabb B-JI and Cabb-S isolates, splicing frequently leads to different fusion between ORFs, particularly between ORF I and II. The corresponding P1P2 fusion proteins expressed in E. coli interact with viral proteins P2 and P3 in vitro. However, they are detected neither during infection nor upon transient expression in planta, which suggests rapid degradation after synthesis and no important biological role in the CaMV infectious cycle. To gain a better understanding of the functional relevance of 35S RNA alternative splicing in CaMV infectivity, we inactivated the previously described splice sites. All the splicing mutants were as pathogenic as the corresponding wild-type isolate. Through RT-PCR-based analysis we demonstrate that CaMV 35S RNA exhibits a complex splicing pattern, as we identify new splice donor and acceptor sites whose selection leads to more than thirteen 35S RNA isoforms in infected turnip plants. Inactivating splice donor or acceptor sites is not lethal for the virus, since disrupted sites are systematically rescued by the activation of cryptic and/or seldom used splice sites. Taken together, our data depict a conserved, complex and flexible process, involving multiple sites, that ensures splicing of 35S RNA. PMID:26162084

  2. Unfolding the mystery of alternative splicing through a unique method of in vivo selection.

    PubMed

    Singh, Ravindra N

    2007-05-01

    Alternative splicing of pre-messenger RNA (pre-mRNA) is a fundamental mechanism of gene regulation in higher eukaryotes. In addition to creating protein diversity, alternative splicing provides the safest mode of gene evolution. Of late, more and more forms of alternatively spliced transcripts (mRNAs) are being discovered for key genes. Some of the alternatively spliced transcripts are also associated with major human diseases. This has created a sense of urgency to find the methods by which regulation of alternative splicing of specific exons could be best understood. Here I review a powerful in vivo selection method that uses a combinatorial library of partially random sequences. Several advantages of this method include in vivo analysis of large sequences, identification of unique sequence motifs, determination of relative strength of splice sites and identification of long-distance interactions including role of RNA structures. This unique method could be applied to identify tissue-specific cis-elements. Similarly, the method is suitable to find cis-elements that become active in response to specific treatments of cells. Considering this unbiased method uses in vivo conditions, it has potential to identify critical regulatory elements as therapeutic targets for a growing number of splicing-associated diseases.

  3. Regulation of alternative splicing in human obesity loci.

    PubMed

    Kaminska, Dorota; Käkelä, Pirjo; Nikkola, Elina; Venesmaa, Sari; Ilves, Imre; Herzig, Karl-Heinz; Kolehmainen, Marjukka; Karhunen, Leila; Kuusisto, Johanna; Gylling, Helena; Pajukanta, Päivi; Laakso, Markku; Pihlajamäki, Jussi

    2016-10-01

    Multiple obesity susceptibility loci have been identified by genome-wide association studies, yet the mechanisms by which these loci influence obesity remain unclear. Alternative splicing could contribute to obesity by regulating the transcriptomic and proteomic diversity of genes in these loci. Based on a database search, 72 of the 136 genes at the 13 obesity loci encoded multiple protein isoforms. Thus, alternative splicing of these genes in adipose tissue samples was analyzed from the Metabolic Syndrome in Men population-based study and from two weight loss intervention studies (surgical and very low calorie diet). Alternative splicing was confirmed in 11 genes with PCR capillary electrophoresis in human subcutaneous adipose tissue. Interestingly, differential splicing of TRA2B, BAG6, and MSH5 was observed between lean individuals with normoglycemia and overweight individuals with type 2 diabetes. Of these genes, we detected fat depot-dependent splicing of TRA2B and BAG6 and weight loss-induced regulation of MSH5 splicing in the intervention studies. Finally, body mass index was a major determinant of TRA2B, BAG6, and MSH5 splicing in the combined data. This study provides evidence for alternative splicing in obesity loci, suggesting that alternative splicing at least in part mediates the obesity-associated risk in these loci. © 2016 The Obesity Society.

  4. Alternative Splicing of STAT3 Is Affected by RNA Editing.

    PubMed

    Goldberg, Lior; Abutbul-Amitai, Mor; Paret, Gideon; Nevo-Caspi, Yael

    2017-03-09

    A-to-I RNA editing, carried out by adenosine deaminase acting on RNA (ADAR) enzymes, is an epigenetic phenomenon of posttranscriptional modifications on pre-mRNA. RNA editing in intronic sequences may influence alternative splicing of flanking exons. We have previously shown that conditions that induce editing result in elevated expression of signal transducer and activator of transcription 3 (STAT3), preferentially the alternatively-spliced STAT3β isoform. Mechanisms regulating alternative splicing of STAT3 have not been elucidated. STAT3 undergoes A-to-I RNA editing in an intron residing in proximity to the alternatively spliced exon. We hypothesized that RNA editing plays a role in regulating alternative splicing toward STAT3β. In this study we extend our observation connecting RNA editing to the preferential induction of STAT3β expression. We study the involvement of ADAR1 in STAT3 editing and reveal the connection between editing and alternative splicing of STAT3. Deferoaxamine treatment caused the induction in STAT3 RNA editing and STAT3β expression. Silencing ADAR1 caused a decrease in STAT3 editing and expression with a preferential decrease in STAT3β. Cells transfected with a mutated minigene showed preferential splicing toward the STAT3β transcript. Editing in the STAT3 intron is performed by ADAR1 and affects STAT3 alternative splicing. These results suggest that RNA editing is one of the molecular mechanisms regulating the expression of STAT3β.

  5. Specific interactions between proteins implicated in splice site selection and regulated alternative splicing.

    PubMed

    Wu, J Y; Maniatis, T

    1993-12-17

    Specific recognition and pairing of the 5' and 3' splice sites are critical steps in pre-mRNA splicing. We report that the splicing factors SC35 and SF2/ASF specifically interact with both the integral U1 small nuclear ribonucleoprotein (snRNP U1-70K) and with the 35 kd subunit of the splicing factor U2AF (U2AF35). Previous studies indicated that the U1 snRNP binds specifically to the 5' splice site, while U2AF35-U2AF65 heterodimer binds to the 3' splice site. Together, these observations suggest that SC35 and other members of the SR family of splicing factors may function in splice site selection by acting as a bridge between components bound to the 5' and 3' splice sites. Interestingly, SC35, SF2/ASF, and U2AF35 also interact with the Drosophila splicing regulators Transformer (Tra) and Transformer-2 (Tra2), suggesting that protein-protein interactions mediated by SR proteins may also play an important role in regulating alternative splicing.

  6. Quaking and PTB control overlapping splicing regulatory networks during muscle cell differentiation.

    PubMed

    Hall, Megan P; Nagel, Roland J; Fagg, W Samuel; Shiue, Lily; Cline, Melissa S; Perriman, Rhonda J; Donohue, John Paul; Ares, Manuel

    2013-05-01

    Alternative splicing contributes to muscle development, but a complete set of muscle-splicing factors and their combinatorial interactions are unknown. Previous work identified ACUAA ("STAR" motif) as an enriched intron sequence near muscle-specific alternative exons such as Capzb exon 9. Mass spectrometry of myoblast proteins selected by the Capzb exon 9 intron via RNA affinity chromatography identifies Quaking (QK), a protein known to regulate mRNA function through ACUAA motifs in 3' UTRs. We find that QK promotes inclusion of Capzb exon 9 in opposition to repression by polypyrimidine tract-binding protein (PTB). QK depletion alters inclusion of 406 cassette exons whose adjacent intron sequences are also enriched in ACUAA motifs. During differentiation of myoblasts to myotubes, QK levels increase two- to threefold, suggesting a mechanism for QK-responsive exon regulation. Combined analysis of the PTB- and QK-splicing regulatory networks during myogenesis suggests that 39% of regulated exons are under the control of one or both of these splicing factors. This work provides the first evidence that QK is a global regulator of splicing during muscle development in vertebrates and shows how overlapping splicing regulatory networks contribute to gene expression programs during differentiation.

  7. RNA-dependent dynamic histone acetylation regulates MCL1 alternative splicing

    PubMed Central

    Khan, Dilshad H.; Gonzalez, Carolina; Cooper, Charlton; Sun, Jian-Min; Chen, Hou Yu; Healy, Shannon; Xu, Wayne; Smith, Karen T.; Workman, Jerry L.; Leygue, Etienne; Davie, James R.

    2014-01-01

    Histone deacetylases (HDACs) and lysine acetyltransferases (KATs) catalyze dynamic histone acetylation at regulatory and coding regions of transcribed genes. Highly phosphorylated HDAC2 is recruited within corepressor complexes to regulatory regions, while the nonphosphorylated form is associated with the gene body. In this study, we characterized the nonphosphorylated HDAC2 complexes recruited to the transcribed gene body and explored the function of HDAC-complex-mediated dynamic histone acetylation. HDAC1 and 2 were coimmunoprecipitated with several splicing factors, including serine/arginine-rich splicing factor 1 (SRSF1) which has roles in alternative splicing. The co-chromatin immunoprecipitation of HDAC1/2 and SRSF1 to the gene body was RNA-dependent. Inhibition of HDAC activity and knockdown of HDAC1, HDAC2 or SRSF1 showed that these proteins were involved in alternative splicing of MCL1. HDAC1/2 and KAT2B were associated with nascent pre-mRNA in general and with MCL1 pre-mRNA specifically. Inhibition of HDAC activity increased the occupancy of KAT2B and acetylation of H3 and H4 of the H3K4 methylated alternative MCL1 exon 2 nucleosome. Thus, nonphosphorylated HDAC1/2 is recruited to pre-mRNA by splicing factors to act at the RNA level with KAT2B and other KATs to catalyze dynamic histone acetylation of the MCL1 alternative exon and alter the splicing of MCL1 pre-mRNA. PMID:24234443

  8. Differential Impacts of Alternative Splicing Networks on Apoptosis

    PubMed Central

    Lin, Jung-Chun; Tsao, Mei-Fen; Lin, Ying-Ju

    2016-01-01

    Apoptosis functions as a common mechanism to eliminate unnecessary or damaged cells during cell renewal and tissue development in multicellular organisms. More than 200 proteins constitute complex networks involved in apoptotic regulation. Imbalanced expressions of apoptosis-related factors frequently lead to malignant diseases. The biological functions of several apoptotic factors are manipulated through alternative splicing mechanisms which expand gene diversity by generating discrete variants from one messenger RNA precursor. It is widely observed that alternatively-spliced variants encoded from apoptosis-related genes exhibit differential effects on apoptotic regulation. Alternative splicing events are meticulously regulated by the interplay between trans-splicing factors and cis-responsive elements surrounding the regulated exons. The major focus of this review is to highlight recent studies that illustrate the influences of alternative splicing networks on apoptotic regulation which participates in diverse cellular processes and diseases. PMID:27983653

  9. Alternative Splicing in Next Generation Sequencing Data of Saccharomyces cerevisiae

    PubMed Central

    Schreiber, Konrad; Csaba, Gergely; Haslbeck, Martin; Zimmer, Ralf

    2015-01-01

    mRNA splicing is required in about 4% of protein coding genes in Saccharomyces cerevisiae. The gene structure of those genes is simple, generally comprising two exons and one intron. In order to characterize the impact of alternative splicing on the S. cerevisiae transcriptome, we perform a systematic analysis of mRNA sequencing data. We find evidence of a pervasive use of alternative splice sites and detect several novel introns both within and outside protein coding regions. We also find a predominance of alternative splicing on the 3’ side of introns, a finding which is consistent with existing knowledge on conservation of exon-intron boundaries in S. cerevisiae. Some of the alternatively spliced transcripts allow for a translation into different protein products. PMID:26469855

  10. Connecting the dots: chromatin and alternative splicing in EMT.

    PubMed

    Warns, Jessica A; Davie, James R; Dhasarathy, Archana

    2016-02-01

    Nature has devised sophisticated cellular machinery to process mRNA transcripts produced by RNA Polymerase II, removing intronic regions and connecting exons together, to produce mature RNAs. This process, known as splicing, is very closely linked to transcription. Alternative splicing, or the ability to produce different combinations of exons that are spliced together from the same genomic template, is a fundamental means of regulating protein complexity. Similar to transcription, both constitutive and alternative splicing can be regulated by chromatin and its associated factors in response to various signal transduction pathways activated by external stimuli. This regulation can vary between different cell types, and interference with these pathways can lead to changes in splicing, often resulting in aberrant cellular states and disease. The epithelial to mesenchymal transition (EMT), which leads to cancer metastasis, is influenced by alternative splicing events of chromatin remodelers and epigenetic factors such as DNA methylation and non-coding RNAs. In this review, we will discuss the role of epigenetic factors including chromatin, chromatin remodelers, DNA methyltransferases, and microRNAs in the context of alternative splicing, and discuss their potential involvement in alternative splicing during the EMT process.

  11. Connecting the dots: chromatin and alternative splicing in EMT

    PubMed Central

    Warns, Jessica A.; Davie, James R.; Dhasarathy, Archana

    2015-01-01

    Nature has devised sophisticated cellular machinery to process mRNA transcripts produced by RNA Polymerase II, removing intronic regions and connecting exons together, to produce mature RNAs. This process, known as splicing, is very closely linked to transcription. Alternative splicing, or the ability to produce different combinations of exons that are spliced together from the same genomic template, is a fundamental means of regulating protein complexity. Similar to transcription, both constitutive and alternative splicing can be regulated by chromatin and its associated factors in response to various signal transduction pathways activated by external stimuli. This regulation can vary between different cell types, and interference with these pathways can lead to changes in splicing, often resulting in aberrant cellular states and disease. The epithelial to mesenchymal transition (EMT), which leads to cancer metastasis, is influenced by alternative splicing events of chromatin remodelers and epigenetic factors such as DNA methylation and non-coding RNAs. In this review, we will discuss the role of epigenetic factors including chromatin, chromatin remodelers, DNA methyltransferases and microRNAs in the context of alternative splicing, and discuss their potential involvement in alternative splicing during the EMT process. PMID:26291837

  12. TassDB: a database of alternative tandem splice sites.

    PubMed

    Hiller, Michael; Nikolajewa, Swetlana; Huse, Klaus; Szafranski, Karol; Rosenstiel, Philip; Schuster, Stefan; Backofen, Rolf; Platzer, Matthias

    2007-01-01

    Subtle alternative splice events at tandem splice sites are frequent in eukaryotes and substantially increase the complexity of transcriptomes and proteomes. We have developed a relational database, TassDB (TAndem Splice Site DataBase), which stores extensive data about alternative splice events at GYNGYN donors and NAGNAG acceptors. These splice events are of subtle nature since they mostly result in the insertion/deletion of a single amino acid or the substitution of one amino acid by two others. Currently, TassDB contains 114 554 tandem splice sites of eight species, 5209 of which have EST/mRNA evidence for alternative splicing. In addition, human SNPs that affect NAGNAG acceptors are annotated. The database provides a user-friendly interface to search for specific genes or for genes containing tandem splice sites with specific features as well as the possibility to download large datasets. This database should facilitate further experimental studies and large-scale bioinformatics analyses of tandem splice sites. The database is available at http://helios.informatik.uni-freiburg.de/TassDB/.

  13. Violating the splicing rules: TG dinucleotides function as alternative 3' splice sites in U2-dependent introns.

    PubMed

    Szafranski, Karol; Schindler, Stefanie; Taudien, Stefan; Hiller, Michael; Huse, Klaus; Jahn, Niels; Schreiber, Stefan; Backofen, Rolf; Platzer, Matthias

    2007-01-01

    Despite some degeneracy of sequence signals that govern splicing of eukaryotic pre-mRNAs, it is an accepted rule that U2-dependent introns exhibit the 3' terminal dinucleotide AG. Intrigued by anecdotal evidence for functional non-AG 3' splice sites, we carried out a human genome-wide screen. We identified TG dinucleotides functioning as alternative 3' splice sites in 36 human genes. The TG-derived splice variants were experimentally validated with a success rate of 92%. Interestingly, ratios of alternative splice variants are tissue-specific for several introns. TG splice sites and their flanking intron sequences are substantially conserved between orthologous vertebrate genes, even between human and frog, indicating functional relevance. Remarkably, TG splice sites are exclusively found as alternative 3' splice sites, never as the sole 3' splice site for an intron, and we observed a distance constraint for TG-AG splice site tandems. Since TGs splice sites are exclusively found as alternative 3' splice sites, the U2 spliceosome apparently accomplishes perfect specificity for 3' AGs at an early splicing step, but may choose 3' TGs during later steps. Given the tiny fraction of TG 3' splice sites compared to the vast amount of non-viable TGs, cis-acting sequence signals must significantly contribute to splice site definition. Thus, we consider TG-AG 3' splice site tandems as promising subjects for studies on the mechanisms of 3' splice site selection.

  14. Identifying splicing regulatory elements with de Bruijn graphs.

    PubMed

    Badr, Eman; Heath, Lenwood S

    2014-12-01

    Splicing regulatory elements (SREs) are short, degenerate sequences on pre-mRNA molecules that enhance or inhibit the splicing process via the binding of splicing factors, proteins that regulate the functioning of the spliceosome. Existing methods for identifying SREs in a genome are either experimental or computational. Here, we propose a formalism based on de Bruijn graphs that combines genomic structure, word count enrichment analysis, and experimental evidence to identify SREs found in exons. In our approach, SREs are not restricted to a fixed length (i.e., k-mers, for a fixed k). As a result, we identify 2001 putative exonic enhancers and 3080 putative exonic silencers for human genes, with lengths varying from 6 to 15 nucleotides. Many of the predicted SREs overlap with experimentally verified binding sites. Our model provides a novel method to predict variable length putative regulatory elements computationally for further experimental investigation.

  15. Cytoplasmic Drosha activity generated by alternative splicing

    PubMed Central

    Dai, Lisheng; Chen, Kevin; Youngren, Brenda; Kulina, Julia; Yang, Acong; Guo, Zhengyu; Li, Jin; Yu, Peng; Gu, Shuo

    2016-01-01

    RNase III enzyme Drosha interacts with DGCR8 to form the Microprocessor, initiating canonical microRNA (miRNA) maturation in the nucleus. Here, we re-evaluated where Drosha functions in cells using Drosha and/or DGCR8 knock out (KO) cells and cleavage reporters. Interestingly, a truncated Drosha mutant located exclusively in the cytoplasm cleaved pri-miRNA effectively in a DGCR8-dependent manner. In addition, we demonstrated that in vitro generated pri-miRNAs when transfected into cells could be processed to mature miRNAs in the cytoplasm. These results indicate the existence of cytoplasmic Drosha (c-Drosha) activity. Although a subset of endogenous pri-miRNAs become enriched in the cytoplasm of Drosha KO cells, it remains unclear whether pri-miRNA processing is the main function of c-Drosha. We identified two novel in-frame Drosha isoforms generated by alternative splicing in both HEK293T and HeLa cells. One isoform loses the putative nuclear localization signal, generating c-Drosha. Further analysis indicated that the c-Drosha isoform is abundant in multiple cell lines, dramatically variable among different human tissues and upregulated in multiple tumors, suggesting that c-Drosha plays a unique role in gene regulation. Our results reveal a new layer of regulation on the miRNA pathway and provide novel insights into the ever-evolving functions of Drosha. PMID:27471035

  16. Contextual fear conditioning induces differential alternative splicing.

    PubMed

    Poplawski, Shane G; Peixoto, Lucia; Porcari, Giulia S; Wimmer, Mathieu E; McNally, Anna G; Mizuno, Keiko; Giese, K Peter; Chatterjee, Snehajyoti; Koberstein, John N; Risso, Davide; Speed, Terence P; Abel, Ted

    2016-10-01

    The process of memory consolidation requires transcription and translation to form long-term memories. Significant effort has been dedicated to understanding changes in hippocampal gene expression after contextual fear conditioning. However, alternative splicing by differential transcript regulation during this time period has received less attention. Here, we use RNA-seq to determine exon-level changes in expression after contextual fear conditioning and retrieval. Our work reveals that a short variant of Homer1, Ania-3, is regulated by contextual fear conditioning. The ribosome biogenesis regulator Las1l, small nucleolar RNA Snord14e, and the RNA-binding protein Rbm3 also change specific transcript usage after fear conditioning. The changes in Ania-3 and Las1l are specific to either the new context or the context-shock association, while the changes in Rbm3 occur after context or shock only. Our analysis revealed novel transcript regulation of previously undetected changes after learning, revealing the importance of high throughput sequencing approaches in the study of gene expression changes after learning. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Evolutionary Character of Alternative Splicing in Plants

    PubMed Central

    Zhang, Chengjun; Yang, Hong; Yang, Huizhao

    2015-01-01

    Alternative splicing (AS) is one of the most important ways to enhance the functional diversity of genes. Huge amounts of data have been produced by microarray, expressed sequence tag, and RNA-seq, and plenty of methods have been developed specifically for this task. The most frequently asked questions in previous research were as follows. What is the content rate of AS genes among the whole gene set? How many AS types are presented in the genome, and which type is dominant? How about the conservation ability of AS among different species? Which kinds of isoforms from some genes have the environmental response to help individual adaptation? Based on this background, we collected analysis results from 17 species to try to map out the landscape of AS studies in plants. We have noted the shortages of previous results, and we appeal to all scientists working in the AS field to make a standard protocol so that analyses between different projects are comparable. PMID:26819552

  18. Lineage-specific splicing of a brain-enriched alternative exon promotes glioblastoma progression

    PubMed Central

    Ferrarese, Roberto; Harsh, Griffith R.; Yadav, Ajay K.; Bug, Eva; Maticzka, Daniel; Reichardt, Wilfried; Dombrowski, Stephen M.; Miller, Tyler E.; Masilamani, Anie P.; Dai, Fangping; Kim, Hyunsoo; Hadler, Michael; Scholtens, Denise M.; Yu, Irene L.Y.; Beck, Jürgen; Srinivasasainagendra, Vinodh; Costa, Fabrizio; Baxan, Nicoleta; Pfeifer, Dietmar; von Elverfeldt, Dominik; Backofen, Rolf; Weyerbrock, Astrid; Duarte, Christine W.; He, Xiaolin; Prinz, Marco; Chandler, James P.; Vogel, Hannes; Chakravarti, Arnab; Rich, Jeremy N.; Carro, Maria S.; Bredel, Markus

    2014-01-01

    Tissue-specific alternative splicing is critical for the emergence of tissue identity during development, yet the role of this process in malignant transformation is undefined. Tissue-specific splicing involves evolutionarily conserved, alternative exons that represent only a minority of the total alternative exons identified. Many of these conserved exons have functional features that influence signaling pathways to profound biological effect. Here, we determined that lineage-specific splicing of a brain-enriched cassette exon in the membrane-binding tumor suppressor annexin A7 (ANXA7) diminishes endosomal targeting of the EGFR oncoprotein, consequently enhancing EGFR signaling during brain tumor progression. ANXA7 exon splicing was mediated by the ribonucleoprotein PTBP1, which is normally repressed during neuronal development. PTBP1 was highly expressed in glioblastomas due to loss of a brain-enriched microRNA (miR-124) and to PTBP1 amplification. The alternative ANXA7 splicing trait was present in precursor cells, suggesting that glioblastoma cells inherit the trait from a potential tumor-initiating ancestor and that these cells exploit this trait through accumulation of mutations that enhance EGFR signaling. Our data illustrate that lineage-specific splicing of a tissue-regulated alternative exon in a constituent of an oncogenic pathway eliminates tumor suppressor functions and promotes glioblastoma progression. This paradigm may offer a general model as to how tissue-specific regulatory mechanisms can reprogram normal developmental processes into oncogenic ones. PMID:24865424

  19. Alternative splicing in angiogenesis: the vascular endothelial growth factor paradigm.

    PubMed

    Ladomery, Michael R; Harper, Steven J; Bates, David O

    2007-05-08

    Alternative splicing, first discovered in the 1970s, has emerged as one of the key generators of proteomic diversity. Not surprisingly, alternative splicing is increasingly linked to the etiology of cancer. This is illustrated by vascular endothelial growth factor (VEGF), the dominant angiogenic factor. Recently, an antiangiogenic family of VEGF isoforms was discovered, and termed VEGF(xxx)b. VEGF(xxx)b isoforms arise from an alternative 3' splice site in exon 8, and differ by a mere six amino acids at the C-terminus. These alternative six amino acids radically change the functional properties of VEGF. VEGF(xxx)b isoform expression is regulated in human tissues and development, and disregulated in many pathological states including cancer. Understanding what regulates VEGF(xxx)b alternative splicing, and therefore the balance of pro- and antiangiogenic isoforms is of great importance and will be explored in detail over the next few years.

  20. Designing oligo libraries taking alternative splicing into account

    NASA Astrophysics Data System (ADS)

    Shoshan, Avi; Grebinskiy, Vladimir; Magen, Avner; Scolnicov, Ariel; Fink, Eyal; Lehavi, David; Wasserman, Alon

    2001-06-01

    We have designed sequences for DNA microarrays and oligo libraries, taking alternative splicing into account. Alternative splicing is a common phenomenon, occurring in more than 25% of the human genes. In many cases, different splice variants have different functions, are expressed in different tissues or may indicate different stages of disease. When designing sequences for DNA microarrays or oligo libraries, it is very important to take into account the sequence information of all the mRNA transcripts. Therefore, when a gene has more than one transcript (as a result of alternative splicing, alternative promoter sites or alternative poly-adenylation sites), it is very important to take all of them into account in the design. We have used the LEADS transcriptome prediction system to cluster and assemble the human sequences in GenBank and design optimal oligonucleotides for all the human genes with a known mRNA sequence based on the LEADS predictions.

  1. Identification of common genetic variation that modulates alternative splicing.

    PubMed

    Hull, Jeremy; Campino, Susana; Rowlands, Kate; Chan, Man-Suen; Copley, Richard R; Taylor, Martin S; Rockett, Kirk; Elvidge, Gareth; Keating, Brendan; Knight, Julian; Kwiatkowski, Dominic

    2007-06-01

    Alternative splicing of genes is an efficient means of generating variation in protein function. Several disease states have been associated with rare genetic variants that affect splicing patterns. Conversely, splicing efficiency of some genes is known to vary between individuals without apparent ill effects. What is not clear is whether commonly observed phenotypic variation in splicing patterns, and hence potential variation in protein function, is to a significant extent determined by naturally occurring DNA sequence variation and in particular by single nucleotide polymorphisms (SNPs). In this study, we surveyed the splicing patterns of 250 exons in 22 individuals who had been previously genotyped by the International HapMap Project. We identified 70 simple cassette exon alternative splicing events in our experimental system; for six of these, we detected consistent differences in splicing pattern between individuals, with a highly significant association between splice phenotype and neighbouring SNPs. Remarkably, for five out of six of these events, the strongest correlation was found with the SNP closest to the intron-exon boundary, although the distance between these SNPs and the intron-exon boundary ranged from 2 bp to greater than 1,000 bp. Two of these SNPs were further investigated using a minigene splicing system, and in each case the SNPs were found to exert cis-acting effects on exon splicing efficiency in vitro. The functional consequences of these SNPs could not be predicted using bioinformatic algorithms. Our findings suggest that phenotypic variation in splicing patterns is determined by the presence of SNPs within flanking introns or exons. Effects on splicing may represent an important mechanism by which SNPs influence gene function.

  2. An alternative splicing program promotes adipose tissue thermogenesis

    PubMed Central

    Vernia, Santiago; Edwards, Yvonne JK; Han, Myoung Sook; Cavanagh-Kyros, Julie; Barrett, Tamera; Kim, Jason K; Davis, Roger J

    2016-01-01

    Alternative pre-mRNA splicing expands the complexity of the transcriptome and controls isoform-specific gene expression. Whether alternative splicing contributes to metabolic regulation is largely unknown. Here we investigated the contribution of alternative splicing to the development of diet-induced obesity. We found that obesity-induced changes in adipocyte gene expression include alternative pre-mRNA splicing. Bioinformatics analysis associated part of this alternative splicing program with sequence specific NOVA splicing factors. This conclusion was confirmed by studies of mice with NOVA deficiency in adipocytes. Phenotypic analysis of the NOVA-deficient mice demonstrated increased adipose tissue thermogenesis and improved glycemia. We show that NOVA proteins mediate a splicing program that suppresses adipose tissue thermogenesis. Together, these data provide quantitative analysis of gene expression at exon-level resolution in obesity and identify a novel mechanism that contributes to the regulation of adipose tissue function and the maintenance of normal glycemia. DOI: http://dx.doi.org/10.7554/eLife.17672.001 PMID:27635635

  3. Myc and SAGA rewire an alternative splicing network during early somatic cell reprogramming

    PubMed Central

    Hirsch, Calley L.; Coban Akdemir, Zeynep; Wang, Li; Jayakumaran, Gowtham; Trcka, Dan; Weiss, Alexander; Hernandez, J. Javier; Pan, Qun; Han, Hong; Xu, Xueping; Xia, Zheng; Salinger, Andrew P.; Wilson, Marenda; Vizeacoumar, Frederick; Datti, Alessandro; Li, Wei; Cooney, Austin J.; Barton, Michelle C.; Blencowe, Benjamin J.

    2015-01-01

    Embryonic stem cells are maintained in a self-renewing and pluripotent state by multiple regulatory pathways. Pluripotent-specific transcriptional networks are sequentially reactivated as somatic cells reprogram to achieve pluripotency. How epigenetic regulators modulate this process and contribute to somatic cell reprogramming is not clear. Here we performed a functional RNAi screen to identify the earliest epigenetic regulators required for reprogramming. We identified components of the SAGA histone acetyltransferase complex, in particular Gcn5, as critical regulators of reprogramming initiation. Furthermore, we showed in mouse pluripotent stem cells that Gcn5 strongly associates with Myc and that, upon initiation of somatic reprogramming, Gcn5 and Myc form a positive feed-forward loop that activates a distinct alternative splicing network and the early acquisition of pluripotency-associated splicing events. These studies expose a Myc–SAGA pathway that drives expression of an essential alternative splicing regulatory network during somatic cell reprogramming. PMID:25877919

  4. Myc and SAGA rewire an alternative splicing network during early somatic cell reprogramming.

    PubMed

    Hirsch, Calley L; Coban Akdemir, Zeynep; Wang, Li; Jayakumaran, Gowtham; Trcka, Dan; Weiss, Alexander; Hernandez, J Javier; Pan, Qun; Han, Hong; Xu, Xueping; Xia, Zheng; Salinger, Andrew P; Wilson, Marenda; Vizeacoumar, Frederick; Datti, Alessandro; Li, Wei; Cooney, Austin J; Barton, Michelle C; Blencowe, Benjamin J; Wrana, Jeffrey L; Dent, Sharon Y R

    2015-04-15

    Embryonic stem cells are maintained in a self-renewing and pluripotent state by multiple regulatory pathways. Pluripotent-specific transcriptional networks are sequentially reactivated as somatic cells reprogram to achieve pluripotency. How epigenetic regulators modulate this process and contribute to somatic cell reprogramming is not clear. Here we performed a functional RNAi screen to identify the earliest epigenetic regulators required for reprogramming. We identified components of the SAGA histone acetyltransferase complex, in particular Gcn5, as critical regulators of reprogramming initiation. Furthermore, we showed in mouse pluripotent stem cells that Gcn5 strongly associates with Myc and that, upon initiation of somatic reprogramming, Gcn5 and Myc form a positive feed-forward loop that activates a distinct alternative splicing network and the early acquisition of pluripotency-associated splicing events. These studies expose a Myc-SAGA pathway that drives expression of an essential alternative splicing regulatory network during somatic cell reprogramming.

  5. Temporal regulation of adenovirus major late alternative RNA splicing.

    PubMed

    Akusjarvi, Goran

    2008-05-01

    Adenovirus makes extensive use of alternative RNA splicing to produce a complex set of spliced mRNAs during replication. The accumulation of viral mRNAs is subjected to a temporal regulation, a mechanism that ensures that proteins that are needed at certain stages of the virus life cycle are produced in a timely fashion. The complex interactions between the virus and the host cell RNA splicing machinery has been studied in detail during the last decade. These studies have resulted in the characterization of two viral proteins, E4-ORF4 and L4-33K, that adenovirus uses to remodel the host cell RNA splicing machinery. Here I will review the current knowledge of how mRNA expression from the adenovirus major late transcription unit is controlled with a particular emphasis on how cis-acting sequence element, trans-acting factors and mechanisms regulating adenovirus major late L1 alternative RNA splicing is controlled.

  6. A long noncoding way to alternative splicing in plant development.

    PubMed

    Kornblihtt, Alberto R

    2014-07-28

    In this issue of Developmental Cell, Bardou et al. (2014) elucidate how long, highly structured noncoding RNAs control alternative splicing regulators that specifically mediate the action of the hormone auxin in the promotion of lateral root growth in Arabidopsis.

  7. Regulation of Alternative Splicing in Tumor Metastasis

    DTIC Science & Technology

    2001-10-01

    2001. Multiple interactions between SRm160 and SR family proteins in enhancer-dependent splicing and development of Caenorhabditis elegans . 11... Caenorhabditis 36 Lorson, C.L. et al. (1999) A single nucleotide in the Biol. 77, 277-291 elegans . Nature 402, 835-838 SMN gene regulates splicing and is...terminate (Birse et al., 1998; Proudfoot, 2000), mutation of the poly(A) signal resulted in the accumulation to high levels in the nuclear fraction of

  8. Computational Analysis of an Evolutionarily Conserved VertebrateMuscle Alternative Splicing Program

    SciTech Connect

    Das, Debopriya; Clark, Tyson A.; Schweitzer, Anthony; Marr,Henry; Yamamoto, Miki L.; Parra, Marilyn K.; Arribere, Josh; Minovitsky,Simon; Dubchak, Inna; Blume, John E.; Conboy, John G.

    2006-06-15

    A novel exon microarray format that probes gene expression with single exon resolution was employed to elucidate critical features of a vertebrate muscle alternative splicing program. A dataset of 56 microarray-defined, muscle-enriched exons and their flanking introns were examined computationally in order to investigate coordination of the muscle splicing program. Candidate intron regulatory motifs were required to meet several stringent criteria: significant over-representation near muscle-enriched exons, correlation with muscle expression, and phylogenetic conservation among genomes of several vertebrate orders. Three classes of regulatory motifs were identified in the proximal downstream intron, within 200nt of the target exons: UGCAUG, a specific binding site for Fox-1 related splicing factors; ACUAAC, a novel branchpoint-like element; and UG-/UGC-rich elements characteristic of binding sites for CELF splicing factors. UGCAUG was remarkably enriched, being present in nearly one-half of all cases. These studies suggest that Fox and CELF splicing factors play a major role in enforcing the muscle-specific alternative splicing program, facilitating expression of a set of unique isoforms of cytoskeletal proteins that are critical to muscle cell differentiation. Supplementary materials: There are four supplementary tables and one supplementary figure. The tables provide additional detailed information concerning the muscle-enriched datasets, and about over-represented oligonucleotide sequences in the flanking introns. The supplementary figure shows RT-PCR data confirming the muscle-enriched expression of exons predicted from the microarray analysis.

  9. Exploration of alternative splicing events in ten different grapevine cultivars.

    PubMed

    Potenza, Emilio; Racchi, Milvia Luisa; Sterck, Lieven; Coller, Emanuela; Asquini, Elisa; Tosatto, Silvio C E; Velasco, Riccardo; Van de Peer, Yves; Cestaro, Alessandro

    2015-09-17

    The complex dynamics of gene regulation in plants are still far from being fully understood. Among many factors involved, alternative splicing (AS) in particular is one of the least well documented. For many years, AS has been considered of less relevant in plants, especially when compared to animals, however, since the introduction of next generation sequencing techniques the number of plant genes believed to be alternatively spliced has increased exponentially. Here, we performed a comprehensive high-throughput transcript sequencing of ten different grapevine cultivars, which resulted in the first high coverage atlas of the grape berry transcriptome. We also developed findAS, a software tool for the analysis of alternatively spliced junctions. We demonstrate that at least 44% of multi-exonic genes undergo AS and a large number of low abundance splice variants is present within the 131.622 splice junctions we have annotated from Pinot noir. Our analysis shows that ~70% of AS events have relatively low expression levels, furthermore alternative splice sites seem to be enriched near the constitutive ones in some extent showing the noise of the splicing mechanisms. However, AS seems to be extensively conserved among the 10 cultivars.

  10. [Alternative mRNA splicing, pathology and molecular therapeutics].

    PubMed

    Corcos, Laurent; Solier, Stéphanie

    2005-03-01

    Pre-mRNA splicing operates towards at least 95 % of the transcript pool. It is subjected to a large number of variations, collectively regrouped under the term of alternative mRNA splicing, which occurs, on average, 6 to 8 times per pre-mRNA molecule. Consequently, many more proteins may be encoded from a single gene, which may satisfy a physiological need, or mark a pathological adaptation. The identification of mutations in sequences required for splicing, both constitutive and alternative, or for their control, has permitted to determine the causes of qualitative or quantitative variations in transcript levels associated with inherited diseases or cancer development. A number of molecular approaches have been undertaken to try to compensate for the effect of deleterious splicing mutations and to restore, at least in part, sufficient amounts of either the normal or a surrogate transcript. These include overexpression of splicing proteins, improvement of their activity by post-translational modification, splice-site increased or decreased usage, and RNA-mediated trans-splicing. Using such approaches, phenotypic improvements have been obtained in animal models, carrying new hopes for the development of therapeutic strategies aimed at correcting both inherited and acquired diseases that involve pre-mRNA splicing defects.

  11. AltTrans: transcript pattern variants annotated for both alternative splicing and alternative polyadenylation.

    PubMed

    Le Texier, Vincent; Riethoven, Jean-Jack; Kumanduri, Vasudev; Gopalakrishnan, Chellappa; Lopez, Fabrice; Gautheret, Daniel; Thanaraj, Thangavel Alphonse

    2006-03-23

    The three major mechanisms that regulate transcript formation involve the selection of alternative sites for transcription start (TS), splicing, and polyadenylation. Currently there are efforts that collect data & annotation individually for each of these variants. It is important to take an integrated view of these data sets and to derive a data set of alternate transcripts along with consolidated annotation. We have been developing in the past computational pipelines that generate value-added data at genome-scale on individual variant types; these include AltSplice on splicing and AltPAS on polyadenylation. We now extend these pipelines and integrate the resultant data sets to facilitate an integrated view of the contributions from splicing and polyadenylation in the formation of transcript variants. The AltSplice pipeline examines gene-transcript alignments and delineates alternative splice events and splice patterns; this pipeline is extended as AltTrans to delineate isoform transcript patterns for each of which both introns/exons and 'terminating' polyA site are delineated; EST/mRNA sequences that qualify the transcript pattern confirm both the underlying splicing and polyadenylation. The AltPAS pipeline examines gene-transcript alignments and delineates all potential polyA sites irrespective of underlying splicing patterns. Resultant polyA sites from both AltTrans and AltPAS are merged. The generated database reports data on alternative splicing, alternative polyadenylation and the resultant alternate transcript patterns; the basal data is annotated for various biological features. The data (named as integrated AltTrans data) generated for both the organisms of human and mouse is made available through the Alternate Transcript Diversity web site at http://www.ebi.ac.uk/atd/. The reported data set presents alternate transcript patterns that are annotated for both alternative splicing and alternative polyadenylation. Results based on current transcriptome data

  12. AltTrans: Transcript pattern variants annotated for both alternative splicing and alternative polyadenylation

    PubMed Central

    Le Texier, Vincent; Riethoven, Jean-Jack; Kumanduri, Vasudev; Gopalakrishnan, Chellappa; Lopez, Fabrice; Gautheret, Daniel; Thanaraj, Thangavel Alphonse

    2006-01-01

    Background The three major mechanisms that regulate transcript formation involve the selection of alternative sites for transcription start (TS), splicing, and polyadenylation. Currently there are efforts that collect data & annotation individually for each of these variants. It is important to take an integrated view of these data sets and to derive a data set of alternate transcripts along with consolidated annotation. We have been developing in the past computational pipelines that generate value-added data at genome-scale on individual variant types; these include AltSplice on splicing and AltPAS on polyadenylation. We now extend these pipelines and integrate the resultant data sets to facilitate an integrated view of the contributions from splicing and polyadenylation in the formation of transcript variants. Description The AltSplice pipeline examines gene-transcript alignments and delineates alternative splice events and splice patterns; this pipeline is extended as AltTrans to delineate isoform transcript patterns for each of which both introns/exons and 'terminating' polyA site are delineated; EST/mRNA sequences that qualify the transcript pattern confirm both the underlying splicing and polyadenylation. The AltPAS pipeline examines gene-transcript alignments and delineates all potential polyA sites irrespective of underlying splicing patterns. Resultant polyA sites from both AltTrans and AltPAS are merged. The generated database reports data on alternative splicing, alternative polyadenylation and the resultant alternate transcript patterns; the basal data is annotated for various biological features. The data (named as integrated AltTrans data) generated for both the organisms of human and mouse is made available through the Alternate Transcript Diversity web site at . Conclusion The reported data set presents alternate transcript patterns that are annotated for both alternative splicing and alternative polyadenylation. Results based on current

  13. Alternative splicing regulation of APP exon 7 by RBFox proteins.

    PubMed

    Alam, Shafiul; Suzuki, Hitoshi; Tsukahara, Toshifumi

    2014-12-01

    RBFox proteins are well-known alternative splicing regulators. We have shown previously that during neuronal differentiation of P19 cells induced by all-trans retinoic acid and cell aggregation, RBFox1 shows markedly increased temporal expression. To find its key splicing regulation, we examined the effect of RBFox1 on 33 previously reported and validated neuronal splicing events of P19 cells. We observed that alternative splicing of three genes, specifically, amyloid precursor protein (APP), disks large homolog 3 (DLG3), and G protein, alpha activating activity polypeptide O (GNAO1), was altered by transient RBFox1 expression in HEK293 and HeLa cells. Moreover, an RBFox1 mutant (RBFox1FA) that was unable to bind the target RNA sequence ((U)GCAUG) did not induce these splicing events. APP generates amyloid beta peptides that are involved in the pathology of Alzheimer's disease, and therefore we examined APP alternative splicing regulation by RBFox1 and other splicing regulators. Our results indicated that RBFox proteins promote the skipping of APP exon 7, but not the inclusion of exon 8. We made APP6789 minigenes and observed that two (U)GCAUG sequences, located upstream of exon 7 and in exon 7, functioned to induce skipping of exon 7 by RBFox proteins. Overall, RBFox proteins may shift APP from exon 7 containing isoforms, APP770 and APP751, toward the exon 7 lacking isoform, APP695, which is predominant in neural tissues.

  14. Alternative Splicing of Type II Procollagen: IIB or not IIB?

    PubMed Central

    McAlinden, Audrey

    2015-01-01

    Over two decades ago, two isoforms of the type II procollagen gene (COL2A1) were discovered. These isoforms, named IIA and IIB, are generated in a developmentally-regulated manner by alternative splicing of exon 2. Chondroprogenitor cells synthesize predominantly IIA isoforms (containing exon 2) while differentiated chondrocytes produce mainly IIB transcripts (devoid of exon 2). Importantly, this IIA-to-IIB alternative splicing switch occurs only during chondrogenesis. More recently, two other isoforms have been reported (IIC and IID) that also involve splicing of exon 2; these findings highlight the complexities involving regulation of COL2A1 expression. The biological significance of why different isoforms of COL2A1 exist within the context of skeletal development and maintenance is still not completely understood. This review will provide current knowledge on COL2A1 isoform expression during chondrocyte differentiation and what is known about some of the mechanisms that control exon 2 alternative splicing. Utilization of mouse models to address the biological significance of Col2a1 alternative splicing in vivo will also be discussed. From the knowledge acquired to date, some new questions and concepts are now being proposed on the importance of Col2a1 alternative splicing in regulating extracellular matrix assembly and how this may subsequently affect cartilage and endochondral bone quality and function. PMID:24669942

  15. Effects of airborne particulate matter on alternative pre-mRNA splicing in colon cancer cells.

    PubMed

    Buggiano, Valeria; Petrillo, Ezequiel; Alló, Mariano; Lafaille, Celina; Redal, María Ana; Alghamdi, Mansour A; Khoder, Mamdouh I; Shamy, Magdy; Muñoz, Manuel J; Kornblihtt, Alberto R

    2015-07-01

    Alternative pre-mRNA splicing plays key roles in determining tissue- and species-specific cell differentiation as well as in the onset of hereditary disease and cancer, being controlled by multiple post- and co-transcriptional regulatory mechanisms. We report here that airborne particulate matter, resulting from industrial pollution, inhibits expression and specifically affects alternative splicing at the 5' untranslated region of the mRNA encoding the bone morphogenetic protein BMP4 in human colon cells in culture. These effects are consistent with a previously reported role for BMP4 in preventing colon cancer development, suggesting that ingestion of particulate matter could contribute to the onset of colon cell proliferation. We also show that the underlying mechanism might involve changes in transcriptional elongation. This is the first study to demonstrate that particulate matter causes non-pleiotropic changes in alternative splicing.

  16. [Alternative splicing regulation: implications in cancer diagnosis and treatment].

    PubMed

    Martínez-Montiel, Nancy; Rosas-Murrieta, Nora; Martínez-Contreras, Rebeca

    2015-04-08

    The accurate expression of the genetic information is regulated by processes like mRNA splicing, proposed after the discoveries of Phil Sharp and Richard Roberts, who demonstrated the existence of intronic sequences, present in almost every structural eukaryotic gene, which should be precisely removed. This intron removal is called "splicing", which generates different proteins from a single mRNA, with different or even antagonistic functions. We currently know that alternative splicing is the most important source of protein diversity, given that 70% of the human genes undergo splicing and that mutations causing defects in this process could originate up to 50% of genetic diseases, including cancer. When these defects occur in genes involved in cell adhesion, proliferation and cell cycle regulation, there is an impact on cancer progression, rising the opportunity to diagnose and treat some types of cancer according to a particular splicing profile.

  17. Alternative pre-mRNA splicing switches modulate gene expression in late erythropoiesis

    SciTech Connect

    Yamamoto, Miki L.; Clark, Tyson A.; Gee, Sherry L.; Kang, Jeong-Ah; Schweitzer, Anthony C.; Wickrema, Amittha; Conboy, John G.

    2009-02-03

    Differentiating erythroid cells execute a unique gene expression program that insures synthesis of the appropriate proteome at each stage of maturation. Standard expression microarrays provide important insight into erythroid gene expression but cannot detect qualitative changes in transcript structure, mediated by RNA processing, that alter structure and function of encoded proteins. We analyzed stage-specific changes in the late erythroid transcriptome via use of high-resolution microarrays that detect altered expression of individual exons. Ten differentiation-associated changes in erythroblast splicing patterns were identified, including the previously known activation of protein 4.1R exon 16 splicing. Six new alternative splicing switches involving enhanced inclusion of internal cassette exons were discovered, as well as 3 changes in use of alternative first exons. All of these erythroid stage-specific splicing events represent activated inclusion of authentic annotated exons, suggesting they represent an active regulatory process rather than a general loss of splicing fidelity. The observation that 3 of the regulated transcripts encode RNA binding proteins (SNRP70, HNRPLL, MBNL2) may indicate significant changes in the RNA processing machinery of late erythroblasts. Together, these results support the existence of a regulated alternative pre-mRNA splicing program that is critical for late erythroid differentiation.

  18. CUG-BP1 regulates RyR1 ASI alternative splicing in skeletal muscle atrophy.

    PubMed

    Tang, Yinglong; Wang, Huiwen; Wei, Bin; Guo, Yuting; Gu, Lei; Yang, Zhiguang; Zhang, Qing; Wu, Yanyun; Yuan, Qi; Zhao, Gang; Ji, Guangju

    2015-11-04

    RNA binding protein is identified as an important mediator of aberrant alternative splicing in muscle atrophy. The altered splicing of calcium channels, such as ryanodine receptors (RyRs), plays an important role in impaired excitation-contraction (E-C) coupling in muscle atrophy; however, the regulatory mechanisms of ryanodine receptor 1 (RyR1) alternative splicing leading to skeletal muscle atrophy remains to be investigated. In this study we demonstrated that CUG binding protein 1 (CUG-BP1) was up-regulated and the alternative splicing of RyR1 ASI (exon70) was aberrant during the process of neurogenic muscle atrophy both in human patients and mouse models. The gain and loss of function experiments in vivo demonstrated that altered splicing pattern of RyR1 ASI was directly mediated by an up-regulated CUG-BP1 function. Furthermore, we found that CUG-BP1 affected the calcium release activity in single myofibers and the extent of atrophy was significantly reduced upon gene silencing of CUG-BP1 in atrophic muscle. These findings improve our understanding of calcium signaling related biological function of CUG-BP1 in muscle atrophy. Thus, we provide an intriguing perspective of involvement of mis-regulated RyR1 splicing in muscular disease.

  19. Histone H3 lysine 36 methylation affects temperature-induced alternative splicing and flowering in plants.

    PubMed

    Pajoro, A; Severing, E; Angenent, G C; Immink, R G H

    2017-06-01

    Global warming severely affects flowering time and reproductive success of plants. Alternative splicing of pre-messenger RNA (mRNA) is an important mechanism underlying ambient temperature-controlled responses in plants, yet its regulation is poorly understood. An increase in temperature promotes changes in plant morphology as well as the transition from the vegetative to the reproductive phase in Arabidopsis thaliana via changes in splicing of key regulatory genes. Here we investigate whether a particular histone modification affects ambient temperature-induced alternative splicing and flowering time. We use a genome-wide approach and perform RNA-sequencing (RNA-seq) analyses and histone H3 lysine 36 tri-methylation (H3K36me3) chromatin immunoprecipitation sequencing (ChIP-seq) in plants exposed to different ambient temperatures. Analysis and comparison of these datasets reveal that temperature-induced differentially spliced genes are enriched in H3K36me3. Moreover, we find that reduction of H3K36me3 deposition causes alteration in temperature-induced alternative splicing. We also show that plants with mutations in H3K36me3 writers, eraser, or readers have altered high ambient temperature-induced flowering. Our results show a key role for the histone mark H3K36me3 in splicing regulation and plant plasticity to fluctuating ambient temperature. Our findings open new perspectives for the breeding of crops that can better cope with environmental changes due to climate change.

  20. Conserved RNA cis-elements regulate alternative splicing of Lepidopteran doublesex.

    PubMed

    Wang, Xiu-Ye; Zheng, Zeng-Zhang; Song, Hong-Sheng; Xu, Yong-Zhen

    2014-01-01

    propose that the 5' splice sites at intron 3 are weak, resulting in multiple alternative splicing events in intron 3 of female Lepidoptera dsx. Activation of the 5' splice site requires regulatory cis-elements in exons 3 for female-specific splicing of Lepidoptera dsx.

  1. Re-splicing of mature mRNA in cancer cells promotes activation of distant weak alternative splice sites

    PubMed Central

    Kameyama, Toshiki; Suzuki, Hitoshi; Mayeda, Akila

    2012-01-01

    Transcripts of the human tumor susceptibility gene 101 (TSG101) are aberrantly spliced in many cancers. A major aberrant splicing event on the TSG101 pre-mRNA involves joining of distant alternative 5′ and 3′ splice sites within exon 2 and exon 9, respectively, resulting in the extensive elimination of the mRNA. The estimated strengths of the alternative splice sites are much lower than those of authentic splice sites. We observed that the equivalent aberrant mRNA could be generated from an intron-less TSG101 gene expressed ectopically in breast cancer cells. Remarkably, we identified a pathway-specific endogenous lariat RNA consisting solely of exonic sequences, predicted to be generated by a re-splicing between exon 2 and exon 9 on the spliced mRNA. Our results provide evidence for a two-step splicing pathway in which the initial constitutive splicing removes all 14 authentic splice sites, thereby bringing the weak alternative splice sites into close proximity. We also demonstrate that aberrant multiple-exon skipping of the fragile histidine triad (FHIT) pre-mRNA in cancer cells occurs via re-splicing of spliced FHIT mRNA. The re-splicing of mature mRNA can potentially generate mutation-independent diversity in cancer transcriptomes. Conversely, a mechanism may exist in normal cells to prevent potentially deleterious mRNA re-splicing events. PMID:22675076

  2. ASTD: The Alternative Splicing and Transcript Diversity database.

    PubMed

    Koscielny, Gautier; Le Texier, Vincent; Gopalakrishnan, Chellappa; Kumanduri, Vasudev; Riethoven, Jean-Jack; Nardone, Francesco; Stanley, Eleanor; Fallsehr, Christine; Hofmann, Oliver; Kull, Meelis; Harrington, Eoghan; Boué, Stéphanie; Eyras, Eduardo; Plass, Mireya; Lopez, Fabrice; Ritchie, William; Moucadel, Virginie; Ara, Takeshi; Pospisil, Heike; Herrmann, Alexander; G Reich, Jens; Guigó, Roderic; Bork, Peer; Doeberitz, Magnus von Knebel; Vilo, Jaak; Hide, Winston; Apweiler, Rolf; Thanaraj, Thangavel Alphonse; Gautheret, Daniel

    2009-03-01

    The Alternative Splicing and Transcript Diversity database (ASTD) gives access to a vast collection of alternative transcripts that integrate transcription initiation, polyadenylation and splicing variant data. Alternative transcripts are derived from the mapping of transcribed sequences to the complete human, mouse and rat genomes using an extension of the computational pipeline developed for the ASD (Alternative Splicing Database) and ATD (Alternative Transcript Diversity) databases, which are now superseded by ASTD. For the human genome, ASTD identifies splicing variants, transcription initiation variants and polyadenylation variants in 68%, 68% and 62% of the gene set, respectively, consistent with current estimates for transcription variation. Users can access ASTD through a variety of browsing and query tools, including expression state-based queries for the identification of tissue-specific isoforms. Participating laboratories have experimentally validated a subset of ASTD-predicted alternative splice forms and alternative polyadenylation forms that were not previously reported. The ASTD database can be accessed at http://www.ebi.ac.uk/astd.

  3. Genome-Wide Survey of Cold Stress Regulated Alternative Splicing in Arabidopsis thaliana with Tiling Microarray

    PubMed Central

    Leviatan, Noam; Alkan, Noam; Leshkowitz, Dena; Fluhr, Robert

    2013-01-01

    Alternative splicing plays a major role in expanding the potential informational content of eukaryotic genomes. It is an important post-transcriptional regulatory mechanism that can increase protein diversity and affect mRNA stability. Alternative splicing is often regulated in a tissue-specific and stress-responsive manner. Cold stress, which adversely affects plant growth and development, regulates the transcription and splicing of plant splicing factors. This can affect the pre-mRNA processing of many genes. To identify cold regulated alternative splicing we applied Affymetrix Arabidopsis tiling arrays to survey the transcriptome under cold treatment conditions. A novel algorithm was used for detection of statistically relevant changes in intron expression within a transcript between control and cold growth conditions. A reverse transcription polymerase chain reaction (RT-PCR) analysis of a number of randomly selected genes confirmed the changes in splicing patterns under cold stress predicted by tiling array. Our analysis revealed new types of cold responsive genes. While their expression level remains relatively unchanged under cold stress their splicing pattern shows detectable changes in the relative abundance of isoforms. The majority of cold regulated alternative splicing introduced a premature termination codon (PTC) into the transcripts creating potential targets for degradation by the nonsense mediated mRNA decay (NMD) process. A number of these genes were analyzed in NMD-defective mutants by RT-PCR and shown to evade NMD. This may result in new and truncated proteins with altered functions or dominant negative effects. The results indicate that cold affects both quantitative and qualitative aspects of gene expression. PMID:23776682

  4. Genome-wide survey of cold stress regulated alternative splicing in Arabidopsis thaliana with tiling microarray.

    PubMed

    Leviatan, Noam; Alkan, Noam; Leshkowitz, Dena; Fluhr, Robert

    2013-01-01

    Alternative splicing plays a major role in expanding the potential informational content of eukaryotic genomes. It is an important post-transcriptional regulatory mechanism that can increase protein diversity and affect mRNA stability. Alternative splicing is often regulated in a tissue-specific and stress-responsive manner. Cold stress, which adversely affects plant growth and development, regulates the transcription and splicing of plant splicing factors. This can affect the pre-mRNA processing of many genes. To identify cold regulated alternative splicing we applied Affymetrix Arabidopsis tiling arrays to survey the transcriptome under cold treatment conditions. A novel algorithm was used for detection of statistically relevant changes in intron expression within a transcript between control and cold growth conditions. A reverse transcription polymerase chain reaction (RT-PCR) analysis of a number of randomly selected genes confirmed the changes in splicing patterns under cold stress predicted by tiling array. Our analysis revealed new types of cold responsive genes. While their expression level remains relatively unchanged under cold stress their splicing pattern shows detectable changes in the relative abundance of isoforms. The majority of cold regulated alternative splicing introduced a premature termination codon (PTC) into the transcripts creating potential targets for degradation by the nonsense mediated mRNA decay (NMD) process. A number of these genes were analyzed in NMD-defective mutants by RT-PCR and shown to evade NMD. This may result in new and truncated proteins with altered functions or dominant negative effects. The results indicate that cold affects both quantitative and qualitative aspects of gene expression.

  5. Identification of an alternative splicing isoform of chicken Lmbr1.

    PubMed

    Huang, Yanqun; Chen, Wen; Li, Ning; Deng, Xuemei; Kang, Xiangtao; Liu, Xiaojun

    2011-10-01

    Lmbr1 is the key candidate gene for limb development. Until now, at least five and four alternative splicing isoforms of Lmbr1 gene have been found in human and mouse, respectively. However, only two alternative splicing isoforms of this homologous gene have been reported in chicken. In the present study, one novel chicken Lmbr1 transcript variant (designated Lmbr1-1) was identified by 5' RACE and RT-PCR. Chicken Lmbr1-1 possesses one novel transcription start site different from Lmbr1-N, and was predicted to encode one 192 amino acid protein with length variation in comparison with chicken LMBR1-N protein, which was produced by 5' spliced site variation of chicken Lmbr1-N exon 10. Comparing with Lmbr1-N transcript, chicken Lmbr1-1 exhibited restricted tissue distribution of the expression. Comparative sequence analysis revealed a highly conservative intron element between chicken and mammalians from the intron 9 of chicken Lmbr1-N, indicating their possible importance as intronic elements in the regulation of alternative splicing of Lmbr1 in vertebrates. By direct PCR sequencing the exon 10 and its flanking sequences in chicken Lmbr1-N, four variation sites/haplotypes were identified from six chicken breeds. One 797A/G nonsynonymous mutation (266Arg/Gln) locating in exon 10 of chicken Lmbr1-N was predicted to affect the exon splice enhancer motif for serine/arginine-rich protein recognition. These data demonstrated that chicken Lmbr1 was alternatively spliced to generate multiple splice forms, as was the case in mammals and each of the alternative splicing isoforms might function differentially.

  6. Regulation of Alternative Splicing in Tumor Metastasis

    DTIC Science & Technology

    2000-10-01

    erythematosus and sarcoidosis . Arthritis Rheum. 41: 1505-15 10. Eldridge A.G., Y. Li, P.A. Sharp, and B.J. Blencowe. 1999. The SRml60/300 splicing coactivator...J. Hum. Genet. 59:279-286. erythematosus and sarcoidosis . Arthritis Rheum. 41:1505-1510. Matsumoto, K., K.M. Wassarman, and A.P. Wolffe. 1998. Nuclear

  7. The determinants of alternative RNA splicing in human cells.

    PubMed

    Ramanouskaya, Tatsiana V; Grinev, Vasily V

    2017-07-13

    Alternative splicing represents an important level of the regulation of gene function in eukaryotic organisms. It plays a critical role in virtually every biological process within an organism, including regulation of cell division and cell death, differentiation of tissues in the embryo and the adult organism, as well as in cellular response to diverse environmental factors. In turn, studies of the last decade have shown that alternative splicing itself is controlled by different mechanisms. Unfortunately, there is no clear understanding of how these diverse mechanisms, or determinants, regulate and constrain the set of alternative RNA species produced from any particular gene in every cell of the human body. Here, we provide a consolidated overview of alternative splicing determinants including RNA-protein interactions, epigenetic regulation via chromatin remodeling, coupling of transcription-to-alternative splicing, effect of secondary structures in pre-RNA, and function of the RNA quality control systems. We also extensively and critically discuss some mechanistic insights on coordinated inclusion/exclusion of exons during the formation of mature RNA molecules. We conclude that the final structure of RNA is pre-determined by a complex interplay between cis- and trans-acting factors. Altogether, currently available empirical data significantly expand our understanding of the functioning of the alternative splicing machinery of cells in normal and pathological conditions. On the other hand, there are still many blind spots that require further deep investigations.

  8. CASH: a constructing comprehensive splice site method for detecting alternative splicing events.

    PubMed

    Wu, Wenwu; Zong, Jie; Wei, Ning; Cheng, Jian; Zhou, Xuexia; Cheng, Yuanming; Chen, Dai; Guo, Qinghua; Zhang, Bo; Feng, Ying

    2017-04-06

    RNA-sequencing (RNA-seq) can generate millions of reads to provide clues for analyzing novel or abnormal alternative splicing (AS) events in cells. However, current methods for exploring AS events are still far from being satisfactory. Here, we present Comprehensive AS Hunting (CASH), which constructs comprehensive splice sites including known and novel AS sites in cells, and identifies differentially AS events between cells. We illuminated the versatility of CASH on RNA-seq data from a wide range of species and also on simulated in silico data, validated the advantages of CASH over other AS predictors and exhibited novel differentially AS events. Moreover, we used CASH to identify SRSF10-regulated AS events and investigated the evolution of SRSF10-regulated splicing. The results showed that SRSF10-regulated splicing events are highly evolvable from chickens, mice to humans. However, SRSF10-regulated splicing model was observed to be immutable, in which SRSF10 binding to cassette exon promotes exon inclusion while binding to downstream exon induces exon skipping. Altogether, CASH can significantly improve the detection of AS events and facilitate the study of AS regulation and function in cells; the SRSF10 data first demonstrate a flexibility of SRSF10 with their regulated splicing events but an immutability of SRSF10-regulated splicing model to produce opposite AS outcomes in vertebrates. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  9. Effects of airborne particulate matter on alternative pre-mRNA splicing in colon cancer cells

    SciTech Connect

    Buggiano, Valeria; Petrillo, Ezequiel; Alló, Mariano; Lafaille, Celina; Redal, María Ana; Alghamdi, Mansour A.; Khoder, Mamdouh I.; Shamy, Magdy; Muñoz, Manuel J.; and others

    2015-07-15

    Alternative pre-mRNA splicing plays key roles in determining tissue- and species-specific cell differentiation as well as in the onset of hereditary disease and cancer, being controlled by multiple post- and co-transcriptional regulatory mechanisms. We report here that airborne particulate matter, resulting from industrial pollution, inhibits expression and specifically affects alternative splicing at the 5′ untranslated region of the mRNA encoding the bone morphogenetic protein BMP4 in human colon cells in culture. These effects are consistent with a previously reported role for BMP4 in preventing colon cancer development, suggesting that ingestion of particulate matter could contribute to the onset of colon cell proliferation. We also show that the underlying mechanism might involve changes in transcriptional elongation. This is the first study to demonstrate that particulate matter causes non-pleiotropic changes in alternative splicing. - Highlights: • Airborne particulate matter (PM10) affects alternative splicing in colon cells. • PM10 upregulates one of the two mRNA variants of the growth factor BMP-4. • This variant has a longer 5′ unstranslated region and introduces an upstream AUG. • By regulating BMP-4 mRNA splicing PM10 inhibits total expression of BMP-4 protein. • BMP-4 downregulation was previously reported to be associated to colon cancer.

  10. Intragenic epigenetic changes modulate NCAM alternative splicing in neuronal differentiation.

    PubMed

    Schor, Ignacio E; Fiszbein, Ana; Petrillo, Ezequiel; Kornblihtt, Alberto R

    2013-08-14

    Alternative splicing contributes to cell type-specific transcriptomes. Here, we show that changes in intragenic chromatin marks affect NCAM (neural cell adhesion molecule) exon 18 (E18) alternative splicing during neuronal differentiation. An increase in the repressive marks H3K9me2 and H3K27me3 along the gene body correlated with inhibition of polymerase II elongation in the E18 region, but without significantly affecting total mRNA levels. Treatment with the general DNA methylation inhibitor 5-azacytidine and BIX 01294, a specific inhibitor of H3K9 dimethylation, inhibited the differentiation-induced E18 inclusion, pointing to a role for repressive marks in sustaining NCAM splicing patterns typical of mature neurons. We demonstrate that intragenic deployment of repressive chromatin marks, induced by intronic small interfering RNAs targeting NCAM intron 18, promotes E18 inclusion in undifferentiated N2a cells, confirming the chromatin changes observed upon differentiation to be sufficient to induce alternative splicing. Combined with previous evidence that neuronal depolarization causes H3K9 acetylation and subsequent E18 skipping, our results show how two alternative epigenetic marks regulate NCAM alternative splicing and E18 levels in different cellular contexts.

  11. Intragenic epigenetic changes modulate NCAM alternative splicing in neuronal differentiation

    PubMed Central

    Schor, Ignacio E; Fiszbein, Ana; Petrillo, Ezequiel; Kornblihtt, Alberto R

    2013-01-01

    Alternative splicing contributes to cell type-specific transcriptomes. Here, we show that changes in intragenic chromatin marks affect NCAM (neural cell adhesion molecule) exon 18 (E18) alternative splicing during neuronal differentiation. An increase in the repressive marks H3K9me2 and H3K27me3 along the gene body correlated with inhibition of polymerase II elongation in the E18 region, but without significantly affecting total mRNA levels. Treatment with the general DNA methylation inhibitor 5-azacytidine and BIX 01294, a specific inhibitor of H3K9 dimethylation, inhibited the differentiation-induced E18 inclusion, pointing to a role for repressive marks in sustaining NCAM splicing patterns typical of mature neurons. We demonstrate that intragenic deployment of repressive chromatin marks, induced by intronic small interfering RNAs targeting NCAM intron 18, promotes E18 inclusion in undifferentiated N2a cells, confirming the chromatin changes observed upon differentiation to be sufficient to induce alternative splicing. Combined with previous evidence that neuronal depolarization causes H3K9 acetylation and subsequent E18 skipping, our results show how two alternative epigenetic marks regulate NCAM alternative splicing and E18 levels in different cellular contexts. PMID:23892457

  12. Singular Value Decomposition-based Alternative Splicing Detection

    PubMed Central

    Hu, Jianhua; He, Xuming; Cote, Gilbert J.; Krahe, Ralf

    2009-01-01

    SUMMARY Altered alternative splicing has been identified as an important factor in tumorigenesis. The Affymetrix exon tiling array is designed for detecting alternative splicing events in a transcriptome-wide fashion; however, there are currently few analysis tools that are well studied for effective detection of alternative splicing events. We propose a new screening procedure based on singular value decomposition (SVD) of the residual matrix from a robust additive model fit to probe selection region (PSR) data. With this approach, we analyze the exon tiling array data from a brain cancer study conducted at the M. D. Anderson Cancer Center, and show that the proposed SVD-based approach is able to better accommodate outlying measures and capitalize on the multidimensional group-by-PSR gene expression profiles for more effective detection of group-specific alternative splicing events as well as the PSRs that are most likely associated with the alternative splicing. Lab validation confirmed some of our findings, but the list of candidates detected with our proposed method may provide a better signpost to guide further investigations. PMID:20305737

  13. AR Alternative Splicing and Prostate Cancer Progression

    DTIC Science & Technology

    2013-07-01

    receptor stabilization in recurrent prostate cancer is associated with hypersensitivity to low androgen. Cancer Res 2001; 61: 2892 - 2898. Supplementary...cells were maintained in RMPI 1640 (Invitrogen) with 10% fetal bovine serum (FBS), 100 units/ml penicillin , and 100g/ml streptomy- cin in a 5% CO2... hypersensitivity to low androgen. Cancer Res 2001;61: 2892–8. 15. Dehm SM, Schmidt LJ, Heemers HV, Vessella RL, Tindall DJ. Splicing of a novel androgen

  14. Detecting alternative gene structures from spliced ESTs: a computational approach.

    PubMed

    Bonizzoni, Paola; Mauri, Giancarlo; Pesole, Graziano; Picardi, Ernesto; Pirola, Yuri; Rizzi, Raffaella

    2009-01-01

    Alternative splicing (AS) is currently considered as one of the main mechanisms able to explain the huge gap between the number of predicted genes and the high complexity of the proteome in humans. The rapid growth of Expressed Sequence Tag (EST) data has encouraged the development of computational methods to predict alternative splicing from the analysis of EST alignment to genome sequences. EST data are also a valuable source to reconstruct the different transcript isoforms that derive from the same gene structure as a consequence of AS, as indeed EST sequences are obtained by fragmenting mRNAs from the same gene. The most recent studies on alternative splice sites detection have revealed that this topic is a quite challenging computational problem, far from a solution. The main computational issues related to the problem of detecting alternative splicing are investigated in this paper, and we analyze algorithmic solutions for this problem. We first formalize an optimization problem related to the prediction of constitutive and alternative splicing sites from EST sequences, the Minimum Exons ESTs Factorization problem (in short, MEF), and show that it is Np-hard, even for restricted instances. This problem leads us to define sets of spliced EST, that is, a set of EST factorized into their constitutive exons with respect to a gene. Then we investigate the computational problem of predicting transcript isoforms from spliced EST sequences. We propose a graph algorithm for the problem that is linear in the number of predicted isoforms and size of the graph. Finally, an experimental analysis of the method is performed to assess the reliability of the predictions.

  15. Aberrant and alternative splicing in skeletal system disease.

    PubMed

    Fan, Xin; Tang, Liling

    2013-10-01

    The main function of skeletal system is to support the body and help movement. A variety of factors can lead to skeletal system disease, including age, exercise, and of course genetic makeup and expression. Pre-mRNA splicing plays a crucial role in gene expression, by creating multiple protein variants with different biological functions. The recent studies show that several skeletal system diseases are related to pre-mRNA splicing. This review focuses on the relationship between pre-mRNA splicing and skeletal system disease. On the one hand, splice site mutation that leads to aberrant splicing often causes genetic skeletal system disease, like COL1A1, SEDL and LRP5. On the other hand, alternative splicing without genomic mutation may generate some marker protein isoforms, for example, FN, VEGF and CD44. Therefore, understanding the relationship between pre-mRNA splicing and skeletal system disease will aid in uncovering the mechanism of disease and contribute to the future development of gene therapy. © 2013 Elsevier B.V. All rights reserved.

  16. Modulators of alternative splicing as novel therapeutics in cancer.

    PubMed

    Oltean, Sebastian

    2015-10-10

    Alternative splicing (AS), the process of removing introns from pre-mRNA and re-arrangement of exons to give several types of mature transcripts, has been described more than 40 years ago. However, until recently, it has not been clear how extensive it is. Genome-wide studies have now conclusively shown that more than 90% of genes are alternatively spliced in humans. This makes AS one of the main drivers of proteomic diversity and, consequently, determinant of cellular function repertoire. Unsurprisingly, given its extent, numerous splice isoforms have been described to be associated with several diseases including cancer. Many of them have antagonistic functions, e.g., pro- and anti-angiogenic or pro- and anti-apoptotic. Additionally several splice factors have been recently described to have oncogene or tumour suppressors activities, like SF3B1 which is frequently mutated in myelodysplastic syndromes. Beside the implications for cancer pathogenesis, de-regulated AS is recognized as one of the novel areas of cell biology where therapeutic manipulations may be designed. This editorial discusses the possibilities of manipulation of AS for therapeutic benefit in cancer. Approaches involving the use of oligonucleotides as well as small molecule splicing modulators are presented as well as thoughts on how specificity might be accomplished in splicing therapeutics.

  17. Alternative splicing of the androgen receptor in polycystic ovary syndrome

    PubMed Central

    Wang, Fangfang; Pan, Jiexue; Liu, Ye; Meng, Qing; Lv, Pingping; Qu, Fan; Ding, Guo-Lian; Klausen, Christian; Leung, Peter C. K.; Chan, Hsiao Chang; Yao, Weimiao; Zhou, Cai-Yun; Shi, Biwei; Zhang, Junyu; Sheng, Jianzhong; Huang, Hefeng

    2015-01-01

    Polycystic ovary syndrome (PCOS) is one of the most common female endocrine disorders and a leading cause of female subfertility. The mechanism underlying the pathophysiology of PCOS remains to be illustrated. Here, we identify two alternative splice variants (ASVs) of the androgen receptor (AR), insertion and deletion isoforms, in granulosa cells (GCs) in ∼62% of patients with PCOS. AR ASVs are strongly associated with remarkable hyperandrogenism and abnormalities in folliculogenesis, and are absent from all control subjects without PCOS. Alternative splicing dramatically alters genome-wide AR recruitment and androgen-induced expression of genes related to androgen metabolism and folliculogenesis in human GCs. These findings establish alternative splicing of AR in GCs as the major pathogenic mechanism for hyperandrogenism and abnormal folliculogenesis in PCOS. PMID:25825716

  18. A procedure for identifying homologous alternative splicing events

    PubMed Central

    Talavera, David; Hospital, Adam; Orozco, Modesto; de la Cruz, Xavier

    2007-01-01

    Background The study of the functional role of alternative splice isoforms of a gene is a very active area of research in biology. The difficulty of the experimental approach (in particular, in its high-throughput version) leaves ample room for the development of bioinformatics tools that can provide a useful first picture of the problem. Among the possible approaches, one of the simplest is to follow classical protein function annotation protocols and annotate target alternative splice events with the information available from conserved events in other species. However, the application of this protocol requires a procedure capable of recognising such events. Here we present a simple but accurate method developed for this purpose. Results We have developed a method for identifying homologous, or equivalent, alternative splicing events, based on the combined use of neural networks and sequence searches. The procedure comprises four steps: (i) BLAST search for homologues of the two isoforms defining the target alternative splicing event; (ii) construction of all possible candidate events; (iii) scoring of the latter with a series of neural networks; and (iv) filtering of the results. When tested in a set of 473 manually annotated pairs of homologous events, our method showed a good performance, with an accuracy of 0.99, a precision of 0.98 and a sensitivity of 0.93. When no candidates were available, the specificity of our method varied between 0.81 and 0.91. Conclusion The method described in this article allows the identification of homologous alternative splicing events, with a good success rate, indicating that such method could be used for the development of functional annotation of alternative splice isoforms. PMID:17640387

  19. CDK12 regulates alternative last exon mRNA splicing and promotes breast cancer cell invasion.

    PubMed

    Tien, Jerry F; Mazloomian, Alborz; Cheng, S-W Grace; Hughes, Christopher S; Chow, Christalle C T; Canapi, Leanna T; Oloumi, Arusha; Trigo-Gonzalez, Genny; Bashashati, Ali; Xu, James; Chang, Vicky C-D; Shah, Sohrab P; Aparicio, Samuel; Morin, Gregg B

    2017-06-20

    CDK12 (cyclin-dependent kinase 12) is a regulatory kinase with evolutionarily conserved roles in modulating transcription elongation. Recent tumor genome studies of breast and ovarian cancers highlighted recurrent CDK12 mutations, which have been shown to disrupt DNA repair in cell-based assays. In breast cancers, CDK12 is also frequently co-amplified with the HER2 (ERBB2) oncogene. The mechanisms underlying functions of CDK12 in general and in cancer remain poorly defined. Based on global analysis of mRNA transcripts in normal and breast cancer cell lines with and without CDK12 amplification, we demonstrate that CDK12 primarily regulates alternative last exon (ALE) splicing, a specialized subtype of alternative mRNA splicing, that is both gene- and cell type-specific. These are unusual properties for spliceosome regulatory factors, which typically regulate multiple forms of alternative splicing in a global manner. In breast cancer cells, regulation by CDK12 modulates ALE splicing of the DNA damage response activator ATM and a DNAJB6 isoform that influences cell invasion and tumorigenesis in xenografts. We found that there is a direct correlation between CDK12 levels, DNAJB6 isoform levels and the migration capacity and invasiveness of breast tumor cells. This suggests that CDK12 gene amplification can contribute to the pathogenesis of the cancer. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. CDK12 regulates alternative last exon mRNA splicing and promotes breast cancer cell invasion

    PubMed Central

    Mazloomian, Alborz; Cheng, S.-W. Grace; Hughes, Christopher S.; Chow, Christalle C.T.; Canapi, Leanna T.; Oloumi, Arusha; Trigo-Gonzalez, Genny; Bashashati, Ali; Xu, James; Chang, Vicky C.-D.; Shah, Sohrab P.; Aparicio, Samuel

    2017-01-01

    Abstract CDK12 (cyclin-dependent kinase 12) is a regulatory kinase with evolutionarily conserved roles in modulating transcription elongation. Recent tumor genome studies of breast and ovarian cancers highlighted recurrent CDK12 mutations, which have been shown to disrupt DNA repair in cell-based assays. In breast cancers, CDK12 is also frequently co-amplified with the HER2 (ERBB2) oncogene. The mechanisms underlying functions of CDK12 in general and in cancer remain poorly defined. Based on global analysis of mRNA transcripts in normal and breast cancer cell lines with and without CDK12 amplification, we demonstrate that CDK12 primarily regulates alternative last exon (ALE) splicing, a specialized subtype of alternative mRNA splicing, that is both gene- and cell type-specific. These are unusual properties for spliceosome regulatory factors, which typically regulate multiple forms of alternative splicing in a global manner. In breast cancer cells, regulation by CDK12 modulates ALE splicing of the DNA damage response activator ATM and a DNAJB6 isoform that influences cell invasion and tumorigenesis in xenografts. We found that there is a direct correlation between CDK12 levels, DNAJB6 isoform levels and the migration capacity and invasiveness of breast tumor cells. This suggests that CDK12 gene amplification can contribute to the pathogenesis of the cancer. PMID:28334900

  1. A chloroplast retrograde signal regulates nuclear alternative splicing

    PubMed Central

    Petrillo, Ezequiel; Herz, Micaela A. Godoy; Fuchs, Armin; Reifer, Dominik; Fuller, John; Yanovsky, Marcelo J.; Simpson, Craig; Brown, John W. S.; Barta, Andrea; Kalyna, Maria; Kornblihtt, Alberto R.

    2015-01-01

    Light is a source of energy and also a regulator of plant physiological adaptations. We show here that light/dark conditions affect alternative splicing of a subset of Arabidopsis genes preferentially encoding proteins involved in RNA processing. The effect requires functional chloroplasts and is also observed in roots when the communication with the photosynthetic tissues is not interrupted, suggesting that a signaling molecule travels through the plant. Using photosynthetic electron transfer inhibitors with different mechanisms of action we deduce that the reduced pool of plastoquinones initiates a chloroplast retrograde signaling that regulates nuclear alternative splicing and is necessary for proper plant responses to varying light conditions. PMID:24763593

  2. Alternative splicing impairs soluble guanylyl cyclase function in aortic aneurysm.

    PubMed

    Martin, Emil; Golunski, Eva; Laing, Susan T; Estrera, Anthony L; Sharina, Iraida G

    2014-12-01

    Nitric oxide (NO) receptor soluble guanylyl cyclase (sGC) is a key regulator of several important vascular functions and is important for maintaining cardiovascular homeostasis and vascular plasticity. Diminished sGC expression and function contributes to pathogenesis of several cardiovascular diseases. However, the processes that control sGC expression in vascular tissue remain poorly understood. Previous work in animal and cell models revealed the complexity of alternative splicing of sGC genes and demonstrated its importance in modulation of sGC function. The aim of this study was to examine the role of alternative splicing of α1 and β1 sGC in healthy and diseased human vascular tissue. Our study found a variety of α1 and β1 sGC splice forms expressed in human aorta. Their composition and abundance were different between samples of aortic tissue removed during surgical repair of aortic aneurysm and samples of aortas without aneurysm. Aortas with aneurysm demonstrated decreased sGC activity, which correlated with increased expression of dysfunctional sGC splice variants. In addition, the expression of 55-kDa oxidation-resistant α1 isoform B sGC (α1-IsoB) was significantly lower in aortic samples with aneurysm. The α1-IsoB splice variant was demonstrated to support sGC activity in aortic lysates. Together, our results suggest that alternative splicing contributes to diminished sGC function in vascular dysfunction. Precise understanding of sGC splicing regulation could help to design new therapeutic interventions and to personalize sGC-targeting therapies in treatments of vascular disease. Copyright © 2014 the American Physiological Society.

  3. The complete local genotype–phenotype landscape for the alternative splicing of a human exon

    PubMed Central

    Julien, Philippe; Miñana, Belén; Baeza-Centurion, Pablo; Valcárcel, Juan; Lehner, Ben

    2016-01-01

    The properties of genotype–phenotype landscapes are crucial for understanding evolution but are not characterized for most traits. Here, we present a >95% complete local landscape for a defined molecular function—the alternative splicing of a human exon (FAS/CD95 exon 6, involved in the control of apoptosis). The landscape provides important mechanistic insights, revealing that regulatory information is dispersed throughout nearly every nucleotide in an exon, that the exon is more robust to the effects of mutations than its immediate neighbours in genotype space, and that high mutation sensitivity (evolvability) will drive the rapid divergence of alternative splicing between species unless it is constrained by selection. Moreover, the extensive epistasis in the landscape predicts that exonic regulatory sequences may diverge between species even when exon inclusion levels are functionally important and conserved by selection. PMID:27161764

  4. Alternative splicing regulates pluripotent state in pluripotent stem cells.

    PubMed

    He, Ling; Bai, Qiang; Tang, Liling

    2015-01-01

    Alternative splicing (AS) generates multiple mature mRNAs from a single pre-mRNA, so AS is the main contributor for the diversity of the proteins, participating in most of the cellular processes. For pluripotent stem cells (PSCs), great effort has been made to search for pluripotency-related genes and their regulatory mechanisms. However, the sophisticated regulation still remains to be clear. Recent studies indicate that stem cells undergo a unique AS pattern and have a different protein expression profile from differentiated cells, giving a new clue that AS switching or AS itself may play a significant role in the processes of differentiation and somatic reprogramming. Indeed, accumulating evidences prove that AS plays critical roles in maintaining pluripotent homeostasis in PSCs. In this review, we summarized recent researches on AS in ESCs and iPSCs, including some distinct AS events in pluripotent cells, and then discussed the new progress on mechanisms for AS in ESCs and iPSCs differentiation and somatic reprogramming.

  5. Transcriptome analysis of alternative splicing events regulated by SRSF10 reveals position-dependent splicing modulation.

    PubMed

    Zhou, Xuexia; Wu, Wenwu; Li, Huang; Cheng, Yuanming; Wei, Ning; Zong, Jie; Feng, Xiaoyan; Xie, Zhiqin; Chen, Dai; Manley, James L; Wang, Hui; Feng, Ying

    2014-04-01

    Splicing factor SRSF10 is known to function as a sequence-specific splicing activator. Here, we used RNA-seq coupled with bioinformatics analysis to identify the extensive splicing network regulated by SRSF10 in chicken cells. We found that SRSF10 promoted both exon inclusion and exclusion. Motif analysis revealed that SRSF10 binding to cassette exons was associated with exon inclusion, whereas the binding of SRSF10 within downstream constitutive exons was associated with exon exclusion. This positional effect was further demonstrated by the mutagenesis of potential SRSF10 binding motifs in two minigene constructs. Functionally, many of SRSF10-verified alternative exons are linked to pathways of stress and apoptosis. Consistent with this observation, cells depleted of SRSF10 expression were far more susceptible to endoplasmic reticulum stress-induced apoptosis than control cells. Importantly, reconstituted SRSF10 in knockout cells recovered wild-type splicing patterns and considerably rescued the stress-related defects. Together, our results provide mechanistic insight into SRSF10-regulated alternative splicing events in vivo and demonstrate that SRSF10 plays a crucial role in cell survival under stress conditions.

  6. Novel mutations in the GH gene (GH1) uncover putative splicing regulatory elements.

    PubMed

    Babu, Deepak; Mellone, Simona; Fusco, Ileana; Petri, Antonella; Walker, Gillian E; Bellone, Simonetta; Prodam, Flavia; Momigliano-Richiardi, Patricia; Bona, Gianni; Giordano, Mara

    2014-05-01

    Mutations affecting exon 3 splicing are the main cause of autosomal dominant Isolated GH Deficiency II (IGHDII) by increasing the level of exon 3-skipped mRNA encoding the functionally inactive dominant-negative 17.5-kDa isoform. The exons and introns of the gene encoding GH (GH1) were screened for the presence of mutations in 103 sporadic isolated GH deficiency cases. Four different variations within exon 3 were identified in 3 patients. One carried c.261C>T (p.Pro87Pro) and c.272A>T (p.Glu91Val), the second c.255G>A (p.Pro85Pro) and c.261 C>T, and the third c.246G>C (p.Glu82Asp). All the variants were likely generated by gene conversion from an homologous gene in the GH1 cluster. In silico analysis predicted that positions c.255 and c.272 were included within 2 putative novel exon splicing enhancers (ESEs). Their effect on splicing was confirmed in vitro. Constructs bearing these 2 variants induced consistently higher levels both of transcript and protein corresponding to the 17.5-kDa isoform. When c.255 and c.272 were combined in cis with the c.261 variant, as in our patients, their effect was weaker. In conclusion, we identified 2 variations, c.255G>A and c.272A>T, located in 2 novel putative exon splicing enhancers and affecting GH1 splicing in vitro by increasing the production of alternatively spliced isoforms. The amount of aberrant isoforms is further regulated by the presence in cis of the c.261 variant. Thus, our results evidenced novel putative splicing regulatory elements within exon 3, confirming the crucial role of this exon in mRNA processing.

  7. Alternative splicing and evolution: diversification, exon definition and function.

    PubMed

    Keren, Hadas; Lev-Maor, Galit; Ast, Gil

    2010-05-01

    Over the past decade, it has been shown that alternative splicing (AS) is a major mechanism for the enhancement of transcriptome and proteome diversity, particularly in mammals. Splicing can be found in species from bacteria to humans, but its prevalence and characteristics vary considerably. Evolutionary studies are helping to address questions that are fundamental to understanding this important process: how and when did AS evolve? Which AS events are functional? What are the evolutionary forces that shaped, and continue to shape, AS? And what determines whether an exon is spliced in a constitutive or alternative manner? In this Review, we summarize the current knowledge of AS and evolution and provide insights into some of these unresolved questions.

  8. Discovery of Novel Splice Variants and Regulatory Mechanisms for Microsomal Triglyceride Transfer Protein in Human Tissues

    PubMed Central

    Suzuki, Takashi; Swift, Larry L.

    2016-01-01

    Microsomal triglyceride transfer protein (MTP) is a unique lipid transfer protein essential for the assembly of triglyceride-rich lipoproteins by the liver and intestine. Previous studies in mice identified a splice variant of MTP with an alternate first exon. Splice variants of human MTP have not been reported. Using PCR approaches we have identified two splice variants in human tissues, which we have named MTP-B and MTP-C. MTP-B has a unique first exon (Ex1B) located 10.5 kb upstream of the first exon (Ex1A) for canonical MTP (MTP-A); MTP-C contains both first exons for MTP-A and MTP-B. MTP-B was found in a number of tissues, whereas MTP-C was prominent in brain and testis. MTP-B does not encode a protein; MTP-C encodes the same protein encoded by MTP-A, although MTP-C translation is strongly inhibited by regulatory elements within its 5′-UTR. Using luciferase assays, we demonstrate that the promoter region upstream of exon 1B is quite adequate to drive expression of MTP. We conclude that alternate splicing plays a key role in regulating cellular MTP levels by introducing distinct promoter regions and unique 5′-UTRs, which contain elements that alter translation efficiency, enabling the cell to optimize MTP activity. PMID:27256115

  9. Discovery of Novel Splice Variants and Regulatory Mechanisms for Microsomal Triglyceride Transfer Protein in Human Tissues.

    PubMed

    Suzuki, Takashi; Swift, Larry L

    2016-06-03

    Microsomal triglyceride transfer protein (MTP) is a unique lipid transfer protein essential for the assembly of triglyceride-rich lipoproteins by the liver and intestine. Previous studies in mice identified a splice variant of MTP with an alternate first exon. Splice variants of human MTP have not been reported. Using PCR approaches we have identified two splice variants in human tissues, which we have named MTP-B and MTP-C. MTP-B has a unique first exon (Ex1B) located 10.5 kb upstream of the first exon (Ex1A) for canonical MTP (MTP-A); MTP-C contains both first exons for MTP-A and MTP-B. MTP-B was found in a number of tissues, whereas MTP-C was prominent in brain and testis. MTP-B does not encode a protein; MTP-C encodes the same protein encoded by MTP-A, although MTP-C translation is strongly inhibited by regulatory elements within its 5'-UTR. Using luciferase assays, we demonstrate that the promoter region upstream of exon 1B is quite adequate to drive expression of MTP. We conclude that alternate splicing plays a key role in regulating cellular MTP levels by introducing distinct promoter regions and unique 5'-UTRs, which contain elements that alter translation efficiency, enabling the cell to optimize MTP activity.

  10. MBNL and CELF proteins regulate alternative splicing of the skeletal muscle chloride channel CLCN1.

    PubMed

    Kino, Yoshihiro; Washizu, Chika; Oma, Yoko; Onishi, Hayato; Nezu, Yuriko; Sasagawa, Noboru; Nukina, Nobuyuki; Ishiura, Shoichi

    2009-10-01

    The expression and function of the skeletal muscle chloride channel CLCN1/ClC-1 is regulated by alternative splicing. Inclusion of the CLCN1 exon 7A is aberrantly elevated in myotonic dystrophy (DM), a genetic disorder caused by the expansion of a CTG or CCTG repeat. Increased exon 7A inclusion leads to a reduction in CLCN1 function, which can be causative of myotonia. Two RNA-binding protein families--muscleblind-like (MBNL) and CUG-BP and ETR-3-like factor (CELF) proteins--are thought to mediate the splicing misregulation in DM. Here, we have identified multiple factors that regulate the alternative splicing of a mouse Clcn1 minigene. The inclusion of exon 7A was repressed by MBNL proteins while promoted by an expanded CUG repeat or CELF4, but not by CUG-BP. Mutation analyses suggested that exon 7A and its flanking region mediate the effect of MBNL1, whereas another distinct region in intron 6 mediates that of CELF4. An exonic splicing enhancer essential for the inclusion of exon 7A was identified at the 5' end of this exon, which might be inhibited by MBNL1. Collectively, these results provide a mechanistic model for the regulation of Clcn1 splicing, and reveal novel regulatory properties of MBNL and CELF proteins.

  11. Light regulates alternative splicing of hydroxypyruvate reductase in pumpkin.

    PubMed

    Mano, S; Hayashi, M; Nishimura, M

    1999-02-01

    Hydroxypyruvate reductase (HPR) is a leaf peroxisomal enzyme that functions in the glycolate pathway of photorespiration in plants. We have obtained two highly similar cDNAs for pumpkin HPR (HPR1 and HPR2). It has been revealed that two HPR mRNAs might be produced by alternative splicing from a single type of pre-mRNA. The HPR1 protein, but not the HPR2 protein, was found to have a targeting sequence into leaf peroxisomes at the C-terminus, suggesting that alternative splicing controls the subcellular localization of the two HPR proteins. Immunoblot analysis and subcellular fractionation experiments showed that HPR1 and HPR2 proteins are localized in leaf peroxisomes and the cytosol, respectively. Moreover, indirect fluorescence microscopy and analyses of transgenic tobacco cultured cells and Arabidopsis thaliana expressing fusion proteins with green fluorescent protein (GFP) revealed the different subcellular localizations of the two HPR proteins. Both mRNAs were induced developmentally and by light, but with quantitative differences. Almost equal amounts of the mRNAs were detected in pumpkin cotyledons grown in darkness, but treatment with light greatly enhanced the production of HPR2 mRNA. These findings indicate that light regulates alternative splicing of HPR mRNA, suggesting the presence of a novel mechanism of mRNA maturation, namely light-regulated alternative splicing, in higher plants.

  12. Rbfox2-coordinated alternative splicing of Mef2d and Rock2 controls myoblast fusion during myogenesis

    PubMed Central

    Bland, Christopher S.; Kalsotra, Auinash; Scavuzzo, Marissa A.; Curk, Tomaz; Ule, Jernej; Li, Wei; Cooper, Thomas A.

    2014-01-01

    Summary Alternative splicing plays important regulatory roles during periods of physiological change. During development a large number of genes coordinately express protein isoform transitions regulated by alternative splicing, however, the mechanisms that coordinate splicing and the functional integration of the resultant tissue-specific protein isoforms are typically unknown. Here we show that the conserved Rbfox2 RNA binding protein regulates 30% of the splicing transitions observed during myogenesis and is required for the specific step of myoblast fusion. Integration of Rbfox2-dependent splicing outcomes from RNA-seq with Rbfox2 iCLIP data identified Mef2d and Rock2 as Rbfox2 splicing targets. Restored activities of Mef2d and Rock2 rescued myoblast fusion in Rbfox2 depleted cultures demonstrating functional cooperation of protein isoforms generated by coordinated alterative splicing. The results demonstrate that coordinated alternative splicing by a single RNA binding protein modulates transcription (Mef2d) and cell signaling (Rock2) programs to drive tissue-specific functions (cell fusion) to promote a developmental transition. PMID:25087874

  13. Intrasplicing coordinates alternative first exons with alternative splicing in the protein 4.1R gene

    SciTech Connect

    Conboy, John G.; Parra, Marilyn K.; Tan, Jeff S.; Mohandas, Narla; Conboy, John G.

    2008-11-07

    In the protein 4.1R gene, alternative first exons splice differentially to alternative 3' splice sites far downstream in exon 2'/2 (E2'/2). We describe a novel intrasplicing mechanism by which exon 1A (E1A) splices exclusively to the distal E2'/2 acceptor via two nested splicing reactions regulated by novel properties of exon 1B (E1B). E1B behaves as an exon in the first step, using its consensus 5' donor to splice to the proximal E2'/2 acceptor. A long region of downstream intron is excised, juxtaposing E1B with E2'/2 to generate a new composite acceptor containing the E1B branchpoint/pyrimidine tract and E2 distal 3' AG-dinucleotide. Next, the upstream E1A splices over E1B to this distal acceptor, excising the remaining intron plus E1B and E2' to form mature E1A/E2 product. We mapped branch points for both intrasplicing reactions and demonstrated that mutation of the E1B 5' splice site or branchpoint abrogates intrasplicing. In the 4.1R gene, intrasplicing ultimately determines N-terminal protein structure and function. More generally, intrasplicing represents a new mechanism whereby alternative promoters can be coordinated with downstream alternative splicing.

  14. HIV-1 splicing is controlled by local RNA structure and binding of splicing regulatory proteins at the major 5' splice site.

    PubMed

    Mueller, Nancy; Berkhout, Ben; Das, Atze T

    2015-07-01

    The 5' leader region of the human immunodeficiency virus 1 (HIV-1) RNA genome contains the major 5' splice site (ss) that is used in the production of the many spliced viral RNAs. This splice-donor (SD) region can fold into a stable stem-loop structure and the thermodynamic stability of this RNA hairpin influences splicing efficiency. In addition, splicing may be modulated by binding of splicing regulatory (SR) proteins, in particular SF2/ASF (SRSF1), SC35 (SRSF2), SRp40 (SRSF5) and SRp55 (SRSF6), to sequence elements in the SD region. The role of RNA structure and SR protein binding in splicing control was previously studied by functional analysis of mutant SD sequences. The interpretation of these studies was complicated by the fact that most mutations simultaneously affect both structure and sequence elements. We therefore tried to disentangle the contribution of these two variables by designing more precise SD region mutants with a single effect on either the sequence or the structure. The current analysis indicates that HIV-1 splicing at the major 5'ss is modulated by both the stability of the local RNA structure and the binding of splicing regulatory proteins.

  15. The Nuclear-Retained Noncoding RNA MALAT1 Regulates Alternative Splicing by Modulating SR Splicing Factor Phosphorylation

    PubMed Central

    Tripathi, Vidisha; Ellis, Jonathan D.; Shen, Zhen; Song, David Y.; Pan, Qun; Watt, Andrew T.; Freier, Susan M.; Bennett, C. Frank; Sharma, Alok; Bubulya, Paula A.; Blencowe, Benjamin J.; Prasanth, Supriya G.; Prasanth, Kannanganattu V.

    2014-01-01

    SUMMARY Alternative splicing (AS) of pre-mRNA is utilized by higher eukaryotes to achieve increased transcriptome and proteomic complexity. The serine/arginine (SR) splicing factors regulate tissue- or cell-type-specific AS in a concentration- and phosphorylation-dependent manner. However, the mechanisms that modulate the cellular levels of active SR proteins remain to be elucidated. In the present study, we provide evidence for a role for the long nuclear-retained regulatory RNA (nrRNA), MALAT1 in AS regulation. MALAT1 interacts with SR proteins and influences the distribution of these and other splicing factors in nuclear speckle domains. Depletion of MALAT1 or overexpression of an SR protein changes the AS of a similar set of endogenous pre-mRNAs. Furthermore, MALAT1 regulates cellular levels of phosphorylated forms of SR proteins. Taken together, our results suggest that MALAT1 regulates AS by modulating the levels of active SR proteins. Our results further highlight the role for an nrRNA in the regulation of gene expression. PMID:20797886

  16. Alternatively spliced, spliceosomal twin introns in Helminthosporium solani.

    PubMed

    Ág, Norbert; Flipphi, Michel; Karaffa, Levente; Scazzocchio, Claudio; Fekete, Erzsébet

    2015-12-01

    Spliceosomal twin introns, "stwintrons", have been defined as complex intervening sequences that carry a second intron ("internal intron") interrupting one of the conserved sequence domains necessary for their correct splicing via consecutive excision events. Previously, we have described and experimentally verified stwintrons in species of Sordariomycetes, where an "internal intron" interrupted the donor sequence of an "external intron". Here we describe and experimentally verify two novel stwintrons of the potato pathogen Helminthosporium solani. One instance involves alternative splicing of an internal intron interrupting the donor domain of an external intron and a second one interrupting the acceptor domain of an overlapping external intron, both events leading to identical mature mRNAs. In the second case, an internal intron interrupts the donor domain of the external intron, while an alternatively spliced intron leads to an mRNA carrying a premature chain termination codon. We thus extend the stwintron concept to the acceptor domain and establish a link of the occurrence of stwintrons with that of alternative splicing.

  17. Body Temperature Cycles Control Rhythmic Alternative Splicing in Mammals.

    PubMed

    Preußner, Marco; Goldammer, Gesine; Neumann, Alexander; Haltenhof, Tom; Rautenstrauch, Pia; Müller-McNicoll, Michaela; Heyd, Florian

    2017-08-03

    The core body temperature of all mammals oscillates with the time of the day. However, direct molecular consequences of small, physiological changes in body temperature remain largely elusive. Here we show that body temperature cycles drive rhythmic SR protein phosphorylation to control an alternative splicing (AS) program. A temperature change of 1°C is sufficient to induce a concerted splicing switch in a large group of functionally related genes, rendering this splicing-based thermometer much more sensitive than previously described temperature-sensing mechanisms. AS of two exons in the 5' UTR of the TATA-box binding protein (Tbp) highlights the general impact of this mechanism, as it results in rhythmic TBP protein levels with implications for global gene expression in vivo. Together our data establish body temperature-driven AS as a core clock-independent oscillator in mammalian peripheral clocks. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Genome-wide association between DNA methylation and alternative splicing in an invertebrate.

    PubMed

    Flores, Kevin; Wolschin, Florian; Corneveaux, Jason J; Allen, April N; Huentelman, Matthew J; Amdam, Gro V

    2012-09-15

    Gene bodies are the most evolutionarily conserved targets of DNA methylation in eukaryotes. However, the regulatory functions of gene body DNA methylation remain largely unknown. DNA methylation in insects appears to be primarily confined to exons. Two recent studies in Apis mellifera (honeybee) and Nasonia vitripennis (jewel wasp) analyzed transcription and DNA methylation data for one gene in each species to demonstrate that exon-specific DNA methylation may be associated with alternative splicing events. In this study we investigated the relationship between DNA methylation, alternative splicing, and cross-species gene conservation on a genome-wide scale using genome-wide transcription and DNA methylation data. We generated RNA deep sequencing data (RNA-seq) to measure genome-wide mRNA expression at the exon- and gene-level. We produced a de novo transcriptome from this RNA-seq data and computationally predicted splice variants for the honeybee genome. We found that exons that are included in transcription are higher methylated than exons that are skipped during transcription. We detected enrichment for alternative splicing among methylated genes compared to unmethylated genes using fisher's exact test. We performed a statistical analysis to reveal that the presence of DNA methylation or alternative splicing are both factors associated with a longer gene length and a greater number of exons in genes. In concordance with this observation, a conservation analysis using BLAST revealed that each of these factors is also associated with higher cross-species gene conservation. This study constitutes the first genome-wide analysis exhibiting a positive relationship between exon-level DNA methylation and mRNA expression in the honeybee. Our finding that methylated genes are enriched for alternative splicing suggests that, in invertebrates, exon-level DNA methylation may play a role in the construction of splice variants by positively influencing exon inclusion during

  19. SplAdder: identification, quantification and testing of alternative splicing events from RNA-Seq data

    PubMed Central

    Kahles, André; Ong, Cheng Soon; Zhong, Yi; Rätsch, Gunnar

    2016-01-01

    Motivation: Understanding the occurrence and regulation of alternative splicing (AS) is a key task towards explaining the regulatory processes that shape the complex transcriptomes of higher eukaryotes. With the advent of high-throughput sequencing of RNA (RNA-Seq), the diversity of AS transcripts could be measured at an unprecedented depth. Although the catalog of known AS events has grown ever since, novel transcripts are commonly observed when working with less well annotated organisms, in the context of disease, or within large populations. Whereas an identification of complete transcripts is technically challenging and computationally expensive, focusing on single splicing events as a proxy for transcriptome characteristics is fruitful and sufficient for a wide range of analyses. Results: We present SplAdder, an alternative splicing toolbox, that takes RNA-Seq alignments and an annotation file as input to (i) augment the annotation based on RNA-Seq evidence, (ii) identify alternative splicing events present in the augmented annotation graph, (iii) quantify and confirm these events based on the RNA-Seq data and (iv) test for significant quantitative differences between samples. Thereby, our main focus lies on performance, accuracy and usability. Availability: Source code and documentation are available for download at http://github.com/ratschlab/spladder. Example data, introductory information and a small tutorial are accessible via http://bioweb.me/spladder. Contacts: andre.kahles@ratschlab.org or gunnar.ratsch@ratschlab.org Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26873928

  20. LRRTM3 Regulates Excitatory Synapse Development through Alternative Splicing and Neurexin Binding.

    PubMed

    Um, Ji Won; Choi, Tae-Yong; Kang, Hyeyeon; Cho, Yi Sul; Choii, Gayoung; Uvarov, Pavel; Park, Dongseok; Jeong, Daun; Jeon, Sangmin; Lee, Dongmin; Kim, Hyun; Lee, Seung-Hee; Bae, Yong-Chul; Choi, Se-Young; Airaksinen, Matti S; Ko, Jaewon

    2016-02-02

    The four members of the LRRTM family (LRRTM1-4) are postsynaptic adhesion molecules essential for excitatory synapse development. They have also been implicated in neuropsychiatric diseases. Here, we focus on LRRTM3, showing that two distinct LRRTM3 variants generated by alternative splicing regulate LRRTM3 interaction with PSD-95, but not its excitatory synapse-promoting activity. Overexpression of either LRRTM3 variant increased excitatory synapse density in dentate gyrus (DG) granule neurons, whereas LRRTM3 knockdown decreased it. LRRTM3 also controlled activity-regulated AMPA receptor surface expression in an alternative splicing-dependent manner. Furthermore, Lrrtm3-knockout mice displayed specific alterations in excitatory synapse density, excitatory synaptic transmission and excitability in DG granule neurons but not in CA1 pyramidal neurons. Lastly, LRRTM3 required only specific splice variants of presynaptic neurexins for their synaptogenic activity. Collectively, our data highlight alternative splicing and differential presynaptic ligand utilization in the regulation of LRRTMs, revealing key regulatory mechanisms for excitatory synapse development.

  1. Reversion to an embryonic alternative splicing program enhances leukemia stem cell self-renewal

    PubMed Central

    Holm, Frida; Hellqvist, Eva; Mason, Cayla N.; Ali, Shawn A.; Delos-Santos, Nathaniel; Barrett, Christian L.; Chun, Hye-Jung; Minden, Mark D.; Moore, Richard A.; Marra, Marco A.; Runza, Valeria; Frazer, Kelly A.; Sadarangani, Anil; Jamieson, Catriona H. M.

    2015-01-01

    Formative research suggests that a human embryonic stem cell-specific alternative splicing gene regulatory network, which is repressed by Muscleblind-like (MBNL) RNA binding proteins, is involved in cell reprogramming. In this study, RNA sequencing, splice isoform-specific quantitative RT-PCR, lentiviral transduction, and in vivo humanized mouse model studies demonstrated that malignant reprogramming of progenitors into self-renewing blast crisis chronic myeloid leukemia stem cells (BC LSCs) was partially driven by decreased MBNL3. Lentiviral knockdown of MBNL3 resulted in reversion to an embryonic alternative splice isoform program typified by overexpression of CD44 transcript variant 3, containing variant exons 8–10, and BC LSC proliferation. Although isoform-specific lentiviral CD44v3 overexpression enhanced chronic phase chronic myeloid leukemia (CML) progenitor replating capacity, lentiviral shRNA knockdown abrogated these effects. Combined treatment with a humanized pan-CD44 monoclonal antibody and a breakpoint cluster region - ABL proto-oncogene 1, nonreceptor tyrosine kinase (BCR-ABL1) antagonist inhibited LSC maintenance in a niche-dependent manner. In summary, MBNL3 down-regulation–related reversion to an embryonic alternative splicing program, typified by CD44v3 overexpression, represents a previously unidentified mechanism governing malignant progenitor reprogramming in malignant microenvironments and provides a pivotal opportunity for selective BC LSC detection and therapeutic elimination. PMID:26621726

  2. Overexpression of Antiangiogenic Vascular Endothelial Growth Factor Isoform and Splicing Regulatory Factors in Oral, Laryngeal and Pharyngeal Squamous Cell Carcinomas

    PubMed

    Biselli-Chicote, Patrícia Matos; Biselli, Joice Matos; Cunha, Bianca R; Castro, Rodrigo; Maniglia, José Victor; Neto, Dalísio de Santi; Tajara, Eloiza Helena; Góis Filho, José Franscisco de; Fukuyama, Erica Erina; Pavarino, Érika Cristina; Goloni-Bertollo, Eny Maria

    2017-08-27

    Background: Overexpression of proangiogenic vascular endothelial growth factor A family VEGFAxxx is associated with tumor growth and metastasis. The role of the alternatively spliced antiangiogenic family VEGFAxxxb is poorly investigated in head and neck squamous cell carcinomas (HNSCCs). The antiangiogenic isoform binds to bevacizumab and its expression level could influence the treatment response and progression-free survival. In this study, the relative expression of VEGFAxxx and VEGFA165b isoforms and splicing regulatory factors genes was investigated in a series of HNSCCs. Methods: VEGFAxxx, VEGFA165b, SRSF6, SRSF5, SRSF1 and SRPK1 gene expression was quantified by quantitative real time PCR in 53 tissue samples obtained by surgery from HNSCC patients. Protein expression was evaluated by immunohistochemistry. Results: VEGFAxxx and VEGFA165b were overexpressed in HNSCCs. Elevated protein expression was also confirmed. However, VEGFA isoforms demonstrated differential expression according to anatomical sites. VEGFAxxx was overexpressed in pharyngeal tumors while the VEGFA165b isoform was up-regulated in oral tumors. The VEGFA165b isoform was also positively correlated with expression of the splicing regulatory genes SRSF1, SRSF6 and SRSF5. Conclusions: We concluded that VEGFAxxx and VEGFA165b isoforms are overexpressed in HNSCCs and the splicing regulatory factors SRSF1, SRSF6, SRSF5 and SRPK1 may contribute to alternative splicing of the VEGFA gene. The findings for the differential expression of the antiangiogenic isoform in HNSCCs could facilitate effective therapeutic strategies for the management of these tumors. Creative Commons Attribution License

  3. Alternative splicing may be responsible for heterogeneity of thyroglobulin structure.

    PubMed

    Mercken, L; Simons, M J; Brocas, H; Vassart, G

    1989-02-01

    During the cloning of the bovine thyroglobulin cDNA, the restriction map of one of the recombinant plasmids was in disagreement with that of the full-length double-stranded thyroglobulin cDNA. When compared to the bovine Tg mRNA sequence, this cDNA clone exhibits a 333-nucleotide deletion which corresponds precisely to 2 exons of the Tg gene. It is thus likely that alternative processing of the premessenger RNA is at the origin of the deletion. The presence of giant introns in the vicinity of the dispensable exons may also reflect some error level in the splicing mechanism. Together with previous results the alternative splicing described in this study indicates that alternative processing of the Tg transcripts may be at the origin of thyroglobulin isoforms.

  4. SplicePie: a novel analytical approach for the detection of alternative, non-sequential and recursive splicing.

    PubMed

    Pulyakhina, Irina; Gazzoli, Isabella; 't Hoen, Peter A C; Verwey, Nisha; den Dunnen, Johan T; den Dunnen, Johan; Aartsma-Rus, Annemieke; Laros, Jeroen F J

    2015-07-13

    Alternative splicing is a powerful mechanism present in eukaryotic cells to obtain a wide range of transcripts and protein isoforms from a relatively small number of genes. The mechanisms regulating (alternative) splicing and the paradigm of consecutive splicing have recently been challenged, especially for genes with a large number of introns. RNA-Seq, a powerful technology using deep sequencing in order to determine transcript structure and expression levels, is usually performed on mature mRNA, therefore not allowing detailed analysis of splicing progression. Sequencing pre-mRNA at different stages of splicing potentially provides insight into mRNA maturation. Although the number of tools that analyze total and cytoplasmic RNA in order to elucidate the transcriptome composition is rapidly growing, there are no tools specifically designed for the analysis of nuclear RNA (which contains mixtures of pre- and mature mRNA). We developed dedicated algorithms to investigate the splicing process. In this paper, we present a new classification of RNA-Seq reads based on three major stages of splicing: pre-, intermediate- and post-splicing. Applying this novel classification we demonstrate the possibility to analyze the order of splicing. Furthermore, we uncover the potential to investigate the multi-step nature of splicing, assessing various types of recursive splicing events. We provide the data that gives biological insight into the order of splicing, show that non-sequential splicing of certain introns is reproducible and coinciding in multiple cell lines. We validated our observations with independent experimental technologies and showed the reliability of our method. The pipeline, named SplicePie, is freely available at: https://github.com/pulyakhina/splicing_analysis_pipeline. The example data can be found at: https://barmsijs.lumc.nl/HG/irina/example_data.tar.gz.

  5. Myocardial alternative RNA splicing and gene expression profiling in early stage hypoplastic left heart syndrome.

    PubMed

    Ricci, Marco; Xu, Yanji; Hammond, Harriet L; Willoughby, David A; Nathanson, Lubov; Rodriguez, Maria M; Vatta, Matteo; Lipshultz, Steven E; Lincoln, Joy

    2012-01-01

    Hypoplastic Left Heart Syndrome (HLHS) is a congenital defect characterized by underdevelopment of the left ventricle and pathological compensation of the right ventricle. If untreated, HLHS is invariably lethal due to the extensive increase in right ventricular workload and eventual failure. Despite the clinical significance, little is known about the molecular pathobiological state of HLHS. Splicing of mRNA transcripts is an important regulatory mechanism of gene expression. Tissue specific alterations of this process have been associated with several cardiac diseases, however, transcriptional signature profiles related to HLHS are unknown. In this study, we performed genome-wide exon array analysis to determine differentially expressed genes and alternatively spliced transcripts in the right ventricle (RV) of six neonates with HLHS, compared to the RV and left ventricle (LV) from non-diseased control subjects. In HLHS, over 180 genes were differentially expressed and 1800 were differentially spliced, leading to changes in a variety of biological processes involving cell metabolism, cytoskeleton, and cell adherence. Additional hierarchical clustering analysis revealed that differential gene expression and mRNA splicing patterns identified in HLHS are unique compared to non-diseased tissue. Our findings suggest that gene expression and mRNA splicing are broadly dysregulated in the RV myocardium of HLHS neonates. In addition, our analysis identified transcriptome profiles representative of molecular biomarkers of HLHS that could be used in the future for diagnostic and prognostic stratification to improve patient outcome.

  6. Leveraging transcript quantification for fast computation of alternative splicing profiles

    PubMed Central

    Alamancos, Gael P.; Pagès, Amadís; Trincado, Juan L.; Bellora, Nicolás; Eyras, Eduardo

    2015-01-01

    Alternative splicing plays an essential role in many cellular processes and bears major relevance in the understanding of multiple diseases, including cancer. High-throughput RNA sequencing allows genome-wide analyses of splicing across multiple conditions. However, the increasing number of available data sets represents a major challenge in terms of computation time and storage requirements. We describe SUPPA, a computational tool to calculate relative inclusion values of alternative splicing events, exploiting fast transcript quantification. SUPPA accuracy is comparable and sometimes superior to standard methods using simulated as well as real RNA-sequencing data compared with experimentally validated events. We assess the variability in terms of the choice of annotation and provide evidence that using complete transcripts rather than more transcripts per gene provides better estimates. Moreover, SUPPA coupled with de novo transcript reconstruction methods does not achieve accuracies as high as using quantification of known transcripts, but remains comparable to existing methods. Finally, we show that SUPPA is more than 1000 times faster than standard methods. Coupled with fast transcript quantification, SUPPA provides inclusion values at a much higher speed than existing methods without compromising accuracy, thereby facilitating the systematic splicing analysis of large data sets with limited computational resources. The software is implemented in Python 2.7 and is available under the MIT license at https://bitbucket.org/regulatorygenomicsupf/suppa. PMID:26179515

  7. Role of the ubiquitin-like protein Hub1 in splice-site usage and alternative splicing

    PubMed Central

    Mishra, Shravan Kumar; Ammon, Tim; Popowicz, Grzegorz M.; Krajewski, Marcin; Nagel, Roland J.; Ares, Manuel; Holak, Tad A.; Jentsch, Stefan

    2013-01-01

    Alternative splicing of pre-messenger RNAs diversifies gene products in eukaryotes and is guided by factors that enable spliceosomes to recognize particular splice sites. Here we report that alternative splicing of Saccharomyces cerevisiae SRC1 pre-mRNA is promoted by the conserved ubiquitin-like protein Hub1. Structural and biochemical data show that Hub1 binds non-covalently to a conserved element termed HIND, which is present in the spliceosomal protein Snu66 in yeast and mammals, and Prp38 in plants. Hub1 binding mildly alters spliceosomal protein interactions and barely affects general splicing in S. cerevisiae. However, spliceosomes that lack Hub1, or are defective in Hub1–HIND interaction, cannot use certain non-canonical 5′ splice sites and are defective in alternative SRC1 splicing. Hub1 confers alternative splicing not only when bound to HIND, but also when experimentally fused to Snu66, Prp38, or even the core splicing factor Prp8. Our study indicates a novel mechanism for splice site utilization that is guided by non-covalent modification of the spliceosome by an unconventional ubiquitin-like modifier. PMID:21614000

  8. Transcriptome survey reveals increased complexity of the alternative splicing landscape in Arabidopsis

    PubMed Central

    Marquez, Yamile; Brown, John W.S.; Simpson, Craig; Barta, Andrea; Kalyna, Maria

    2012-01-01

    Alternative splicing (AS) is a key regulatory mechanism that contributes to transcriptome and proteome diversity. As very few genome-wide studies analyzing AS in plants are available, we have performed high-throughput sequencing of a normalized cDNA library which resulted in a high coverage transcriptome map of Arabidopsis. We detect ∼150,000 splice junctions derived mostly from typical plant introns, including an eightfold increase in the number of U12 introns (2069). Around 61% of multiexonic genes are alternatively spliced under normal growth conditions. Moreover, we provide experimental validation of 540 AS transcripts (from 256 genes coding for important regulatory factors) using high-resolution RT-PCR and Sanger sequencing. Intron retention (IR) is the most frequent AS event (∼40%), but many IRs have relatively low read coverage and are less well-represented in assembled transcripts. Additionally, ∼51% of Arabidopsis genes produce AS transcripts which do not involve IR. Therefore, the significance of IR in generating transcript diversity was generally overestimated in previous assessments. IR analysis allowed the identification of a large set of cryptic introns inside annotated coding exons. Importantly, a significant fraction of these cryptic introns are spliced out in frame, indicating a role in protein diversity. Furthermore, we show extensive AS coupled to nonsense-mediated decay in AFC2, encoding a highly conserved LAMMER kinase which phosphorylates splicing factors, thus establishing a complex loop in AS regulation. We provide the most comprehensive analysis of AS to date which will serve as a valuable resource for the plant community to study transcriptome complexity and gene regulation. PMID:22391557

  9. Emerging role of alternative splicing of CRF1 receptor in CRF signaling

    PubMed Central

    Ẓmijewski, Michał A.; Slominski, Andrzej T.

    2010-01-01

    Alternative splicing of mRNA is one of the most important mechanisms responsible for an increase of the genomic capacity. Thus the majority of human proteins including G protein-coupled receptors (GPCRs) possess several isoforms as a result of mRNA splicing. The corticotropin-releasing factor (CRF) and its receptors are the most proximal elements of hypothalamic-pituitary-adrenal axis (HPA) — the central machinery of stress response. Moreover, expression of CRF and regulated activity of CRF receptor type 1 (CRF1) can also play an important role in regulation of local stress response in peripheral tissues including skin, gastrointestinal tract or reproductive system. In humans, expression of at least eight variants of CRF1 mRNA (α, β, c, d, e, f, g and h) was detected and alternative splicing was found to be regulated by diverse physiological and pathological factors including: growth conditions, onset of labor, during pregnancy or exposure to ultraviolet irradiation. The pattern of expression of CRF1 isoforms is cell type specific and recently has been linked to observed differences in responsiveness to CRF stimulation. In the proposed model of regulation of CRF-signaling, isoform CRF1α plays a central role. Other isoforms modulate its activity by oligomerization, leading to alteration in receptor trafficking, localization and function. Co-expression of CRF1 isoforms modulates sensitivity of cells to the ligands and influences downstream coupling to G-proteins. The other possible regulatory mechanisms include fast mRNA and/or protein turnover or decoy receptor function of CRF1 isoforms. Taken together, alternative splicing of CRF1 can represent another level of regulation of CRF-mediated stress responses at the central and peripheral levels. Chronic stress or malfunction of the HPA-axis have been linked to numerous human pathologies, suggesting that alternative splicing of CRF1 receptor could represent a promising target for drugs development. PMID:20234885

  10. Emerging role of alternative splicing of CRF1 receptor in CRF signaling.

    PubMed

    Zmijewski, Michał A; Slominski, Andrzej T

    2010-01-01

    Alternative splicing of mRNA is one of the most important mechanisms responsible for an increase of the genomic capacity. Thus the majority of human proteins including G protein-coupled receptors (GPCRs) possess several isoforms as a result of mRNA splicing. The corticotropin-releasing factor (CRF) and its receptors are the most proximal elements of hypothalamic-pituitary-adrenal axis (HPA) - the central machinery of stress response. Moreover, expression of CRF and regulated activity of CRF receptor type 1 (CRF1) can also play an important role in regulation of local stress response in peripheral tissues including skin, gastrointestinal tract or reproductive system. In humans, expression of at least eight variants of CRF1 mRNA (alpha, beta, c, d, e, f, g and h) was detected and alternative splicing was found to be regulated by diverse physiological and pathological factors including: growth conditions, onset of labor, during pregnancy or exposure to ultraviolet irradiation. The pattern of expression of CRF1 isoforms is cell type specific and recently has been linked to observed differences in responsiveness to CRF stimulation. In the proposed model of regulation of CRF-signaling, isoform CRF1alpha plays a central role. Other isoforms modulate its activity by oligomerization, leading to alteration in receptor trafficking, localization and function. Co-expression of CRF1 isoforms modulates sensitivity of cells to the ligands and influences downstream coupling to G-proteins. The other possible regulatory mechanisms include fast mRNA and/or protein turnover or decoy receptor function of CRF1 isoforms. Taken together, alternative splicing of CRF1 can represent another level of regulation of CRF-mediated stress responses at the central and peripheral levels. Chronic stress or malfunction of the HPA-axis have been linked to numerous human pathologies, suggesting that alternative splicing of CRF1 receptor could represent a promising target for drugs development.

  11. Alternative Splicing Governs Cone Cyclic Nucleotide-gated (CNG) Channel Sensitivity to Regulation by Phosphoinositides*

    PubMed Central

    Dai, Gucan; Sherpa, Tshering; Varnum, Michael D.

    2014-01-01

    Precursor mRNA encoding CNGA3 subunits of cone photoreceptor cyclic nucleotide-gated (CNG) channels undergoes alternative splicing, generating isoforms differing in the N-terminal cytoplasmic region of the protein. In humans, four variants arise from alternative splicing, but the functional significance of these changes has been a persistent mystery. Heterologous expression of the four possible CNGA3 isoforms alone or with CNGB3 subunits did not reveal significant differences in basic channel properties. However, inclusion of optional exon 3, with or without optional exon 5, produced heteromeric CNGA3 + CNGB3 channels exhibiting an ∼2-fold greater shift in K1/2,cGMP after phosphatidylinositol 4,5-biphosphate or phosphatidylinositol 3,4,5-trisphosphate application compared with channels lacking the sequence encoded by exon 3. We have previously identified two structural features within CNGA3 that support phosphoinositides (PIPn) regulation of cone CNG channels: N- and C-terminal regulatory modules. Specific mutations within these regions eliminated PIPn sensitivity of CNGA3 + CNGB3 channels. The exon 3 variant enhanced the component of PIPn regulation that depends on the C-terminal region rather than the nearby N-terminal region, consistent with an allosteric effect on PIPn sensitivity because of altered N-C coupling. Alternative splicing of CNGA3 occurs in multiple species, although the exact variants are not conserved across CNGA3 orthologs. Optional exon 3 appears to be unique to humans, even compared with other primates. In parallel, we found that a specific splice variant of canine CNGA3 removes a region of the protein that is necessary for high sensitivity to PIPn. CNGA3 alternative splicing may have evolved, in part, to tune the interactions between cone CNG channels and membrane-bound phosphoinositides. PMID:24675082

  12. Specific CLK Inhibitors from a Novel Chemotype for Regulation of Alternative Splicing

    PubMed Central

    Fedorov, Oleg; Huber, Kilian; Eisenreich, Andreas; Filippakopoulos, Panagis; King, Oliver; Bullock, Alex N.; Szklarczyk, Damian; Jensen, Lars J.; Fabbro, Doriano; Trappe, Jörg; Rauch, Ursula; Bracher, Franz; Knapp, Stefan

    2011-01-01

    Summary There is a growing recognition of the importance of protein kinases in the control of alternative splicing. To define the underlying regulatory mechanisms, highly selective inhibitors are needed. Here, we report the discovery and characterization of the dichloroindolyl enaminonitrile KH-CB19, a potent and highly specific inhibitor of the CDC2-like kinase isoforms 1 and 4 (CLK1/CLK4). Cocrystal structures of KH-CB19 with CLK1 and CLK3 revealed a non-ATP mimetic binding mode, conformational changes in helix αC and the phosphate binding loop and halogen bonding to the kinase hinge region. KH-CB19 effectively suppressed phosphorylation of SR (serine/arginine) proteins in cells, consistent with its expected mechanism of action. Chemical inhibition of CLK1/CLK4 generated a unique pattern of splicing factor dephosphorylation and had at low nM concentration a profound effect on splicing of the two tissue factor isoforms flTF (full-length TF) and asHTF (alternatively spliced human TF). PMID:21276940

  13. Arabidopsis PTB1 and PTB2 proteins negatively regulate splicing of a mini-exon splicing reporter and affect alternative splicing of endogenous genes differentially.

    PubMed

    Simpson, Craig G; Lewandowska, Dominika; Liney, Michele; Davidson, Diane; Chapman, Sean; Fuller, John; McNicol, Jim; Shaw, Paul; Brown, John W S

    2014-07-01

    This paper examines the function of Arabidopsis thaliana AtPTB1 and AtPTB2 as plant splicing factors. The effect on splicing of overexpression of AtPTB1 and AtPTB2 was analysed in an in vivo protoplast transient expression system with a novel mini-exon splicing reporter. A range of mutations in pyrimidine-rich sequences were compared with and without AtPTB and NpU2AF65 overexpression. Splicing analyses of constructs in protoplasts and RNA from overexpression lines used high-resolution reverse transcription polymerase chain reaction (RT-PCR). AtPTB1 and AtPTB2 reduced inclusion/splicing of the potato invertase mini-exon splicing reporter, indicating that these proteins can repress plant intron splicing. Mutation of the polypyrimidine tract and closely associated Cytosine and Uracil-rich (CU-rich) sequences, upstream of the mini-exon, altered repression by AtPTB1 and AtPTB2. Coexpression of a plant orthologue of U2AF65 alleviated the splicing repression of AtPTB1. Mutation of a second CU-rich upstream of the mini-exon 3' splice site led to a decline in mini-exon splicing, indicating the presence of a splicing enhancer sequence. Finally, RT-PCR of AtPTB overexpression lines with c. 90 known alternative splicing (AS) events showed that AtPTBs significantly altered AS of over half the events. AtPTB1 and AtPTB2 are splicing factors that influence alternative splicing. This occurs in the potato invertase mini-exon via the polypyrimidine tract and associated pyrimidine-rich sequence.

  14. Involvement of Alternative Splicing in Barley Seed Germination

    PubMed Central

    Zhang, Qisen; Zhang, Xiaoqi; Wang, Songbo; Tan, Cong; Zhou, Gaofeng; Li, Chengdao

    2016-01-01

    Seed germination activates many new biological processes including DNA, membrane and mitochondrial repairs and requires active protein synthesis and sufficient energy supply. Alternative splicing (AS) regulates many cellular processes including cell differentiation and environmental adaptations. However, limited information is available on the regulation of seed germination at post-transcriptional levels. We have conducted RNA-sequencing experiments to dissect AS events in barley seed germination. We identified between 552 and 669 common AS transcripts in germinating barley embryos from four barley varieties (Hordeum vulgare L. Bass, Baudin, Harrington and Stirling). Alternative 3’ splicing (34%-45%), intron retention (32%-34%) and alternative 5’ splicing (16%-21%) were three major AS events in germinating embryos. The AS transcripts were predominantly mapped onto ribosome, RNA transport machineries, spliceosome, plant hormone signal transduction, glycolysis, sugar and carbon metabolism pathways. Transcripts of these genes were also very abundant in the early stage of seed germination. Correlation analysis of gene expression showed that AS hormone responsive transcripts could also be co-expressed with genes responsible for protein biosynthesis and sugar metabolisms. Our RNA-sequencing data revealed that AS could play important roles in barley seed germination. PMID:27031341

  15. Alternative Splicing of G9a Regulates Neuronal Differentiation.

    PubMed

    Fiszbein, Ana; Giono, Luciana E; Quaglino, Ana; Berardino, Bruno G; Sigaut, Lorena; von Bilderling, Catalina; Schor, Ignacio E; Steinberg, Juliana H Enriqué; Rossi, Mario; Pietrasanta, Lía I; Caramelo, Julio J; Srebrow, Anabella; Kornblihtt, Alberto R

    2016-03-29

    Chromatin modifications are critical for the establishment and maintenance of differentiation programs. G9a, the enzyme responsible for histone H3 lysine 9 dimethylation in mammalian euchromatin, exists as two isoforms with differential inclusion of exon 10 (E10) through alternative splicing. We find that the G9a methyltransferase is required for differentiation of the mouse neuronal cell line N2a and that E10 inclusion increases during neuronal differentiation of cultured cells, as well as in the developing mouse brain. Although E10 inclusion greatly stimulates overall H3K9me2 levels, it does not affect G9a catalytic activity. Instead, E10 increases G9a nuclear localization. We show that the G9a E10(+) isoform is necessary for neuron differentiation and regulates the alternative splicing pattern of its own pre-mRNA, enhancing E10 inclusion. Overall, our findings indicate that by regulating its own alternative splicing, G9a promotes neuron differentiation and creates a positive feedback loop that reinforces cellular commitment to differentiation.

  16. Involvement of Alternative Splicing in Barley Seed Germination.

    PubMed

    Zhang, Qisen; Zhang, Xiaoqi; Wang, Songbo; Tan, Cong; Zhou, Gaofeng; Li, Chengdao

    2016-01-01

    Seed germination activates many new biological processes including DNA, membrane and mitochondrial repairs and requires active protein synthesis and sufficient energy supply. Alternative splicing (AS) regulates many cellular processes including cell differentiation and environmental adaptations. However, limited information is available on the regulation of seed germination at post-transcriptional levels. We have conducted RNA-sequencing experiments to dissect AS events in barley seed germination. We identified between 552 and 669 common AS transcripts in germinating barley embryos from four barley varieties (Hordeum vulgare L. Bass, Baudin, Harrington and Stirling). Alternative 3' splicing (34%-45%), intron retention (32%-34%) and alternative 5' splicing (16%-21%) were three major AS events in germinating embryos. The AS transcripts were predominantly mapped onto ribosome, RNA transport machineries, spliceosome, plant hormone signal transduction, glycolysis, sugar and carbon metabolism pathways. Transcripts of these genes were also very abundant in the early stage of seed germination. Correlation analysis of gene expression showed that AS hormone responsive transcripts could also be co-expressed with genes responsible for protein biosynthesis and sugar metabolisms. Our RNA-sequencing data revealed that AS could play important roles in barley seed germination.

  17. Abnormalities in alternative splicing in diabetes: therapeutic targets.

    PubMed

    Dlamini, Zodwa; Mokoena, Fortunate; Hull, Rodney

    2017-08-01

    Diabetes mellitus (DM) is a non-communicable, metabolic disorder that affects 416 million individuals worldwide. Type 2 diabetes contributes to a vast 85-90% of the diabetes incidences while 10-15% of patients suffer from type 1 diabetes. These two predominant forms of DM cause a significant loss of functional pancreatic β-cell mass causing different degrees of insulin deficiency, most likely, due to increased β-cell apoptosis. Treatment options involve the use of insulin sensitisers, α-glucosidase inhibitors, and β-cell secretagogues which are often expensive, limited in efficacy and carry detrimental adverse effects. Cost-effective options for treatment exists in the form of herbal drugs, however, scientific validations of these widely used medicinal plants are still underway. Alternative splicing (AS) is a co-ordinated post-transcriptional process in which a single gene generates multiple mRNA transcripts which results in increased amounts of functionally different protein isoforms and in some cases aberrant splicing leads to metabolic disease. In this review, we explore the association of AS with metabolic alterations in DM and the biological significance of the abnormal splicing of some pathogenic diabetes-related genes. An understanding of the molecular mechanism behind abnormally spliced transcripts will aid in the development of new diagnostic, prognostic and therapeutic tools. © 2017 Society for Endocrinology.

  18. The Evolutionary Relationship between Alternative Splicing and Gene Duplication.

    PubMed

    Iñiguez, Luis P; Hernández, Georgina

    2017-01-01

    The protein diversity that exists today has resulted from various evolutionary processes. It is well known that gene duplication (GD) along with the accumulation of mutations are responsible, among other factors, for an increase in the number of different proteins. The gene structure in eukaryotes requires the removal of non-coding sequences, introns, to produce mature mRNAs. This process, known as cis-splicing, referred to here as splicing, is regulated by several factors which can lead to numerous splicing arrangements, commonly designated as alternative splicing (AS). AS, producing several transcripts isoforms form a single gene, also increases the protein diversity. However, the evolution and manner for increasing protein variation differs between AS and GD. An important question is how are patterns of AS affected after a GD event. Here, we review the current knowledge of AS and GD, focusing on their evolutionary relationship. These two processes are now considered the main contributors to the increasing protein diversity and therefore their relationship is a relevant, yet understudied, area of evolutionary study.

  19. The Evolutionary Relationship between Alternative Splicing and Gene Duplication

    PubMed Central

    Iñiguez, Luis P.; Hernández, Georgina

    2017-01-01

    The protein diversity that exists today has resulted from various evolutionary processes. It is well known that gene duplication (GD) along with the accumulation of mutations are responsible, among other factors, for an increase in the number of different proteins. The gene structure in eukaryotes requires the removal of non-coding sequences, introns, to produce mature mRNAs. This process, known as cis-splicing, referred to here as splicing, is regulated by several factors which can lead to numerous splicing arrangements, commonly designated as alternative splicing (AS). AS, producing several transcripts isoforms form a single gene, also increases the protein diversity. However, the evolution and manner for increasing protein variation differs between AS and GD. An important question is how are patterns of AS affected after a GD event. Here, we review the current knowledge of AS and GD, focusing on their evolutionary relationship. These two processes are now considered the main contributors to the increasing protein diversity and therefore their relationship is a relevant, yet understudied, area of evolutionary study. PMID:28261262

  20. Modification of Akt by SUMO conjugation regulates alternative splicing and cell cycle

    PubMed Central

    Risso, Guillermo; Pelisch, Federico; Pozzi, Berta; Mammi, Pablo; Blaustein, Matías; Colman-Lerner, Alejandro; Srebrow, Anabella

    2013-01-01

    Akt/PKB is a key signaling molecule in higher eukaryotes and a crucial protein kinase in human health and disease. Phosphorylation, acetylation, and ubiquitylation have been reported as important regulatory post-translational modifications of this kinase. We describe here that Akt is modified by SUMO conjugation, and show that lysine residues 276 and 301 are the major SUMO attachment sites within this protein. We found that phosphorylation and SUMOylation of Akt appear as independent events. However, decreasing Akt SUMOylation levels severely affects the role of this kinase as a regulator of fibronectin and Bcl-x alternative splicing. Moreover, we observed that the Akt mutant (Akt E17K) found in several human tumors displays increased levels of SUMOylation and also an enhanced capacity to regulate fibronectin splicing patterns. This splicing regulatory activity is completely abolished by decreasing Akt E17K SUMO conjugation levels. Additionally, we found that SUMOylation controls Akt regulatory function at G₁/S transition during cell cycle progression. These findings reveal SUMO conjugation as a novel level of regulation for Akt activity, opening new areas of exploration related to the molecular mechanisms involved in the diverse cellular functions of this kinase. PMID:24013425

  1. Alternative splicing as a biomarker and potential target for drug discovery.

    PubMed

    Le, Kai-qin; Prabhakar, Bellur S; Hong, Wan-jin; Li, Liang-cheng

    2015-10-01

    Alternative splicing is a key process of multi-exonic gene expression during pre-mRNA maturation. In this process, particular exons of a gene will be included within or excluded from the final matured mRNA, and the resulting transcripts generate diverse protein isoforms. Recent evidence demonstrates that approximately 95% of human genes with multiple exons undergo alternative splicing during pre-mRNA maturation. Thus, alternative splicing plays a critical role in physiological processes and cell development programs, and.dysregulation of alternative splicing is highly associated with human diseases, such as cancer, diabetes and neurodegenerative diseases. In this review, we discuss the regulation of alternative splicing, examine the relationship between alternative splicing and human diseases, and describe several approaches that modify alternative splicing, which could aid in human disease diagnosis and therapy.

  2. Control of fibroblast fibronectin expression and alternative splicing via the PI3K/Akt/mTOR pathway

    SciTech Connect

    White, Eric S.; Sagana, Rommel L.; Booth, Adam J.; Yan, Mei; Cornett, Ashley M.; Bloomheart, Christopher A.; Tsui, Jessica L.; Wilke, Carol A.; Moore, Bethany B.; Ritzenthaler, Jeffrey D.; Roman, Jesse; Muro, Andres F.

    2010-10-01

    Fibronectin (FN), a ubiquitous glycoprotein that plays critical roles in physiologic and pathologic conditions, undergoes alternative splicing which distinguishes plasma FN (pFN) from cellular FN (cFN). Although both pFN and cFN can be incorporated into the extracellular matrix, a distinguishing feature of cFN is the inclusion of an alternatively spliced exon termed EDA (for extra type III domain A). The molecular steps involved in EDA splicing are well-characterized, but pathways influencing EDA splicing are less clear. We have previously found an obligate role for inhibition of the tumor suppressor phosphatase and tensin homologue on chromosome 10 (PTEN), the primary regulator of the PI3K/Akt pathway, in fibroblast activation. Here we show TGF-{beta}, a potent inducer of both EDA splicing and fibroblast activation, inhibits PTEN expression and activity in mesenchymal cells, corresponding with enhanced PI3K/Akt signaling. In pten{sup -/-} fibroblasts, which resemble activated fibroblasts, inhibition of Akt attenuated FN production and decreased EDA alternative splicing. Moreover, inhibition of mammalian target of rapamycin (mTOR) in pten{sup -/-} cells also blocked FN production and EDA splicing. This effect was due to inhibition of Akt-mediated phosphorylation of the primary EDA splicing regulatory protein SF2/ASF. Importantly, FN silencing in pten{sup -/-} cells resulted in attenuated proliferation and migration. Thus, our results demonstrate that the PI3K/Akt/mTOR axis is instrumental in FN transcription and alternative splicing, which regulates cell behavior.

  3. Computational Identification of Tissue-Specific Splicing Regulatory Elements in Human Genes from RNA-Seq Data

    PubMed Central

    Badr, Eman; ElHefnawi, Mahmoud; Heath, Lenwood S.

    2016-01-01

    Alternative splicing is a vital process for regulating gene expression and promoting proteomic diversity. It plays a key role in tissue-specific expressed genes. This specificity is mainly regulated by splicing factors that bind to specific sequences called splicing regulatory elements (SREs). Here, we report a genome-wide analysis to study alternative splicing on multiple tissues, including brain, heart, liver, and muscle. We propose a pipeline to identify differential exons across tissues and hence tissue-specific SREs. In our pipeline, we utilize the DEXSeq package along with our previously reported algorithms. Utilizing the publicly available RNA-Seq data set from the Human BodyMap project, we identified 28,100 differentially used exons across the four tissues. We identified tissue-specific exonic splicing enhancers that overlap with various previously published experimental and computational databases. A complicated exonic enhancer regulatory network was revealed, where multiple exonic enhancers were found across multiple tissues while some were found only in specific tissues. Putative combinatorial exonic enhancers and silencers were discovered as well, which may be responsible for exon inclusion or exclusion across tissues. Some of the exonic enhancers are found to be co-occurring with multiple exonic silencers and vice versa, which demonstrates a complicated relationship between tissue-specific exonic enhancers and silencers. PMID:27861625

  4. Computational Identification of Tissue-Specific Splicing Regulatory Elements in Human Genes from RNA-Seq Data.

    PubMed

    Badr, Eman; ElHefnawi, Mahmoud; Heath, Lenwood S

    2016-01-01

    Alternative splicing is a vital process for regulating gene expression and promoting proteomic diversity. It plays a key role in tissue-specific expressed genes. This specificity is mainly regulated by splicing factors that bind to specific sequences called splicing regulatory elements (SREs). Here, we report a genome-wide analysis to study alternative splicing on multiple tissues, including brain, heart, liver, and muscle. We propose a pipeline to identify differential exons across tissues and hence tissue-specific SREs. In our pipeline, we utilize the DEXSeq package along with our previously reported algorithms. Utilizing the publicly available RNA-Seq data set from the Human BodyMap project, we identified 28,100 differentially used exons across the four tissues. We identified tissue-specific exonic splicing enhancers that overlap with various previously published experimental and computational databases. A complicated exonic enhancer regulatory network was revealed, where multiple exonic enhancers were found across multiple tissues while some were found only in specific tissues. Putative combinatorial exonic enhancers and silencers were discovered as well, which may be responsible for exon inclusion or exclusion across tissues. Some of the exonic enhancers are found to be co-occurring with multiple exonic silencers and vice versa, which demonstrates a complicated relationship between tissue-specific exonic enhancers and silencers.

  5. Reduced fidelity of branch point recognition and alternative splicing induced by the anti-tumor drug spliceostatin A

    PubMed Central

    Corrionero, Anna; Miñana, Belén; Valcárcel, Juan

    2011-01-01

    Spliceostatin A (SSA) is a stabilized derivative of a Pseudomonas bacterial fermentation product that displays potent anti-proliferative and anti-tumor activities in cancer cells and animal models. The drug inhibits pre-mRNA splicing in vitro and in vivo and binds SF3b, a protein subcomplex of U2 small nuclear ribonucleoprotein (snRNP), which is essential for recognition of the pre-mRNA branch point. We report that SSA prevents interaction of an SF3b 155-kDa subunit with the pre-mRNA, concomitant with nonproductive recruitment of U2 snRNP to sequences 5′ of the branch point. Differences in base-pairing potential with U2 snRNA in this region lead to different sensitivity of 3′ splice sites to SSA, and to SSA-induced changes in alternative splicing. Indeed, rather than general splicing inhibition, splicing-sensitive microarray analyses reveal specific alternative splicing changes induced by the drug that significantly overlap with those induced by knockdown of SF3b 155. These changes lead to down-regulation of genes important for cell division, including cyclin A2 and Aurora A kinase, thus providing an explanation for the anti-proliferative effects of SSA. Our results reveal a mechanism that prevents nonproductive base-pairing interactions in the spliceosome, and highlight the regulatory and cancer therapeutic potential of perturbing the fidelity of splice site recognition. PMID:21363963

  6. Poliovirus 2A protease triggers a selective nucleo-cytoplasmic redistribution of splicing factors to regulate alternative pre-mRNA splicing.

    PubMed

    Álvarez, Enrique; Castelló, Alfredo; Carrasco, Luis; Izquierdo, José M

    2013-01-01

    Poliovirus protease 2A (2A(pro)) obstructs host gene expression by reprogramming transcriptional and post-transcriptional regulatory events during infection. Here we demonstrate that expression of 2A(pro) induces a selective nucleo-cytoplasm translocation of several important RNA binding proteins and splicing factors. Subcellular fractionation studies, together with immunofluorescence microscopy revealed an asymmetric distribution of HuR and TIA1/TIAR in 2A(pro) expressing cells, which modulates splicing of the human Fas exon 6. Consistent with this result, knockdown of HuR or overexpression of TIA1/TIAR, leads to Fas exon 6 inclusion in 2A(pro)-expressing cells. Therefore, poliovirus 2A(pro) can target alternative pre-mRNA splicing by regulating protein shuttling between the nucleus and the cytoplasm.

  7. A general definition and nomenclature for alternative splicing events.

    PubMed

    Sammeth, Michael; Foissac, Sylvain; Guigó, Roderic

    2008-08-08

    Understanding the molecular mechanisms responsible for the regulation of the transcriptome present in eukaryotic cells is one of the most challenging tasks in the postgenomic era. In this regard, alternative splicing (AS) is a key phenomenon contributing to the production of different mature transcripts from the same primary RNA sequence. As a plethora of different transcript forms is available in databases, a first step to uncover the biology that drives AS is to identify the different types of reflected splicing variation. In this work, we present a general definition of the AS event along with a notation system that involves the relative positions of the splice sites. This nomenclature univocally and dynamically assigns a specific "AS code" to every possible pattern of splicing variation. On the basis of this definition and the corresponding codes, we have developed a computational tool (AStalavista) that automatically characterizes the complete landscape of AS events in a given transcript annotation of a genome, thus providing a platform to investigate the transcriptome diversity across genes, chromosomes, and species. Our analysis reveals that a substantial part--in human more than a quarter-of the observed splicing variations are ignored in common classification pipelines. We have used AStalavista to investigate and to compare the AS landscape of different reference annotation sets in human and in other metazoan species and found that proportions of AS events change substantially depending on the annotation protocol, species-specific attributes, and coding constraints acting on the transcripts. The AStalavista system therefore provides a general framework to conduct specific studies investigating the occurrence, impact, and regulation of AS.

  8. A General Definition and Nomenclature for Alternative Splicing Events

    PubMed Central

    Guigó, Roderic

    2008-01-01

    Understanding the molecular mechanisms responsible for the regulation of the transcriptome present in eukaryotic cells is one of the most challenging tasks in the postgenomic era. In this regard, alternative splicing (AS) is a key phenomenon contributing to the production of different mature transcripts from the same primary RNA sequence. As a plethora of different transcript forms is available in databases, a first step to uncover the biology that drives AS is to identify the different types of reflected splicing variation. In this work, we present a general definition of the AS event along with a notation system that involves the relative positions of the splice sites. This nomenclature univocally and dynamically assigns a specific “AS code” to every possible pattern of splicing variation. On the basis of this definition and the corresponding codes, we have developed a computational tool (AStalavista) that automatically characterizes the complete landscape of AS events in a given transcript annotation of a genome, thus providing a platform to investigate the transcriptome diversity across genes, chromosomes, and species. Our analysis reveals that a substantial part—in human more than a quarter—of the observed splicing variations are ignored in common classification pipelines. We have used AStalavista to investigate and to compare the AS landscape of different reference annotation sets in human and in other metazoan species and found that proportions of AS events change substantially depending on the annotation protocol, species-specific attributes, and coding constraints acting on the transcripts. The AStalavista system therefore provides a general framework to conduct specific studies investigating the occurrence, impact, and regulation of AS. PMID:18688268

  9. A network-based analysis of colon cancer splicing changes reveals a tumorigenesis-favoring regulatory pathway emanating from ELK1

    PubMed Central

    Hollander, Dror; Donyo, Maya; Atias, Nir; Mekahel, Keren; Melamed, Zeev; Yannai, Sivan; Lev-Maor, Galit; Shilo, Asaf; Schwartz, Schraga; Barshack, Iris; Sharan, Roded; Ast, Gil

    2016-01-01

    Splicing aberrations are prominent drivers of cancer, yet the regulatory pathways controlling them are mostly unknown. Here we develop a method that integrates physical interaction, gene expression, and alternative splicing data to construct the largest map of transcriptomic and proteomic interactions leading to cancerous splicing aberrations defined to date, and identify driver pathways therein. We apply our method to colon adenocarcinoma and non-small-cell lung carcinoma. By focusing on colon cancer, we reveal a novel tumor-favoring regulatory pathway involving the induction of the transcription factor MYC by the transcription factor ELK1, as well as the subsequent induction of the alternative splicing factor PTBP1 by both. We show that PTBP1 promotes specific RAC1, NUMB, and PKM splicing isoforms that are major triggers of colon tumorigenesis. By testing the pathway's activity in patient tumor samples, we find ELK1, MYC, and PTBP1 to be overexpressed in conjunction with oncogenic KRAS mutations, and show that these mutations increase ELK1 levels via the RAS-MAPK pathway. We thus illuminate, for the first time, a full regulatory pathway connecting prevalent cancerous mutations to functional tumor-inducing splicing aberrations. Our results demonstrate our method is applicable to different cancers to reveal regulatory pathways promoting splicing aberrations. PMID:26860615

  10. Variation in sequence and organization of splicing regulatory elements in vertebrate genes

    PubMed Central

    Yeo, Gene; Hoon, Shawn; Venkatesh, Byrappa; Burge, Christopher B.

    2004-01-01

    Although core mechanisms and machinery of premRNA splicing are conserved from yeast to human, the details of intron recognition often differ, even between closely related organisms. For example, genes from the pufferfish Fugu rubripes generally contain one or more introns that are not properly spliced in mouse cells. Exploiting available genome sequence data, a battery of sequence analysis techniques was used to reach several conclusions about the organization and evolution of splicing regulatory elements in vertebrate genes. The classical splice site and putative branch site signals are completely conserved across the vertebrates studied (human, mouse, pufferfish, and zebrafish), and exonic splicing enhancers also appear broadly conserved in vertebrates. However, another class of splicing regulatory elements, the intronic splicing enhancers, appears to differ substantially between mammals and fish, with G triples (GGG) very abundant in mammalian introns but comparatively rare in fish. Conversely, short repeats of AC and GT are predicted to function as intronic splicing enhancers in fish but are not enriched in mammalian introns. Consistent with this pattern, exonic splicing enhancer-binding SR proteins are highly conserved across all vertebrates, whereas heterogeneous nuclear ribonucleoproteins, which bind many intronic sequences, vary in domain structure and even presence/absence between mammals and fish. Exploiting differences in intronic sequence composition, a statistical model was developed to predict the splicing phenotype of Fugu introns in mammalian systems and was used to engineer the spliceability of a Fugu intron in human cells by insertion of specific sequences, thereby rescuing splicing in human cells. PMID:15505203

  11. ASF/SF2-like maize pre-mRNA splicing factors affect splice site utilization and their transcripts are alternatively spliced.

    PubMed

    Gao, Huirong; Gordon-Kamm, William J; Lyznik, L Alexander

    2004-09-15

    Three ASF/SF2-like alternative splicing genes from maize were identified, cloned, and analyzed. Each of these genes (zmSRp30, zmSRp31, and zmSRp32) contains two RNA binding domains, a signature sequence SWQDLKD, and a characteristic serine/ariginine-rich domain. There is a strong structural similarity to the human ASF/SF2 splicing factor and to the Arabidopsis atSRp34/p30 proteins. Similar to ASF/SF2-like genes in other organisms, the maize pre-mRNA messages are alternatively spliced. They are differentially expressed in maize tissues with relatively uniform levels of zmSRp30 and zmSRp31 messages being observed throughout the plant, while zmSRp32 messages preferentially accumulated in the meristematic regions. Overexpression of zmSRp32 in maize cells leads to the enhanced selection of weak 5' intron splice sites during the processing of pre-mRNA molecules. Overexpression of the zmSRp31 or zmSRp32 gene affects regulation of wheat dwarf virus rep gene pre-mRNA splicing, presumably by interacting with the weak 5' splice site, CCGU. Our results suggest that the described genes are functional homologues of the human ASF/SF2 alternative splicing factor and they indicate a diversity of the ASF/SF2-like alternative splicing factors in monocot plant cells.

  12. Structure of the human gene and two rat cDNAs encoding the alpha chain of GTP-binding regulatory protein Go: two different mRNAs are generated by alternative splicing.

    PubMed Central

    Tsukamoto, T; Toyama, R; Itoh, H; Kozasa, T; Matsuoka, M; Kaziro, Y

    1991-01-01

    Go is a specific class ("other") of signal-transducing heterotrimeric GTP-binding proteins (G proteins) that is expressed in high levels in mammalian brain. We have cloned two different rat cDNAs encoding the alpha subunit of Go (Go alpha-1 and Go alpha-2) and a human Go alpha chromosomal gene. The human Go alpha gene spans more than 100 kilobases and contains 11 exons, including one noncoding exon in the 3' flanking region. The 5' flanking region is highly G + C-rich and contains five G.C boxes (Sp1 binding sites) but no TATA box. Exons 7 and 8 coding for amino acid residues 242-354 of Go alpha protein are duplicated (referred to as exons 7A, 7B, 8A, and 8B). It was found that exons 7A and 8A code for Go alpha-1, and 7B and 8B code for Go alpha-2. This indicates that two different Go alpha mRNAs may be generated by alternative splicing of a single Go alpha gene. The splice sites of the Go alpha-1 and Go alpha-2 genes are completely identical with those encoding human inhibitory G protein alpha subunits Gi2 alpha and Gi3 alpha [Itoh, H., Toyama, R., Kozasa, T., Tsukamoto, T., Matsuoka, M. & Kaziro, Y. (1988) J. Biol. Chem. 263, 6656-6664] and also transducin G protein alpha subunit Gt1 alpha [Raport, C. J., Dere, B. & Hurley, J. (1989) J. Biol. Chem. 264, 7122-7128]. Sequence homology and conservation of the exon-intron organization indicate that the genes coding for Go alpha, Gi2 alpha, Gi3 alpha, Gt1 alpha, and probably Gi1 alpha may be evolved from a common progenitor. Like Go alpha-1, Go alpha-2 is expressed mainly in brain. Images PMID:1901650

  13. Cell-autonomous regulation of fast troponin T pre-mRNA alternative splicing in response to mechanical stretch.

    PubMed

    Schilder, Rudolf J; Kimball, Scot R; Jefferson, Leonard S

    2012-08-01

    How mechanochemical signals induced by the amount of weight borne by the skeletal musculature are translated into modifications to muscle sarcomeres is poorly understood. Our laboratory recently demonstrated that, in response to experimentally induced increases in the weight load borne by a rat, alternative splicing of the fast skeletal muscle troponin T (Tnnt3) pre-mRNA in gastrocnemius was adjusted in a correlated fashion with the amount of added weight. (Schilder RJ, Kimball SR, Marden JH, Jefferson LS. J Exp Biol 214: 1523-1532, 2011). Thus muscle load is perceived quantitatively by the body, and mechanisms that sense it appear to control processes that generate muscle sarcomere composition plasticity, such as alternative pre-mRNA splicing. Here we demonstrate how mechanical stretch (see earlier comment) of C2C12 muscle cells in culture results in changes to Tnnt3 pre-mRNA alternative splicing that are qualitatively similar to those observed in response to added weight in rats. Moreover, inhibition of Akt signaling, but not that of ERK1/2, prevents the stretch-induced effect on Tnnt3 pre-mRNA alternative splicing. These findings suggest that effects of muscle load on Tnnt3 pre-mRNA alternative splicing are controlled by a cell-autonomous mechanism, rather than systemically. They also indicate that, in addition to its regulatory role in protein synthesis and muscle mass plasticity, Akt signaling may regulate muscle sarcomere composition by modulating alternative splicing events in response to load. Manipulation of Tnnt3 pre-mRNA alternative splicing by mechanical stretch of cells in culture provides a model to investigate the biology of weight sensing by skeletal muscles and facilitates identification of mechanisms through which skeletal muscles match their performance and experienced load.

  14. HIV-1 Vpr N-terminal tagging affects alternative splicing of the viral genome

    PubMed Central

    Baeyens, Ann; Naessens, Evelien; Van Nuffel, Anouk; Weening, Karin E.; Reilly, Anne-Marie; Claeys, Eva; Trypsteen, Wim; Vandekerckhove, Linos; Eyckerman, Sven; Gevaert, Kris; Verhasselt, Bruno

    2016-01-01

    To facilitate studies on Vpr function in replicating HIV-1, we aimed to tag the protein in an infectious virus. First we showed that N-, but not C-terminal HA/FLAG tagging of Vpr protein preserves Vpr cytopathicity. Cloning the tags into proviral DNA however ablated viral production and replication. By construction of additional viral variants we could show this defect was not protein- but RNA-dependent and sequence specific, and characterized by oversplicing of the genomic RNA. Simulation of genomic RNA folding suggested that introduction of the tag sequence induced an alternative folding structure in a region enriched in splice sites and splicing regulatory sequences. In silico predictions identified the HA/His6-Vpr tagging in HIV-1 to affect mRNA folding less than HA/FLAG-Vpr tagging. In vitro infectivity and mRNA splice pattern improved but did not reach wild-type values. Thus, sequence-specific insertions may interfere with mRNA splicing, possibly due to altered RNA folding. Our results point to the complexity of viral RNA genome sequence interactions. This should be taken into consideration when designing viral manipulation strategies, for both research as for biological interventions. PMID:27721439

  15. Genome-wide identification of Fas/CD95 alternative splicing regulators reveals links with iron homeostasis.

    PubMed

    Tejedor, J Ramón; Papasaikas, Panagiotis; Valcárcel, Juan

    2015-01-08

    Alternative splicing of Fas/CD95 exon 6 generates either a membrane-bound receptor that promotes, or a soluble isoform that inhibits, apoptosis. Using an automatized genome-wide siRNA screening for alternative splicing regulators of endogenous transcripts in mammalian cells, we identified 200 genes whose knockdown modulates the ratio between Fas/CD95 isoforms. These include classical splicing regulators; core spliceosome components; and factors implicated in transcription and chromatin remodeling, RNA transport, intracellular signaling, and metabolic control. Coherent effects of genes involved in iron homeostasis and pharmacological modulation of iron levels revealed a link between intracellular iron and Fas/CD95 exon 6 inclusion. A splicing regulatory network linked iron levels with reduced activity of the Zinc-finger-containing splicing regulator SRSF7, and in vivo and in vitro assays revealed that iron inhibits SRSF7 RNA binding. Our results uncover numerous links between cellular pathways and RNA processing and a mechanism by which iron homeostasis can influence alternative splicing.

  16. WT1 interacts with the splicing protein RBM4 and regulates its ability to modulate alternative splicing in vivo

    SciTech Connect

    Markus, M. Andrea; Heinrich, Bettina; Raitskin, Oleg; Adams, David J.; Mangs, Helena; Goy, Christine; Ladomery, Michael; Sperling, Ruth; Stamm, Stefan; Morris, Brian J. . E-mail: brianm@medsci.usyd.edu.au

    2006-10-15

    Wilm's tumor protein 1 (WT1), a protein implicated in various cancers and developmental disorders, consists of two major isoforms: WT1(-KTS), a transcription factor, and WT1(+KTS), a post-transcriptional regulator that binds to RNA and can interact with splicing components. Here we show that WT1 interacts with the novel splicing regulator RBM4. Each protein was found to colocalize in nuclear speckles and to cosediment with supraspliceosomes in glycerol gradients. RBM4 conferred dose-dependent and cell-specific regulation of alternative splicing of pre-mRNAs transcribed from several reporter genes. We found that overexpressed WT1(+KTS) abrogated this effect of RBM4 on splice-site selection, whereas WT1(-KTS) did not. We conclude that the (+KTS) form of WT1 is able to inhibit the effect of RBM4 on alternative splicing.

  17. Novel alternative splicing isoform biomarkers identification from high-throughput plasma proteomics profiling of breast cancer

    PubMed Central

    2013-01-01

    Background In the biopharmaceutical industry, biomarkers define molecular taxonomies of patients and diseases and serve as surrogate endpoints in early-phase drug trials. Molecular biomarkers can be much more sensitive than traditional lab tests. Discriminating disease biomarkers by traditional method such as DNA microarray has proved challenging. Alternative splicing isoform represents a new class of diagnostic biomarkers. Recent scientific evidence is demonstrating that the differentiation and quantification of individual alternative splicing isoforms could improve insights into disease diagnosis and management. Identifying and characterizing alternative splicing isoforms are essential to the study of molecular mechanisms and early detection of complex diseases such as breast cancer. However, there are limitations with traditional methods used for alternative splicing isoform determination such as transcriptome-level, low level of coverage and poor focus on alternative splicing. Results Therefore, we presented a peptidomics approach to searching novel alternative splicing isoforms in clinical proteomics. Our results showed that the approach has significant potential in enabling discovery of new types of high-quality alternative splicing isoform biomarkers. Conclusions We developed a peptidomics approach for the proteomics community to analyze, identify, and characterize alternative splicing isoforms from MS-based proteomics experiments with more coverage and exclusive focus on alternative splicing. The approach can help generate novel hypotheses on molecular risk factors and molecular mechanisms of cancer in early stage, leading to identification of potentially highly specific alternative splicing isoform biomarkers for early detection of cancer. PMID:24565027

  18. Alternative splicing and the progesterone receptor in breast cancer

    PubMed Central

    Cork, David MW; Lennard, Thomas WJ; Tyson-Capper, Alison J

    2008-01-01

    Progesterone receptor status is a marker for hormone responsiveness and disease prognosis in breast cancer. Progesterone receptor negative tumours have generally been shown to have a poorer prognosis than progesterone receptor positive tumours. The observed loss of progesterone receptor could be through a range of mechanisms, including the generation of alternatively spliced progesterone receptor variants that are not detectable by current screening methods. Many progesterone receptor mRNA variants have been described with deletions of various whole, multiple or partial exons that encode differing protein functional domains. These variants may alter the progestin responsiveness of a tissue and contribute to the abnormal growth associated with breast cancer. Absence of specific functional domains from these spliced variants may also make them undetectable or indistinguishable from full length progesterone receptor by conventional antibodies. A comprehensive investigation into the expression profile and activity of progesterone receptor spliced variants in breast cancer is required to advance our understanding of tumour hormone receptor status. This, in turn, may aid the development of new biomarkers of disease prognosis and improve adjuvant treatment decisions. PMID:18557990

  19. APPRIS: annotation of principal and alternative splice isoforms

    PubMed Central

    Rodriguez, Jose Manuel; Maietta, Paolo; Ezkurdia, Iakes; Pietrelli, Alessandro; Wesselink, Jan-Jaap; Lopez, Gonzalo; Valencia, Alfonso; Tress, Michael L.

    2013-01-01

    Here, we present APPRIS (http://appris.bioinfo.cnio.es), a database that houses annotations of human splice isoforms. APPRIS has been designed to provide value to manual annotations of the human genome by adding reliable protein structural and functional data and information from cross-species conservation. The visual representation of the annotations provided by APPRIS for each gene allows annotators and researchers alike to easily identify functional changes brought about by splicing events. In addition to collecting, integrating and analyzing reliable predictions of the effect of splicing events, APPRIS also selects a single reference sequence for each gene, here termed the principal isoform, based on the annotations of structure, function and conservation for each transcript. APPRIS identifies a principal isoform for 85% of the protein-coding genes in the GENCODE 7 release for ENSEMBL. Analysis of the APPRIS data shows that at least 70% of the alternative (non-principal) variants would lose important functional or structural information relative to the principal isoform. PMID:23161672

  20. APPRIS: annotation of principal and alternative splice isoforms.

    PubMed

    Rodriguez, Jose Manuel; Maietta, Paolo; Ezkurdia, Iakes; Pietrelli, Alessandro; Wesselink, Jan-Jaap; Lopez, Gonzalo; Valencia, Alfonso; Tress, Michael L

    2013-01-01

    Here, we present APPRIS (http://appris.bioinfo.cnio.es), a database that houses annotations of human splice isoforms. APPRIS has been designed to provide value to manual annotations of the human genome by adding reliable protein structural and functional data and information from cross-species conservation. The visual representation of the annotations provided by APPRIS for each gene allows annotators and researchers alike to easily identify functional changes brought about by splicing events. In addition to collecting, integrating and analyzing reliable predictions of the effect of splicing events, APPRIS also selects a single reference sequence for each gene, here termed the principal isoform, based on the annotations of structure, function and conservation for each transcript. APPRIS identifies a principal isoform for 85% of the protein-coding genes in the GENCODE 7 release for ENSEMBL. Analysis of the APPRIS data shows that at least 70% of the alternative (non-principal) variants would lose important functional or structural information relative to the principal isoform.

  1. Lost in Translation: Pitfalls in Deciphering Plant Alternative Splicing Transcripts

    PubMed Central

    Brown, John W.S.; Simpson, Craig G.; Marquez, Yamile; Gadd, Geoffrey M.; Barta, Andrea; Kalyna, Maria

    2015-01-01

    Transcript annotation in plant databases is incomplete and often inaccurate, leading to misinterpretation. As more and more RNA-seq data are generated, plant scientists need to be aware of potential pitfalls and understand the nature and impact of specific alternative splicing transcripts on protein production. A primary area of concern and the topic of this article is the (mis)annotation of open reading frames and premature termination codons. The basic message is that to adequately address expression and functions of transcript isoforms, it is necessary to be able to predict their fate in terms of whether protein isoforms are generated or specific transcripts are unproductive or degraded. PMID:26286536

  2. Novel IL-15 isoforms generated by alternative splicing are expressed in the intestinal epithelium.

    PubMed

    Tan, X; Lefrançois, L

    2006-07-01

    Previous studies have identified mRNA three isoforms encoding interleukin-15 (IL-15) that are produced through differential splicing and encode for the same mature IL-15 protein with two different signal peptides. Our analysis of mouse intestinal epithelial cells revealed two new IL-15 mRNA isoforms generated by different alternative splicing events. In one form (IL-15DeltaE6), exon 6 is absent, and in the second form the first 48 nt of exon 7 are absent (IL-15DeltaE7) through usage of an alternative 5' splicing site within exon 7. These mRNA isoforms encoded in-frame IL-15 protein variants lacking either 15aa (IL-15DeltaE6) or 16aa (IL-15DeltaE7) both utilizing the normal long signal peptide. Significant structural changes were predicted for these new IL-15 isoforms. RNAse protection assays revealed the highest expression of isoform mRNA in the intestinal epithelium and functional analysis of recombinant IL-15 isoform proteins suggested possible regulatory functions.

  3. Female-specific insect lethality engineered using alternative splicing.

    PubMed

    Fu, Guoliang; Condon, Kirsty C; Epton, Matthew J; Gong, Peng; Jin, Li; Condon, George C; Morrison, Neil I; Dafa'alla, Tarig H; Alphey, Luke

    2007-03-01

    The Sterile Insect Technique is a species-specific and environmentally friendly method of pest control involving mass release of sterilized insects that reduce the wild population through infertile matings. Insects carrying a female-specific autocidal genetic system offer an attractive alternative to conventional sterilization methods while also eliminating females from the release population. We exploited sex-specific alternative splicing in insects to engineer female-specific autocidal genetic systems in the Mediterranean fruit fly, Ceratitis capitata. These rely on the insertion of cassette exons from the C. capitata transformer gene into a heterologous tetracycline-repressible transactivator such that the transactivator transcript is disrupted in male splice variants but not in the female-specific one. As the key components of these systems function across a broad phylogenetic range, this strategy addresses the paucity of sex-specific expression systems (e.g., early-acting, female-specific promoters) in insects other than Drosophila melanogaster. The approach may have wide applicability for regulating gene expression in other organisms, particularly for combinatorial control with appropriate promoters.

  4. Opportunities and methods for studying alternative splicing in cancer with RNA-Seq.

    PubMed

    Feng, Huijuan; Qin, Zhiyi; Zhang, Xuegong

    2013-11-01

    The biogenesis, development and metastases of cancer are associated with many variations in the transcriptome. Alternative splicing of genes is a major post-transcriptional regulation mechanism that is involved in many types of cancer. The next-generation sequencing applied on RNAs (RNA-Seq) provides a new technology for studying transcriptomes. It provides an unprecedented opportunity for quantitatively studying alternative splicing in a systematic way. This mini-review summarizes the current RNA-Seq studies on cancer transcriptomes especially studies on cancer-related alternative splicing, and discusses the strategy for quantitative study of alternative splicing in cancers with RNA-Seq, the bioinformatics methods available and existing questions.

  5. Alternative Splicing, DNA Damage and Modulating Drugs in Radiation Therapy for Cancer.

    PubMed

    Tang, Jen-Yang; Li, Ruei-Nian; Chen, Ping-Ho; Huang, Hurng-Wern; Hou, Ming-Feng; Chang, Hsueh-Wei

    2015-01-01

    Radiotherapy effectively destroys cancer cells in many sites of the body, but several limitations remain. This study investigated alternative splicing, which is a common mechanism of increased diversity in mRNAs and proteins. The relationships of alternative splicing to DNA damage and radiation such as UV and ionizing radiation were analyzed. The DNA damage responses of many genes involved in alternative splicing were compared between non-radiation and radiation treatments. Drugs that affect radioresistence or radiosensitization by modulating the effects of alternative splicing and radiation were also reviewed.

  6. New insights into RNA secondary structure in the alternative splicing of pre-mRNAs.

    PubMed

    Jin, Yongfeng; Yang, Yun; Zhang, Peng

    2011-01-01

    Alternative splicing is an important mechanism in generating proteomic diversity, and RNA secondary structure is an important element in splicing regulation. The use of high-throughput sequencing and other approaches has increased the number of known pre-mRNA secondary structures by several orders of magnitude, and we now have new insights into the role of RNA secondary structure in alternative splicing and the mechanisms involved (e.g., physical competition, long-range RNA pairing, the structural splicing code, and co-transcriptional splicing). Furthermore, an RNA pairing-based mechanism ensures the selection of only one of several available exons (e.g., Dscam splicing). Here we review several recent discoveries related to the role of RNA secondary structure in alternative splicing and the underlying mechanisms.

  7. Gene duplication followed by exon structure divergence substitutes for alternative splicing in zebrafish.

    PubMed

    Lambert, Matthew J; Olsen, Kyle G; Cooper, Cynthia D

    2014-08-10

    In this study we report novel findings regarding the evolutionary relationship between gene duplication and alternative splicing, two processes that increase proteomic diversity. By studying teleost fish, we find that gene duplication followed by exon structure divergence between paralogs, but not gene duplication alone, leads to a significant reduction in alternative splicing, as measured by both the proportion of genes that undergo alternative splicing as well as mean number of transcripts per gene. Additionally, we show that this effect is independent of gene family size and gene function. Furthermore, we provide evidence that the reduction in alternative splicing may be due to the partitioning of ancestral splice forms among the duplicate genes - a form of subfunctionalization. Taken together these results indicate that exon structure evolution subsequent to gene duplication may be a common substitute for alternative splicing.

  8. Real-time imaging of cotranscriptional splicing reveals a kinetic model that reduces noise: implications for alternative splicing regulation

    PubMed Central

    Schmidt, Ute; Robert, Marie-Cécile; Yoshida, Minoru; Villemin, Jean-Philippe; Auboeuf, Didier; Aitken, Stuart

    2011-01-01

    Splicing is a key process that expands the coding capacity of genomes. Its kinetics remain poorly characterized, and the distribution of splicing time caused by the stochasticity of single splicing events is expected to affect regulation efficiency. We conducted a small-scale survey on 40 introns in human cells and observed that most were spliced cotranscriptionally. Consequently, we constructed a reporter system that splices cotranscriptionally and can be monitored in live cells and in real time through the use of MS2–GFP. All small nuclear ribonucleoproteins (snRNPs) are loaded on nascent pre-mRNAs, and spliceostatin A inhibits splicing but not snRNP recruitment. Intron removal occurs in minutes and is best described by a model where several successive steps are rate limiting. Each pre-mRNA molecule is predicted to require a similar time to splice, reducing kinetic noise and improving the regulation of alternative splicing. This model is relevant to other kinetically controlled processes acting on few molecules. PMID:21624952

  9. The Involvement of Splicing Factor hnRNP A1 in UVB-Induced Alternative Splicing of hdm2.

    PubMed

    Feng, Jianguo; Li, Li; Tong, Lingying; Tang, Liling; Wu, Shiyong

    2016-01-12

    Human homolog double minute 2 (hdm2), an oncoprotein, which binds to tumor suppressor p53 to facilitate its degradation, has been known to contribute to tumorigenesis. Its splicing variants are reported to be highly expressed in many cancers and can be induced by ultraviolet B light (UVB). However, the mechanisms of how UVB radiation induces hdm2 alternative splicing still remain unclear. In this study, we investigated the roles of two common splicing factors, heterogeneous nuclear ribonucleoproteins (hnRNP) A1 and serine/arginine-rich splicing factor 1 (SRSF1), in regulating UVB-induced hdm2 splicing. Our study indicated that while the expression of both hnRNP A1 and SRSF1 are induced, only hnRNP A1 is involved in hdm2 alternative splicing upon UVB irradiation. Overexpression of hnRNP A1 resulted in decrease of full-length hdm2 (hdm2-FL) and increase of hdm2B, one of hdm2 alternate-splicing forms; while down-regulated hnRNP A1 expression led to the decrease of the hdm2-FL and hdm2B in HaCaT cells. Protein-mRNA binding assay confirmed that UVB irradiation could increase the binding of hnRNP A1 to hdm2 pre-mRNA. In conclusion, we elucidated that UVB induces alternative splicing of hdm2 via increasing the expression and the binding of hnRNP A1 to hdm2 full-length mRNA. This article is protected by copyright. All rights reserved.

  10. PPS, a large multidomain protein, functions with sex-lethal to regulate alternative splicing in Drosophila.

    PubMed

    Johnson, Matthew L; Nagengast, Alexis A; Salz, Helen K

    2010-03-05

    Alternative splicing controls the expression of many genes, including the Drosophila sex determination gene Sex-lethal (Sxl). Sxl expression is controlled via a negative regulatory mechanism where inclusion of the translation-terminating male exon is blocked in females. Previous studies have shown that the mechanism leading to exon skipping is autoregulatory and requires the SXL protein to antagonize exon inclusion by interacting with core spliceosomal proteins, including the U1 snRNP protein Sans-fille (SNF). In studies begun by screening for proteins that interact with SNF, we identified PPS, a previously uncharacterized protein, as a novel component of the machinery required for Sxl male exon skipping. PPS encodes a large protein with four signature motifs, PHD, BRK, TFS2M, and SPOC, typically found in proteins involved in transcription. We demonstrate that PPS has a direct role in Sxl male exon skipping by showing first that loss of function mutations have phenotypes indicative of Sxl misregulation and second that the PPS protein forms a complex with SXL and the unspliced Sxl RNA. In addition, we mapped the recruitment of PPS, SXL, and SNF along the Sxl gene using chromatin immunoprecipitation (ChIP), which revealed that, like many other splicing factors, these proteins bind their RNA targets while in close proximity to the DNA. Interestingly, while SNF and SXL are specifically recruited to their predicted binding sites, PPS has a distinct pattern of accumulation along the Sxl gene, associating with a region that includes, but is not limited to, the SxlPm promoter. Together, these data indicate that PPS is different from other splicing factors involved in male-exon skipping and suggest, for the first time, a functional link between transcription and SXL-mediated alternative splicing. Loss of zygotic PPS function, however, is lethal to both sexes, indicating that its role may be of broad significance.

  11. Biogenesis of intronic miRNAs located in clusters by independent transcription and alternative splicing

    PubMed Central

    Ramalingam, Pradeep; Palanichamy, Jayanth Kumar; Singh, Anand; Das, Prerna; Bhagat, Mohita; Kassab, Muzaffer Ahmad; Sinha, Subrata; Chattopadhyay, Parthaprasad

    2014-01-01

    miRNAs are generally classified as “intergenic” or “intronic” based upon their genomic location. Intergenic miRNAs are known to be transcribed as independent transcription units, while intronic miRNAs are believed to be processed from the introns of their hosting transcription units and hence share common regulatory mechanisms and expression patterns with its host gene. Recent reports in the literature suggest that some intronic miRNAs, which do not show concordance in expression with their respective host genes, might be transcribed and regulated as independent transcription units. However, there is no direct evidence for the existence of independently transcribed intronic miRNA in humans to date. We have characterized the full-length primary transcripts (pri-miRNAs) of three human intronic miRNAs—miR 106b, miR 93, and miR 24-1—by RNA ligase-mediated RACE and show that human intronic miRNA can indeed be transcribed as independent transcription units. Also, clustered miRNAs are generally believed to arise from a common primary transcript and are expected to have similar expression profiles. However, we have identified several novel alternatively spliced transcripts by RT-PCR, each of which harbors a single pre-miRNA from a cluster of closely located intronic miRNAs. We show that these transcripts represent unique pri-miRNAs for each of these clustered miRNAs. We also report the identification of conserved splice acceptor signals which are responsible for maturation of these novel splice variants. Our results suggest that alternative splicing might play a role in uncoupling the expression of clustered miRNAs from each other, which otherwise are generally believed to be co-transcribed and co-expressed. PMID:24226766

  12. Predicting functional alternative splicing by measuring RNA selection pressure from multigenome alignments.

    PubMed

    Lu, Hongchao; Lin, Lan; Sato, Seiko; Xing, Yi; Lee, Christopher J

    2009-12-01

    High-throughput methods such as EST sequencing, microarrays and deep sequencing have identified large numbers of alternative splicing (AS) events, but studies have shown that only a subset of these may be functional. Here we report a sensitive bioinformatics approach that identifies exons with evidence of a strong RNA selection pressure ratio (RSPR)--i.e., evolutionary selection against mutations that change only the mRNA sequence while leaving the protein sequence unchanged--measured across an entire evolutionary family, which greatly amplifies its predictive power. Using the UCSC 28 vertebrate genome alignment, this approach correctly predicted half to three-quarters of AS exons that are known binding targets of the NOVA splicing regulatory factor, and predicted 345 strongly selected alternative splicing events in human, and 262 in mouse. These predictions were strongly validated by several experimental criteria of functional AS such as independent detection of the same AS event in other species, reading frame-preservation, and experimental evidence of tissue-specific regulation: 75% (15/20) of a sample of high-RSPR exons displayed tissue specific regulation in a panel of ten tissues, vs. only 20% (4/20) among a sample of low-RSPR exons. These data suggest that RSPR can identify exons with functionally important splicing regulation, and provides biologists with a dataset of over 600 such exons. We present several case studies, including both well-studied examples (GRIN1) and novel examples (EXOC7). These data also show that RSPR strongly outperforms other approaches such as standard sequence conservation (which fails to distinguish amino acid selection pressure from RNA selection pressure), or pairwise genome comparison (which lacks adequate statistical power for predicting individual exons).

  13. Extensive alternative splicing transitions during postnatal skeletal muscle development are required for calcium handling functions

    PubMed Central

    Brinegar, Amy E; Xia, Zheng; Loehr, James Anthony; Li, Wei; Rodney, George Gerald

    2017-01-01

    Postnatal development of skeletal muscle is a highly dynamic period of tissue remodeling. Here, we used RNA-seq to identify transcriptome changes from late embryonic to adult mouse muscle and demonstrate that alternative splicing developmental transitions impact muscle physiology. The first 2 weeks after birth are particularly dynamic for differential gene expression and alternative splicing transitions, and calcium-handling functions are significantly enriched among genes that undergo alternative splicing. We focused on the postnatal splicing transitions of the three calcineurin A genes, calcium-dependent phosphatases that regulate multiple aspects of muscle biology. Redirected splicing of calcineurin A to the fetal isoforms in adult muscle and in differentiated C2C12 slows the timing of muscle relaxation, promotes nuclear localization of calcineurin target Nfatc3, and/or affects expression of Nfatc transcription targets. The results demonstrate a previously unknown specificity of calcineurin isoforms as well as the broader impact of alternative splicing during muscle postnatal development. PMID:28826478

  14. Extensive alternative splicing transitions during postnatal skeletal muscle development are required for calcium handling functions.

    PubMed

    Brinegar, Amy E; Xia, Zheng; Loehr, James Anthony; Li, Wei; Rodney, George Gerald; Cooper, Thomas A

    2017-08-11

    Postnatal development of skeletal muscle is a highly dynamic period of tissue remodeling. Here, we used RNA-seq to identify transcriptome changes from late embryonic to adult mouse muscle and demonstrate that alternative splicing developmental transitions impact muscle physiology. The first 2 weeks after birth are particularly dynamic for differential gene expression and alternative splicing transitions, and calcium-handling functions are significantly enriched among genes that undergo alternative splicing. We focused on the postnatal splicing transitions of the three calcineurin A genes, calcium-dependent phosphatases that regulate multiple aspects of muscle biology. Redirected splicing of calcineurin A to the fetal isoforms in adult muscle and in differentiated C2C12 slows the timing of muscle relaxation, promotes nuclear localization of calcineurin target Nfatc3, and/or affects expression of Nfatc transcription targets. The results demonstrate a previously unknown specificity of calcineurin isoforms as well as the broader impact of alternative splicing during muscle postnatal development.

  15. Evolutionarily Dynamic Alternative Splicing of GPR56 Regulates Regional Cerebral Cortical Patterning

    PubMed Central

    Bae, Byoung-Il; Tietjen, Ian; Atabay, Kutay D.; Evrony, Gilad D.; Johnson, Matthew B.; Asare, Ebenezer; Wang, Peter P.; Murayama, Ayako Y.; Im, Kiho; Lisgo, Steven N.; Overman, Lynne; Šestan, Nenad; Chang, Bernard S.; Barkovich, A. James; Grant, P. Ellen; Topçu, Meral; Politsky, Jeffrey; Okano, Hideyuki; Piao, Xianhua; Walsh, Christopher A.

    2015-01-01

    The human neocortex has numerous specialized functional areas whose formation is poorly understood. Here, we describe a 15–base pair deletion mutation in a regulatory element of GPR56 that selectively disrupts human cortex surrounding the Sylvian fissure bilaterally including “Broca’s area,” the primary language area, by disrupting regional GPR56 expression and blocking RFX transcription factor binding. GPR56 encodes a heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptor required for normal cortical development and is expressed in cortical progenitor cells. GPR56 expression levels regulate progenitor proliferation. GPR56 splice forms are highly variable between mice and humans, and the regulatory element of gyrencephalic mammals directs restricted lateral cortical expression. Our data reveal a mechanism by which control of GPR56 expression pattern by multiple alternative promoters can influence stem cell proliferation, gyral patterning, and, potentially, neocortex evolution. PMID:24531968

  16. Alternative splicing enhances transcriptome complexity in desiccating seeds.

    PubMed

    Srinivasan, Arunkumar; Jiménez-Gómez, José M; Fornara, Fabio; Soppe, Wim J J; Brambilla, Vittoria

    2016-12-01

    Before being dispersed in the environment, mature seeds need to be dehydrated. The survival of seeds after dispersal depends on their low hydration in combination with high desiccation tolerance. These characteristics are established during seed maturation. Some key seed maturation genes have been reported to be regulated by alternative splicing (AS). However, so far AS was described only for single genes and a comprehensive analysis of AS during seed maturation has been lacking. We investigated gene expression and AS during Arabidopsis thaliana seed development at a global level, before and after desiccation. Bioinformatics tools were developed to identify differentially spliced regions within genes. Our data suggest the importance and shows the peculiar features of AS during seed desiccation. We identified AS in 34% of genes that are expressed at both timepoints before and after desiccation. Most of these AS transcript variants had not been found before in other tissues. Among the AS genes some seed master regulators could be found. Interestingly, 6% of all expressed transcripts were not transcriptionally regulated during desiccation, but only modified by AS. We propose that AS should be more routinely taken into account in the analysis of transcriptomic data to prevent overlooking potentially important regulators. © 2016 Institute of Botany, Chinese Academy of Sciences.

  17. Comparative cross-species alternative splicing in plants.

    PubMed

    Ner-Gaon, Hadas; Leviatan, Noam; Rubin, Eitan; Fluhr, Robert

    2007-07-01

    Alternative splicing (AS) can add significantly to genome complexity. Plants are thought to exhibit less AS than animals. An algorithm, based on expressed sequence tag (EST) pairs gapped alignment, was developed that takes advantage of the relatively small intron and exon size in plants and directly compares pairs of ESTs to search for AS. EST pairs gapped alignment was first evaluated in Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and tomato (Solanum lycopersicum) for which annotated genome sequence is available and was shown to accurately predict splicing events. The method was then applied to 11 plant species that include 17 cultivars for which enough ESTs are available. The results show a large, 3.7-fold difference in AS rates between plant species with Arabidopsis and rice in the lower range and lettuce (Lactuca sativa) and sorghum (Sorghum bicolor) in the upper range. Hence, compared to higher animals, plants show a much greater degree of variety in their AS rates and in some plant species the rates of animal and plant AS are comparable although the distribution of AS types may differ. In eudicots but not monocots, a correlation between genome size and AS rates was detected, implying that in eudicots the mechanisms that lead to larger genomes are a driving force for the evolution of AS.

  18. My road to alternative splicing control: from simple paths to loops and interconnections.

    PubMed

    Chabot, Benoit

    2015-06-01

    With the functional importance of alternative splicing being validated in nearly every mammalian biological system and implicated in many human diseases, it is now crucial to identify the molecular programs that control the production of splice variants. In this article, I will survey how our knowledge of the basic principles of alternative splicing control evolved over the last 25 years. I will also describe how investigation of the splicing control of an apoptotic regulator led us to identify novel effectors and revealed the existence of converging pathways linking splicing decisions to DNA damage. Finally, I will review how our efforts at developing tools designed to monitor and redirect splicing helped assess the impact of misregulated splicing in cancer.

  19. Regulation of hyaluronidase activity by alternative mRNA splicing.

    PubMed

    Lokeshwar, Vinata B; Schroeder, Grethchen L; Carey, Robert I; Soloway, Mark S; Iida, Naoko

    2002-09-13

    Hyaluronidase is a hyaluronic acid-degrading endoglycosidase that is present in many toxins and the levels of which are elevated in cancer. Increased concentration of HYAL1-type hyaluronidase correlates with tumor progression and is a marker for grade (G) 2 or 3 bladder cancer. Using bladder tissues and cells, prostate cancer cells, and kidney tissues and performing reverse transcription-PCR, cDNA cloning, DNA sequencing, and in vitro translation, we identified splice variants of HYAL1 and HYAL3. HYAL1v1 variant lacks a 30-amino acid (aa) sequence (301-330) present in HYAL1 protein. HYAL1v1, HYAL1v2 (aa 183-435 present in HYAL1 wild type), HYAL1v3 (aa 1-207), HYAL1v4 (aa 260-435), and HYAL1v5 (aa 340-435) are enzymatically inactive and are expressed in normal tissues/cells and G1 bladder tumor tissues. However, HYAL1 wild type is expressed in G2/G3 tumors and in invasive tumor cells. Stable transfection and HYAL1v1-specific antibody confirmed that the HYAL1 sequence from aa 301 to 330 is critical for hyaluronidase activity. All tumor cells and tissues mainly express HYAL3 variants. HYAL3v1 lacks a 30-aa sequence (299-328) present in HYAL3 protein, that is homologous to the 30-aa HYAL1 sequence. HYAL3v1, HYAL3v2 (aa 251-417 present in HYAL3 wild type), and HYAL3v3 (aa 251-417, but lacking aa 299-328), are enzymatically inactive. Although splicing of a single independent exon generates HYAL1v1 and HYAL3v1, internal exon splicing generates the other HYAL1/HYAL3 variants. These results demonstrate that alternative mRNA splicing controls cellular expression of enzymatically active hyaluronidase and may explain the elevated hyaluronidase levels in bladder/prostate cancer.

  20. Alternative splicing, muscle calcium sensitivity, and the modulation of dragonfly flight performance

    PubMed Central

    Marden, James H.; Fitzhugh, Gail H.; Wolf, Melisande R.; Arnold, Kristina D.; Rowan, Barry

    1999-01-01

    Calcium sensitivity of myosin cross-bridge activation in striated muscles commonly varies during ontogeny and in response to alterations in muscle usage, but the consequences for whole-organism physiology are not well known. Here we show that the relative abundances of alternatively spliced transcripts of the calcium regulatory protein troponin T (TnT) vary widely in flight muscle of Libellula pulchella dragonflies, and that the mixture of TnT splice variants explains significant portions of the variation in muscle calcium sensitivity, wing-beat frequency, and an index of aerodynamic power output during free flight. Two size-distinguishable morphs differ in their maturational pattern of TnT splicing, yet they show the same relationship between TnT transcript mixture and calcium sensitivity and between calcium sensitivity and aerodynamic power output. This consistency of effect in different developmental and physiological contexts strengthens the hypothesis that TnT isoform variation modulates muscle calcium sensitivity and whole-organism locomotor performance. Modulating muscle power output appears to provide the ecologically important ability to operate at different points along a tradeoff between performance and energetic cost. PMID:10611380

  1. Novel alternative splice variants of chicken NPAS3 are expressed in the developing central nervous system.

    PubMed

    Shin, Jiheon; Kim, Jaesang

    2013-11-10

    We report isolation of novel splice variants of chicken Neuronal Per-Arnt-Sim domain protein 3 (cNPAS3) gene distinct from the previously predicted cNPAS3 at the 5' end. Newly identified cNPAS3 splice variants feature N-terminus coding sequences with high degrees of homology to human NPAS3 (hNAPS3). We also show that the alternative splicing pattern of NPAS3 is conserved between chicken and human. RNA in situ hybridization indicated that the expression of cNPAS3 in the developing central nervous system (CNS) is limited to the ventricular zone and only partially overlaps with that of chicken Reelin (cReelin), the only known regulatory target gene of NPAS3 in the adult brain. Overexpression of cNPAS3 by in ovo electroporation had little effect on the expression of Sox2, a marker for neural precursors, or of Isl1/2, a marker for early differentiating motor neurons. Taken together with the little effect of cNPAS3 overexpression on cReelin, it is noted that the function of NPAS3 in the developing CNS remains to be determined. Still, identification of proper cDNA sequences for cNPAS3 should represent a solid beginning of the understanding process. © 2013.

  2. Cell type-restricted activity of hnRNPM promotes breast cancer metastasis via regulating alternative splicing.

    PubMed

    Xu, Yilin; Gao, Xin D; Lee, Jae-Hyung; Huang, Huilin; Tan, Haiyan; Ahn, Jaegyoon; Reinke, Lauren M; Peter, Marcus E; Feng, Yue; Gius, David; Siziopikou, Kalliopi P; Peng, Junmin; Xiao, Xinshu; Cheng, Chonghui

    2014-06-01

    Tumor metastasis remains the major cause of cancer-related death, but its molecular basis is still not well understood. Here we uncovered a splicing-mediated pathway that is essential for breast cancer metastasis. We show that the RNA-binding protein heterogeneous nuclear ribonucleoprotein M (hnRNPM) promotes breast cancer metastasis by activating the switch of alternative splicing that occurs during epithelial-mesenchymal transition (EMT). Genome-wide deep sequencing analysis suggests that hnRNPM potentiates TGFβ signaling and identifies CD44 as a key downstream target of hnRNPM. hnRNPM ablation prevents TGFβ-induced EMT and inhibits breast cancer metastasis in mice, whereas enforced expression of the specific CD44 standard (CD44s) splice isoform overrides the loss of hnRNPM and permits EMT and metastasis. Mechanistically, we demonstrate that the ubiquitously expressed hnRNPM acts in a mesenchymal-specific manner to precisely control CD44 splice isoform switching during EMT. This restricted cell-type activity of hnRNPM is achieved by competition with ESRP1, an epithelial splicing regulator that binds to the same cis-regulatory RNA elements as hnRNPM and is repressed during EMT. Importantly, hnRNPM is associated with aggressive breast cancer and correlates with increased CD44s in patient specimens. These findings demonstrate a novel molecular mechanism through which tumor metastasis is endowed by the hnRNPM-mediated splicing program.

  3. Genome-Wide Detection of Condition-Sensitive Alternative Splicing in Arabidopsis Roots1[C][W

    PubMed Central

    Li, Wenfeng; Lin, Wen-Dar; Ray, Prasun; Lan, Ping; Schmidt, Wolfgang

    2013-01-01

    Iron (Fe) deficiency is a world-wide nutritional disorder in both plants and humans, resulting from its restricted bioavailability for plants and, subsequently, low Fe concentration in edible plant parts. Plants have evolved sophisticated mechanisms to alleviate Fe deficiency, with the aim of recalibrating metabolic fluxes and maintaining cellular Fe homeostasis. To analyze condition-sensitive changes in precursor mRNA (pre-mRNA) splicing pattern, we mapped the transcriptome of Fe-deficient and Fe-sufficient Arabidopsis (Arabidopsis thaliana) roots using the RNA sequencing technology and a newly developed software toolbox, the Read Analysis & Comparison Kit in Java (RACKJ). In alternatively spliced genes, stress-related Gene Ontology categories were overrepresented, while housekeeping cellular functions were mainly transcriptionally controlled. Fe deficiency increased the complexity of the splicing pattern and triggered the differential alternative splicing of 313 genes, the majority of which had differentially retained introns. Several genes with important functions in Fe acquisition and homeostasis were both differentially expressed and differentially alternatively spliced upon Fe deficiency, indicating a complex regulation of gene activity in Fe-deficient conditions. A comparison with a data set for phosphate-deficient plants suggests that changes in splicing patterns are nutrient specific and not or not chiefly caused by stochastic fluctuations. In sum, our analysis identified extensive posttranscriptional control, biasing the abundance and activity of proteins in a condition-dependent manner. The production of a mixture of functional and nonfunctional transcripts may provide a means to fine-tune the abundance of transcripts with critical importance in cellular Fe homeostasis. It is assumed that differential gene expression and nutrient deficiency-induced changes in pre-mRNA splicing represent parallel, but potentially interacting, regulatory mechanisms. PMID

  4. MATS: a Bayesian framework for flexible detection of differential alternative splicing from RNA-Seq data

    PubMed Central

    Shen, Shihao; Park, Juw Won; Huang, Jian; Dittmar, Kimberly A.; Lu, Zhi-xiang; Zhou, Qing; Carstens, Russ P.; Xing, Yi

    2012-01-01

    Ultra-deep RNA sequencing has become a powerful approach for genome-wide analysis of pre-mRNA alternative splicing. We develop MATS (multivariate analysis of transcript splicing), a Bayesian statistical framework for flexible hypothesis testing of differential alternative splicing patterns on RNA-Seq data. MATS uses a multivariate uniform prior to model the between-sample correlation in exon splicing patterns, and a Markov chain Monte Carlo (MCMC) method coupled with a simulation-based adaptive sampling procedure to calculate the P-value and false discovery rate (FDR) of differential alternative splicing. Importantly, the MATS approach is applicable to almost any type of null hypotheses of interest, providing the flexibility to identify differential alternative splicing events that match a given user-defined pattern. We evaluated the performance of MATS using simulated and real RNA-Seq data sets. In the RNA-Seq analysis of alternative splicing events regulated by the epithelial-specific splicing factor ESRP1, we obtained a high RT–PCR validation rate of 86% for differential exon skipping events with a MATS FDR of <10%. Additionally, over the full list of RT–PCR tested exons, the MATS FDR estimates matched well with the experimental validation rate. Our results demonstrate that MATS is an effective and flexible approach for detecting differential alternative splicing from RNA-Seq data. PMID:22266656

  5. Alternative splicing in plants – coming of age

    PubMed Central

    Syed, Naeem H.; Kalyna, Maria; Marquez, Yamile; Barta, Andrea; Brown, John W.S.

    2012-01-01

    More than 60% of intron-containing genes undergo alternative splicing (AS) in plants. This number will increase when AS in different tissues, developmental stages, and environmental conditions are explored. Although the functional impact of AS on protein complexity is still understudied in plants, recent examples demonstrate its importance in regulating plant processes. AS also regulates transcript levels and the link with nonsense-mediated decay and generation of unproductive mRNAs illustrate the need for both transcriptional and AS data in gene expression analyses. AS has influenced the evolution of the complex networks of regulation of gene expression and variation in AS contributed to adaptation of plants to their environment and therefore will impact strategies for improving plant and crop phenotypes. PMID:22743067

  6. An EMT–Driven Alternative Splicing Program Occurs in Human Breast Cancer and Modulates Cellular Phenotype

    PubMed Central

    Flytzanis, Nicholas C.; Balsamo, Michele; Condeelis, John S.; Oktay, Maja H.; Burge, Christopher B.; Gertler, Frank B.

    2011-01-01

    Epithelial-mesenchymal transition (EMT), a mechanism important for embryonic development, plays a critical role during malignant transformation. While much is known about transcriptional regulation of EMT, alternative splicing of several genes has also been correlated with EMT progression, but the extent of splicing changes and their contributions to the morphological conversion accompanying EMT have not been investigated comprehensively. Using an established cell culture model and RNA–Seq analyses, we determined an alternative splicing signature for EMT. Genes encoding key drivers of EMT–dependent changes in cell phenotype, such as actin cytoskeleton remodeling, regulation of cell–cell junction formation, and regulation of cell migration, were enriched among EMT–associated alternatively splicing events. Our analysis suggested that most EMT–associated alternative splicing events are regulated by one or more members of the RBFOX, MBNL, CELF, hnRNP, or ESRP classes of splicing factors. The EMT alternative splicing signature was confirmed in human breast cancer cell lines, which could be classified into basal and luminal subtypes based exclusively on their EMT–associated splicing pattern. Expression of EMT–associated alternative mRNA transcripts was also observed in primary breast cancer samples, indicating that EMT–dependent splicing changes occur commonly in human tumors. The functional significance of EMT–associated alternative splicing was tested by expression of the epithelial-specific splicing factor ESRP1 or by depletion of RBFOX2 in mesenchymal cells, both of which elicited significant changes in cell morphology and motility towards an epithelial phenotype, suggesting that splicing regulation alone can drive critical aspects of EMT–associated phenotypic changes. The molecular description obtained here may aid in the development of new diagnostic and prognostic markers for analysis of breast cancer progression. PMID:21876675

  7. Correcting for differential transcript coverage reveals a strong relationship between alternative splicing and organism complexity.

    PubMed

    Chen, Lu; Bush, Stephen J; Tovar-Corona, Jaime M; Castillo-Morales, Atahualpa; Urrutia, Araxi O

    2014-06-01

    What at the genomic level underlies organism complexity? Although several genomic features have been associated with organism complexity, in the case of alternative splicing, which has long been proposed to explain the variation in complexity, no such link has been established. Here, we analyzed over 39 million expressed sequence tags available for 47 eukaryotic species with fully sequenced genomes to obtain a comparable index of alternative splicing estimates, which corrects for the distorting effect of a variable number of transcripts per species--an important obstacle for comparative studies of alternative splicing. We find that alternative splicing has steadily increased over the last 1,400 My of eukaryotic evolution and is strongly associated with organism complexity, assayed as the number of cell types. Importantly, this association is not explained as a by-product of covariance between alternative splicing with other variables previously linked to complexity including gene content, protein length, proteome disorder, and protein interactivity. In addition, we found no evidence to suggest that the relationship of alternative splicing to cell type number is explained by drift due to reduced N(e) in more complex species. Taken together, our results firmly establish alternative splicing as a significant predictor of organism complexity and are, in principle, consistent with an important role of transcript diversification through alternative splicing as a means of determining a genome's functional information capacity.

  8. CTCF, a Novel Regulator of Alternative Splicing | Center for Cancer Research

    Cancer.gov

    Alternative splicing, or the inclusion of different patterns of exons from the same gene, plays an important role in expanding the coding possibilities of a limited genome. The immune system is an ideal system to study this since alternative splicing is used to generate an almost unlimited number of antibodies against any pathogen we might encounter.

  9. FULL-GENOME ANALYSIS OF ALTERNATIVE SPLICING IN MOUSE LIVER AFTER HEPATOTOXICANT EXPOSURE

    EPA Science Inventory

    Alternative splicing plays a role in determining gene function and protein diversity. We have employed whole genome exon profiling using Affymetrix Mouse Exon 1.0 ST arrays to understand the significance of alternative splicing on a genome-wide scale in response to multiple toxic...

  10. Regulation of alternative splicing by the circadian clock and food related cues

    PubMed Central

    2012-01-01

    Background The circadian clock orchestrates daily rhythms in metabolism, physiology and behaviour that allow organisms to anticipate regular changes in their environment, increasing their adaptation. Such circadian phenotypes are underpinned by daily rhythms in gene expression. Little is known, however, about the contribution of post-transcriptional processes, particularly alternative splicing. Results Using Affymetrix mouse exon-arrays, we identified exons with circadian alternative splicing in the liver. Validated circadian exons were regulated in a tissue-dependent manner and were present in genes with circadian transcript abundance. Furthermore, an analysis of circadian mutant Vipr2-/- mice revealed the existence of distinct physiological pathways controlling circadian alternative splicing and RNA binding protein expression, with contrasting dependence on Vipr2-mediated physiological signals. This view was corroborated by the analysis of the effect of fasting on circadian alternative splicing. Feeding is an important circadian stimulus, and we found that fasting both modulates hepatic circadian alternative splicing in an exon-dependent manner and changes the temporal relationship with transcript-level expression. Conclusions The circadian clock regulates alternative splicing in a manner that is both tissue-dependent and concurrent with circadian transcript abundance. This adds a novel temporal dimension to the regulation of mammalian alternative splicing. Moreover, our results demonstrate that circadian alternative splicing is regulated by the interaction between distinct physiological cues, and illustrates the capability of single genes to integrate circadian signals at different levels of regulation. PMID:22721557

  11. Regulation of alternative splicing by the circadian clock and food related cues.

    PubMed

    McGlincy, Nicholas J; Valomon, Amandine; Chesham, Johanna E; Maywood, Elizabeth S; Hastings, Michael H; Ule, Jernej

    2012-06-21

    The circadian clock orchestrates daily rhythms in metabolism, physiology and behaviour that allow organisms to anticipate regular changes in their environment, increasing their adaptation. Such circadian phenotypes are underpinned by daily rhythms in gene expression. Little is known, however, about the contribution of post-transcriptional processes, particularly alternative splicing. Using Affymetrix mouse exon-arrays, we identified exons with circadian alternative splicing in the liver. Validated circadian exons were regulated in a tissue-dependent manner and were present in genes with circadian transcript abundance. Furthermore, an analysis of circadian mutant Vipr2-/- mice revealed the existence of distinct physiological pathways controlling circadian alternative splicing and RNA binding protein expression, with contrasting dependence on Vipr2-mediated physiological signals. This view was corroborated by the analysis of the effect of fasting on circadian alternative splicing. Feeding is an important circadian stimulus, and we found that fasting both modulates hepatic circadian alternative splicing in an exon-dependent manner and changes the temporal relationship with transcript-level expression. The circadian clock regulates alternative splicing in a manner that is both tissue-dependent and concurrent with circadian transcript abundance. This adds a novel temporal dimension to the regulation of mammalian alternative splicing. Moreover, our results demonstrate that circadian alternative splicing is regulated by the interaction between distinct physiological cues, and illustrates the capability of single genes to integrate circadian signals at different levels of regulation.

  12. FULL-GENOME ANALYSIS OF ALTERNATIVE SPLICING IN MOUSE LIVER AFTER HEPATOTOXICANT EXPOSURE

    EPA Science Inventory

    Alternative splicing plays a role in determining gene function and protein diversity. We have employed whole genome exon profiling using Affymetrix Mouse Exon 1.0 ST arrays to understand the significance of alternative splicing on a genome-wide scale in response to multiple toxic...

  13. TIA-1 and TIAR activate splicing of alternative exons with weak 5' splice sites followed by a U-rich stretch on their own pre-mRNAs.

    PubMed

    Le Guiner, C; Lejeune, F; Galiana, D; Kister, L; Breathnach, R; Stévenin, J; Del Gatto-Konczak, F

    2001-11-02

    TIA-1 has recently been shown to activate splicing of specific pre-mRNAs transcribed from transiently transfected minigenes, and of some 5' splice sites in vitro, but has not been shown to activate splicing of any endogenous pre-mRNA. We show here that overexpression of TIA-1 or the related protein TIAR has little effect on splicing of several endogenous pre-mRNAs containing alternative exons, but markedly activates splicing of some normally rarely used alternative exons on the TIA-1 and TIAR pre-mRNAs. These exons have weak 5' splice sites followed by U-rich stretches. When the U-rich stretch following the 5' splice site of a TIA-1 alternative exon was deleted, TIAR overexpression induced use of a cryptic 5' splice site also followed by a U-rich stretch in place of the original splice site. Using in vitro splicing assays, we have shown that TIA-1 is directly involved in activating the 5' splice sites of the TIAR alternative exons. Activation requires a downstream U-rich stretch of at least 10 residues. Our results confirm that TIA-1 activates 5' splice sites followed by U-rich sequences and show that TIAR exerts a similar activity. They suggest that both proteins may autoregulate their expression at the level of splicing.

  14. Identification and analysis of two splice variants of human G2A generated by alternative splicing.

    PubMed

    Ogawa, Ai; Obinata, Hideru; Hattori, Tomoyasu; Kishi, Mikiko; Tatei, Kazuaki; Ishikawa, Osamu; Izumi, Takashi

    2010-02-01

    G2A is a G protein-coupled receptor that can be induced by various stressors. G2A is reported to have proton-sensing activity that mediates intracellular inositol phosphate (IP) accumulation with decreasing pH. We previously showed that G2A is also activated by some oxidized free fatty acids such as 9-hydroxyoctadecadienoic acid (9-HODE). In this study, we identified a novel alternative splice variant of G2A (G2A-b) that has a partially different N terminus compared with the G2A originally reported (G2A-a). The two splice variants of G2A show similar tissue distributions, but G2A-b is expressed more abundantly. There was no difference between the two variants in 9-HODE-induced cellular responses, such as intracellular calcium mobilization and GDP/GTP exchange of Galpha protein, and in proton-sensitive IP accumulation. However, G2A-b showed a higher basal activity in terms of IP accumulation. Mutagenesis study revealed that the difference in the basal activity is attributable to the K7 residue that exists only in G2A-a. We further demonstrated that an R42A mutation largely impaired both the basal and proton-sensing activities, but did not affect the 9-HODE-induced intracellular calcium increase. Taken together, we found an additional novel G2A variant (G2A-b) that is the major transcript with functional response to ligand stimulation as well as G2A-a, and succeeded in discriminating proton-sensing and oxidized fatty acid-sensing activities of G2A.

  15. Alternatively Spliced Homologous Exons Have Ancient Origins and Are Highly Expressed at the Protein Level.

    PubMed

    Abascal, Federico; Ezkurdia, Iakes; Rodriguez-Rivas, Juan; Rodriguez, Jose Manuel; del Pozo, Angela; Vázquez, Jesús; Valencia, Alfonso; Tress, Michael L

    2015-06-01

    Alternative splicing of messenger RNA can generate a wide variety of mature RNA transcripts, and these transcripts may produce protein isoforms with diverse cellular functions. While there is much supporting evidence for the expression of alternative transcripts, the same is not true for the alternatively spliced protein products. Large-scale mass spectroscopy experiments have identified evidence of alternative splicing at the protein level, but with conflicting results. Here we carried out a rigorous analysis of the peptide evidence from eight large-scale proteomics experiments to assess the scale of alternative splicing that is detectable by high-resolution mass spectroscopy. We find fewer splice events than would be expected: we identified peptides for almost 64% of human protein coding genes, but detected just 282 splice events. This data suggests that most genes have a single dominant isoform at the protein level. Many of the alternative isoforms that we could identify were only subtly different from the main splice isoform. Very few of the splice events identified at the protein level disrupted functional domains, in stark contrast to the two thirds of splice events annotated in the human genome that would lead to the loss or damage of functional domains. The most striking result was that more than 20% of the splice isoforms we identified were generated by substituting one homologous exon for another. This is significantly more than would be expected from the frequency of these events in the genome. These homologous exon substitution events were remarkably conserved--all the homologous exons we identified evolved over 460 million years ago--and eight of the fourteen tissue-specific splice isoforms we identified were generated from homologous exons. The combination of proteomics evidence, ancient origin and tissue-specific splicing indicates that isoforms generated from homologous exons may have important cellular roles.

  16. Alternatively Spliced Homologous Exons Have Ancient Origins and Are Highly Expressed at the Protein Level

    PubMed Central

    Abascal, Federico; Ezkurdia, Iakes; Rodriguez-Rivas, Juan; Rodriguez, Jose Manuel; del Pozo, Angela; Vázquez, Jesús; Valencia, Alfonso; Tress, Michael L.

    2015-01-01

    Alternative splicing of messenger RNA can generate a wide variety of mature RNA transcripts, and these transcripts may produce protein isoforms with diverse cellular functions. While there is much supporting evidence for the expression of alternative transcripts, the same is not true for the alternatively spliced protein products. Large-scale mass spectroscopy experiments have identified evidence of alternative splicing at the protein level, but with conflicting results. Here we carried out a rigorous analysis of the peptide evidence from eight large-scale proteomics experiments to assess the scale of alternative splicing that is detectable by high-resolution mass spectroscopy. We find fewer splice events than would be expected: we identified peptides for almost 64% of human protein coding genes, but detected just 282 splice events. This data suggests that most genes have a single dominant isoform at the protein level. Many of the alternative isoforms that we could identify were only subtly different from the main splice isoform. Very few of the splice events identified at the protein level disrupted functional domains, in stark contrast to the two thirds of splice events annotated in the human genome that would lead to the loss or damage of functional domains. The most striking result was that more than 20% of the splice isoforms we identified were generated by substituting one homologous exon for another. This is significantly more than would be expected from the frequency of these events in the genome. These homologous exon substitution events were remarkably conserved—all the homologous exons we identified evolved over 460 million years ago—and eight of the fourteen tissue-specific splice isoforms we identified were generated from homologous exons. The combination of proteomics evidence, ancient origin and tissue-specific splicing indicates that isoforms generated from homologous exons may have important cellular roles. PMID:26061177

  17. QKI5-mediated alternative splicing of the histone variant macroH2A1 regulates gastric carcinogenesis

    PubMed Central

    Li, Feng; Yi, Ping; Pi, Jingnan; Li, Lanfang; Hui, Jingyi; Wang, Fang; Liang, Aihua; Yu, Jia

    2016-01-01

    Alternative pre-mRNA splicing is a key mechanism for increasing proteomic diversity and modulating gene expression. Emerging evidence indicated that the splicing program is frequently dysregulated during tumorigenesis. Cancer cells produce protein isoforms that can promote growth and survival. The RNA-binding protein QKI5 is a critical regulator of alternative splicing in expanding lists of primary human tumors and tumor cell lines. However, its biological role and regulatory mechanism are poorly defined in gastric cancer (GC) development and progression. In this study, we demonstrated that the downregulation of QKI5 was associated with pTNM stage and pM state of GC patients. Re-introduction of QKI5 could inhibit GC cell proliferation, migration, and invasion in vitro and in vivo, which might be due to the altered splicing pattern of macroH2A1 pre-mRNA, leading to the accumulation of macroH2A1.1 isoform. Furthermore, QKI5 could inhibit cyclin L1 expression via promoting macroH2A1.1 production. Thus, this study identified a novel regulatory axis involved in gastric tumorigenesis and provided a new strategy for GC therapy. PMID:27092877

  18. Quantification of alternatively spliced FGFR2 RNAs using the RNA invasive cleavage assay

    PubMed Central

    WAGNER, ERIC J.; CURTIS, MICHELLE L.; ROBSON, NICOLE D.; BARANIAK, ANDREW P.; EIS, PEGGY S.; GARCIA-BLANCO, MARIANO A.

    2003-01-01

    The regulated splicing of fibroblast growth factor receptor-2 (FGFR2) transcripts leads to tissue-specific expression of distinct receptor isoforms. These isoforms contain two different versions of the ligand binding Ig-like domain III, which are encoded by exon IIIb or exon IIIc. The mutually exclusive use of exon IIIb and exon IIIc can be recapitulated in tissue culture using DT3 and AT3 rat prostate carcinoma cells. We used this well-characterized system to evaluate the precision and accuracy of the RNA invasive cleavage assay to specifically measure FGFR2 alternative splicing outcomes. Experiments presented here demonstrated that the RNA invasive cleavage assay could specifically detect isoforms with discrimination levels that ranged from 1 in 5 × 103 to 1 in 105. Moreover the assay could detect close to 0.01 amole of FGFR2 RNAs. The assay detected the expected levels of transcripts containing either exon IIIb or IIIc, but, surprisingly, it detected high levels of IIIb-IIIc double inclusion transcripts. This finding, which has important implications for the role of exon silencing and of mRNA surveillance mechanisms, had been missed by RT-PCR. Additionally, we used the RNA invasive cleavage assay to demonstrate a novel function for the regulatory element IAS2 in repressing exon IIIc inclusion. We also show here that purification of RNA is not necessary for the invasive cleavage assay, because crude cell lysates could be used to accurately measure alternative transcripts. The data presented here indicate that the RNA invasive cleavage assay is an important addition to the repertoire of techniques available for the study of alternative splicing. PMID:14624010

  19. Widespread and subtle: alternative splicing at short-distance tandem sites.

    PubMed

    Hiller, Michael; Platzer, Matthias

    2008-05-01

    Alternative splicing at donor or acceptor sites located just a few nucleotides apart is widespread in many species. It results in subtle changes in the transcripts and often in the encoded proteins. Several of these tandem splice events contribute to the repertoire of functionally different proteins, whereas many are neutral or deleterious. Remarkably, some of the functional events are differentially spliced in tissues or developmental stages, whereas others exhibit constant splicing ratios, indicating that function is not always associated with differential splicing. Stochastic splice site selection seems to play a major role in these processes. Here, we review recent progress in understanding functional and evolutionary aspects as well as the mechanism of splicing at short-distance tandem sites.

  20. Alternative splicing: enhancing ability to cope with stress via transcriptome plasticity.

    PubMed

    Mastrangelo, Anna M; Marone, Daniela; Laidò, Giovanni; De Leonardis, Anna M; De Vita, Pasquale

    2012-04-01

    Alternative splicing is a mechanism for the regulation of gene expression that is widespread in higher eukaryotes. Genome-wide approaches, based on comparison of expressed and genomic sequences, on tiling arrays, and on next-generation sequencing, have provided growing knowledge of the extent, distribution and association of alternative splicing with stress-related genes in plants. The functional meaning of alternative splicing in response to stress has been defined for many genes, and in particular for those involved in the regulation of the stress responses, such as protein kinases, transcription factors, splicing regulators and pathogen-resistance genes. The production of proteins with diverse domain rearrangements from the same gene is the main alternative splicing mechanism for pathogen-resistance genes. The plant response to abiotic stress is also characterized by a second mechanism, which consists of the expression of alternative transcripts that are targeted to nonsense-mediated decay. These quantitatively regulate stress-related gene expression. Many alternative splicing events are well conserved among plant species, and also across kingdoms, especially those observed in response to stress, for genes encoding splicing regulators, and other classes of RNA-binding proteins. Nevertheless, non-conserved events indicate that alternative splicing represents an evolutionary strategy that rapidly increases genome plasticity and develops new gene functions, along with other mechanisms such as gene duplication. Finally, the study of the naturally occurring variability of alternative splicing and the identification of genomic regions involved in the regulation of alternative splicing in crops are proposed as strategies for selecting genotypes with superior performance under adverse environmental conditions. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  1. Integrative analysis of tissue-specific methylation and alternative splicing identifies conserved transcription factor binding motifs

    PubMed Central

    Wan, Jun; Oliver, Verity F.; Zhu, Heng; Zack, Donald J.; Qian, Jiang; Merbs, Shannath L.

    2013-01-01

    The exact role of intragenic DNA methylation in regulating tissue-specific gene regulation is unclear. Recently, the DNA-binding protein CTCF has been shown to participate in the regulation of alternative splicing in a DNA methylation-dependent manner. To globally evaluate the relationship between DNA methylation and tissue-specific alternative splicing, we performed genome-wide DNA methylation profiling of mouse retina and brain. In protein-coding genes, tissue-specific differentially methylated regions (T-DMRs) were preferentially located in exons and introns. Gene ontology and evolutionary conservation analysis suggest that these T-DMRs are likely to be biologically relevant. More than 14% of alternatively spliced genes were associated with a T-DMR. T-DMR-associated genes were enriched for developmental genes, suggesting that a specific set of alternatively spliced genes may be regulated through DNA methylation. Novel DNA sequences motifs overrepresented in T-DMRs were identified as being associated with positive and/or negative regulation of alternative splicing in a position-dependent context. The majority of these evolutionarily conserved motifs contain a CpG dinucleotide. Some transcription factors, which recognize these motifs, are known to be involved in splicing. Our results suggest that DNA methylation-dependent alternative splicing is widespread and lay the foundation for further mechanistic studies of the role of DNA methylation in tissue-specific splicing regulation. PMID:23887936

  2. SKIP Confers Osmotic Tolerance during Salt Stress by Controlling Alternative Gene Splicing in Arabidopsis.

    PubMed

    Feng, Jinlin; Li, Jingjing; Gao, Zhaoxu; Lu, Yaru; Yu, Junya; Zheng, Qian; Yan, Shuning; Zhang, Wenjiao; He, Hang; Ma, Ligeng; Zhu, Zhengge

    2015-07-01

    Deciphering the mechanisms underlying plant responses to abiotic stress is key for improving plant stress resistance. Much is known about the regulation of gene expression in response to salt stress at the transcriptional level; however, little is known about this process at the posttranscriptional level. Recently, we demonstrated that SKIP is a component of spliceosome that interacts with clock gene pre-mRNAs and is essential for regulating their alternative splicing and mRNA maturation. In this study, we found that skip-1 plants are hypersensitive to both salt and osmotic stresses, and that SKIP is required for the alternative splicing and mRNA maturation of several salt-tolerance genes, including NHX1, CBL1, P5CS1, RCI2A, and PAT10. A genome-wide analysis revealed that SKIP mediates the alternative splicing of many genes under salt-stress conditions, and that most of the alternative splicing events in skip-1 involve intron retention and can generate a premature termination codon in the transcribed mRNA. SKIP also controls alternative splicing by modulating the recognition or cleavage of 5' and 3' splice donor and acceptor sites under salt-stress conditions. Therefore, this study addresses the fundamental question of how the mRNA splicing machinery in plants contributes to salt-stress responses at the posttranscriptional level, and provides a link between alternative splicing and salt tolerance.

  3. Alternative splicing of the FMR1 gene in human fetal brain neurons

    SciTech Connect

    Tao Huang; Yan Shen; Xue-bin Qin; Guan-Yun Wu

    1996-08-09

    The alternative splicing expression of the FMR1 gene was reported in several human and mouse tissues. Five regions of FMR1 gene can be alternatively spliced, but the combination of them has not been investigated fully. We reported here the analysis of alternative splicing pattern of the FMR1 gene in cultured fetal human neurons, using a RT-PCR and cloning strategy. Eleven splicing types were cloned and different isoforms were not equally represented. The dominant isoform represents nearly 40%, and the other isoforms were relatively rare. One isoform has a different carboxyl-terminus. Most of the alternative spliced regions appear hydrophilic; thus, they may locate on the surface of the FMR1 protein. 16 refs., 2 figs.

  4. A novel intra-U1 snRNP cross-regulation mechanism: alternative splicing switch links U1C and U1-70K expression.

    PubMed

    Rösel-Hillgärtner, Tanja Dorothe; Hung, Lee-Hsueh; Khrameeva, Ekaterina; Le Querrec, Patrick; Gelfand, Mikhail S; Bindereif, Albrecht

    2013-01-01

    The U1 small nuclear ribonucleoprotein (snRNP)-specific U1C protein participates in 5' splice site recognition and regulation of pre-mRNA splicing. Based on an RNA-Seq analysis in HeLa cells after U1C knockdown, we found a conserved, intra-U1 snRNP cross-regulation that links U1C and U1-70K expression through alternative splicing and U1 snRNP assembly. To investigate the underlying regulatory mechanism, we combined mutational minigene analysis, in vivo splice-site blocking by antisense morpholinos, and in vitro binding experiments. Alternative splicing of U1-70K pre-mRNA creates the normal (exons 7-8) and a non-productive mRNA isoform, whose balance is determined by U1C protein levels. The non-productive isoform is generated through a U1C-dependent alternative 3' splice site, which requires an adjacent cluster of regulatory 5' splice sites and binding of intact U1 snRNPs. As a result of nonsense-mediated decay (NMD) of the non-productive isoform, U1-70K mRNA and protein levels are down-regulated, and U1C incorporation into the U1 snRNP is impaired. U1-70K/U1C-deficient particles are assembled, shifting the alternative splicing balance back towards productive U1-70K splicing, and restoring assembly of intact U1 snRNPs. Taken together, we established a novel feedback regulation that controls U1-70K/U1C homeostasis and ensures correct U1 snRNP assembly and function.

  5. Analysis of Alternative Pre-RNA Splicing in the Mouse Retina Using a Fluorescent Reporter.

    PubMed

    Murphy, Daniel; Kolandaivelu, Saravanan; Ramamurthy, Visvanathan; Stoilov, Peter

    2016-01-01

    In vivo alternative splicing is controlled in a tissue and cell type specific manner. Often individual cellular components of complex tissues will express different splicing programs. Thus, when studying splicing in multicellular organisms it is critical to determine the exon inclusion levels in individual cells positioned in the context of their native tissue or organ. Here we describe how a fluorescent splicing reporter in combination with in vivo electroporation can be used to visualize alternative splicing in individual cells within mature tissues. In a test case we show how the splicing of a photoreceptor specific exon can be visualized within the mouse retina. The retina was chosen as an example of a complex tissue that is fragile and whose cells cannot be studied in culture. With minor modifications to the injection and electroporation procedure, the protocol we outline can be applied to other tissues and organs.

  6. Oncogenic fusion protein EWS-FLI1 is a network hub that regulates alternative splicing.

    PubMed

    Selvanathan, Saravana P; Graham, Garrett T; Erkizan, Hayriye V; Dirksen, Uta; Natarajan, Thanemozhi G; Dakic, Aleksandra; Yu, Songtao; Liu, Xuefeng; Paulsen, Michelle T; Ljungman, Mats E; Wu, Cathy H; Lawlor, Elizabeth R; Üren, Aykut; Toretsky, Jeffrey A

    2015-03-17

    The synthesis and processing of mRNA, from transcription to translation initiation, often requires splicing of intragenic material. The final mRNA composition varies based on proteins that modulate splice site selection. EWS-FLI1 is an Ewing sarcoma (ES) oncoprotein with an interactome that we demonstrate to have multiple partners in spliceosomal complexes. We evaluate the effect of EWS-FLI1 on posttranscriptional gene regulation using both exon array and RNA-seq. Genes that potentially regulate oncogenesis, including CLK1, CASP3, PPFIBP1, and TERT, validate as alternatively spliced by EWS-FLI1. In a CLIP-seq experiment, we find that EWS-FLI1 RNA-binding motifs most frequently occur adjacent to intron-exon boundaries. EWS-FLI1 also alters splicing by directly binding to known splicing factors including DDX5, hnRNP K, and PRPF6. Reduction of EWS-FLI1 produces an isoform of γ-TERT that has increased telomerase activity compared with wild-type (WT) TERT. The small molecule YK-4-279 is an inhibitor of EWS-FLI1 oncogenic function that disrupts specific protein interactions, including helicases DDX5 and RNA helicase A (RHA) that alters RNA-splicing ratios. As such, YK-4-279 validates the splicing mechanism of EWS-FLI1, showing alternatively spliced gene patterns that significantly overlap with EWS-FLI1 reduction and WT human mesenchymal stem cells (hMSC). Exon array analysis of 75 ES patient samples shows similar isoform expression patterns to cell line models expressing EWS-FLI1, supporting the clinical relevance of our findings. These experiments establish systemic alternative splicing as an oncogenic process modulated by EWS-FLI1. EWS-FLI1 modulation of mRNA splicing may provide insight into the contribution of splicing toward oncogenesis, and, reciprocally, EWS-FLI1 interactions with splicing proteins may inform the splicing code.

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

    PubMed Central

    Llorian, Miriam; Gooding, Clare; Bellora, Nicolas; Hallegger, Martina; Buckroyd, Adrian; Wang, Xiao; Rajgor, Dipen; Kayikci, Melis; Feltham, Jack; Ule, Jernej; Eyras, Eduardo; Smith, Christopher W.J.

    2016-01-01

    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. PMID:27317697

  8. The emerging role of alternative splicing in senescence and aging.

    PubMed

    Deschênes, Mathieu; Chabot, Benoit

    2017-07-13

    Deregulation of precursor mRNA splicing is associated with many illnesses and has been linked to age-related chronic diseases. Here we review recent progress documenting how defects in the machinery that performs intron removal and controls splice site selection contribute to cellular senescence and organismal aging. We discuss the functional association linking p53, IGF-1, SIRT1, and ING-1 splice variants with senescence and aging, and review a selection of splicing defects occurring in accelerated aging (progeria), vascular aging, and Alzheimer's disease. Overall, it is becoming increasingly clear that changes in the activity of splicing factors and in the production of key splice variants can impact cellular senescence and the aging phenotype. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  9. Computational analysis of translational readthrough proteins in Drosophila and yeast reveals parallels to alternative splicing

    PubMed Central

    Pancsa, Rita; Macossay-Castillo, Mauricio; Kosol, Simone; Tompa, Peter

    2016-01-01

    In translational readthrough (TR) the ribosome continues extending the nascent protein beyond the first in-frame termination codon. Due to the lack of dedicated analyses of eukaryotic TR cases, the associated functional-evolutionary advantages are still unclear. Here, based on a variety of computational methods, we describe the structural and functional properties of previously proposed D. melanogaster and S. cerevisiae TR proteins and extensions. We found that in D. melanogaster TR affects long proteins in mainly regulatory roles. Their TR-extensions are structurally disordered and rich in binding motifs, which, together with their cell-type- and developmental stage-dependent inclusion, suggest that similarly to alternatively spliced exons they rewire cellular interaction networks in a temporally and spatially controlled manner. In contrast, yeast TR proteins are rather short and fulfil mainly housekeeping functions, like translation. Yeast extensions usually lack disorder and linear motifs, which precludes elucidating their functional relevance with sufficient confidence. Therefore we propose that by being much more restricted and by lacking clear functional hallmarks in yeast as opposed to fruit fly, TR shows remarkable parallels with alternative splicing. Additionally, the lack of conservation of TR extensions among orthologous TR proteins suggests that TR-mediated functions may be generally specific to lower taxonomic levels. PMID:27561673

  10. Alternative splicing acting as a bridge in evolution

    PubMed Central

    Salamov, Asaf; Kuo, Alan; Aerts, Andrea L.; Kong, Xiangyang; Grigoriev, Igor V.

    2015-01-01

    Background Alternative splicing (AS) regulates diverse cellular and developmental functions through alternative protein structures of different isoforms. Alternative exons dominate AS in vertebrates; however, very little is known about the extent and function of AS in lower eukaryotes. To understand the role of introns in gene evolution, we examined AS from a green algal and five fungal genomes using a novel EST-based gene-modeling algorithm (COMBEST). Methods AS from each genome was classified with COMBEST that maps EST sequences to genomes to build gene models. Various aspects of AS were analyzed through statistical methods. The interplay of intron 3n length, phase, coding property, and intron retention (RI) were examined with Chi-square testing. Results With 3 to 834 times EST coverage, we identified up to 73% of AS in intron-containing genes and found preponderance of RI among 11 types of AS. The number of exons, expression level, and maximum intron length correlated with number of AS per gene (NAG), and intron-rich genes suppressed AS. Genes with AS were more ancient, and AS was conserved among fungal genomes. Among stopless introns, non-retained introns (NRI) avoided, but major RI preferred 3n length. In contrast, stop-containing introns showed uniform distribution among 3n, 3n+1, and 3n+2 lengths. We found a clue to the intron phase enigma: it was the coding function of introns involved in AS that dictates the intron phase bias. Conclusions Majority of AS is non-functional, and the extent of AS is suppressed for intron-rich genes. RI through 3n length, stop codon, and phase bias bridges the transition from functionless to functional alternative isoforms. PMID:27358887

  11. TassDB2 - A comprehensive database of subtle alternative splicing events.

    PubMed

    Sinha, Rileen; Lenser, Thorsten; Jahn, Niels; Gausmann, Ulrike; Friedel, Swetlana; Szafranski, Karol; Huse, Klaus; Rosenstiel, Philip; Hampe, Jochen; Schuster, Stefan; Hiller, Michael; Backofen, Rolf; Platzer, Matthias

    2010-04-29

    Subtle alternative splicing events involving tandem splice sites separated by a short (2-12 nucleotides) distance are frequent and evolutionarily widespread in eukaryotes, and a major contributor to the complexity of transcriptomes and proteomes. However, these events have been either omitted altogether in databases on alternative splicing, or only the cases of experimentally confirmed alternative splicing have been reported. Thus, a database which covers all confirmed cases of subtle alternative splicing as well as the numerous putative tandem splice sites (which might be confirmed once more transcript data becomes available), and allows to search for tandem splice sites with specific features and download the results, is a valuable resource for targeted experimental studies and large-scale bioinformatics analyses of tandem splice sites. Towards this goal we recently set up TassDB (Tandem Splice Site DataBase, version 1), which stores data about alternative splicing events at tandem splice sites separated by 3 nt in eight species. We have substantially revised and extended TassDB. The currently available version 2 contains extensive information about tandem splice sites separated by 2-12 nt for the human and mouse transcriptomes including data on the conservation of the tandem motifs in five vertebrates. TassDB2 offers a user-friendly interface to search for specific genes or for genes containing tandem splice sites with specific features as well as the possibility to download result datasets. For example, users can search for cases of alternative splicing where the proportion of EST/mRNA evidence supporting the minor isoform exceeds a specific threshold, or where the difference in splice site scores is specified by the user. The predicted impact of each event on the protein is also reported, along with information about being a putative target for the nonsense-mediated decay (NMD) pathway. Links are provided to the UCSC genome browser and other external resources

  12. Relationship between nucleosome positioning and progesterone-induced alternative splicing in breast cancer cells.

    PubMed

    Iannone, Camilla; Pohl, Andy; Papasaikas, Panagiotis; Soronellas, Daniel; Vicent, Guillermo P; Beato, Miguel; ValcáRcel, Juan

    2015-03-01

    Splicing of mRNA precursors can occur cotranscriptionally and it has been proposed that chromatin structure influences splice site recognition and regulation. Here we have systematically explored potential links between nucleosome positioning and alternative splicing regulation upon progesterone stimulation of breast cancer cells. We confirm preferential nucleosome positioning in exons and report four distinct profiles of nucleosome density around alternatively spliced exons, with RNA polymerase II accumulation closely following nucleosome positioning. Hormone stimulation induces switches between profile classes, correlating with a subset of alternative splicing changes. Hormone-induced exon inclusion often correlates with higher nucleosome occupancy at the exon or the preceding intronic region and with higher RNA polymerase II accumulation. In contrast, exons skipped upon hormone stimulation display low nucleosome densities even before hormone treatment, suggesting that chromatin structure primes alternative splicing regulation. Skipped exons frequently harbor binding sites for hnRNP AB, a hormone-induced splicing regulator whose knock down prevents some hormone-induced skipping events. Collectively, our results argue that a variety of chromatin architecture mechanisms can influence alternative splicing decisions.

  13. Genome-Wide Analysis of Alternative Splicing during Development and Drought Stress in Maize.

    PubMed

    Thatcher, Shawn R; Danilevskaya, Olga N; Meng, Xin; Beatty, Mary; Zastrow-Hayes, Gina; Harris, Charlotte; Van Allen, Brandon; Habben, Jeffrey; Li, Bailin

    2016-01-01

    Alternative splicing plays a crucial role in plant development as well as stress responses. Although alternative splicing has been studied during development and in response to stress, the interplay between these two factors remains an open question. To assess the effects of drought stress on developmentally regulated splicing in maize (Zea mays), 94 RNA-seq libraries from ear, tassel, and leaf of the B73 public inbred line were constructed at four developmental stages under both well-watered and drought conditions. This analysis was supplemented with a publicly available series of 53 libraries from developing seed, embryo, and endosperm. More than 48,000 novel isoforms, often with stage- or condition-specific expression, were uncovered, suggesting that developmentally regulated alternative splicing occurs in thousands of genes. Drought induced large developmental splicing changes in leaf and ear but relatively few in tassel. Most developmental stage-specific splicing changes affected by drought were tissue dependent, whereas stage-independent changes frequently overlapped between leaf and ear. A linear relationship was found between gene expression changes in splicing factors and alternative spicing of other genes during development. Collectively, these results demonstrate that alternative splicing is strongly associated with tissue type, developmental stage, and stress condition. © 2016 American Society of Plant Biologists. All Rights Reserved.

  14. Genome-Wide Analysis of Alternative Splicing during Development and Drought Stress in Maize1[OPEN

    PubMed Central

    Thatcher, Shawn R.; Meng, Xin; Beatty, Mary; Zastrow-Hayes, Gina; Harris, Charlotte; Habben, Jeffrey; Li, Bailin

    2016-01-01

    Alternative splicing plays a crucial role in plant development as well as stress responses. Although alternative splicing has been studied during development and in response to stress, the interplay between these two factors remains an open question. To assess the effects of drought stress on developmentally regulated splicing in maize (Zea mays), 94 RNA-seq libraries from ear, tassel, and leaf of the B73 public inbred line were constructed at four developmental stages under both well-watered and drought conditions. This analysis was supplemented with a publicly available series of 53 libraries from developing seed, embryo, and endosperm. More than 48,000 novel isoforms, often with stage- or condition-specific expression, were uncovered, suggesting that developmentally regulated alternative splicing occurs in thousands of genes. Drought induced large developmental splicing changes in leaf and ear but relatively few in tassel. Most developmental stage-specific splicing changes affected by drought were tissue dependent, whereas stage-independent changes frequently overlapped between leaf and ear. A linear relationship was found between gene expression changes in splicing factors and alternative spicing of other genes during development. Collectively, these results demonstrate that alternative splicing is strongly associated with tissue type, developmental stage, and stress condition. PMID:26582726

  15. ABERRANT SPLICING OF A BRAIN-ENRICHED ALTERNATIVE EXON ELIMINATES TUMOR SUPPRESSOR FUNCTION AND PROMOTES ONCOGENE FUNCTION DURING BRAIN TUMORIGENESIS

    PubMed Central

    Bredel, Markus; Ferrarese, Roberto; Harsh, Griffith R.; Yadav, Ajay K.; Bug, Eva; Maticzka, Daniel; Reichardt, Wilfried; Masilamani, Anie P.; Dai, Fangping; Kim, Hyunsoo; Hadler, Michael; Scholtens, Denise M.; Yu, Irene L.Y.; Beck, Jürgen; Srinivasasainagendra, Vinodh; Costa, Fabrizio; Baxan, Nicoleta; Pfeifer, Dietmar; Elverfeldt, Dominik v.; Backofen, Rolf; Weyerbrock, Astrid; Duarte, Christine W.; He, Xiaolin; Prinz, Marco; Chandler, James P.; Vogel, Hannes; Chakravarti, Arnab; Rich, Jeremy N.; Carro, Maria S.

    2014-01-01

    . CONCLUSIONS: Our data illustrate how anomalous splicing of a tissue-regulated exon in a constituent of an oncogenic signaling pathway eliminates its tumor suppressor function and promotes tumorigenesis. This paradigm of malignant glial transformation as a consequence of tissue-specific alternative exon splicing in a tumor suppressor, may have widespread applicability in explaining how changes in critical tissue-specific regulatory mechanisms reprogram normal development to oncogenesis. SECONDARY CATEGORY: n/a.

  16. Mutually exclusive splicing regulates the Nav 1.6 sodium channel function through a combinatorial mechanism that involves three distinct splicing regulatory elements and their ligands

    PubMed Central

    Zubović, Lorena; Baralle, Marco; Baralle, Francisco E.

    2012-01-01

    Mutually exclusive splicing is a form of alternative pre-mRNA processing that consists in the use of only one of a set of two or more exons. We have investigated the mechanisms involved in this process for exon 18 of the Nav 1.6 sodium channel transcript and its significance regarding gene-expression regulation. The 18N exon (neonatal form) has a stop codon in phase and although the mRNA can be detected by amplification methods, the truncated protein has not been observed. The switch from 18N to 18A (adult form) occurs only in a restricted set of neural tissues producing the functional channel while other tissues display the mRNA with the 18N exon also in adulthood. We demonstrate that the mRNA species carrying the stop codon is subjected to Nonsense-Mediated Decay, providing a control mechanism of channel expression. We also map a string of cis-elements within the mutually exclusive exons and in the flanking introns responsible for their strict tissue and temporal specificity. These elements bind a series of positive (RbFox-1, SRSF1, SRSF2) and negative (hnRNPA1, PTB, hnRNPA2/B1, hnRNPD-like JKTBP) splicing regulatory proteins. These splicing factors, with the exception of RbFox-1, are ubiquitous but their levels vary during development and differentiation, ensuing unique sets of tissue and temporal levels of splicing factors. The combinatorial nature of these elements is highlighted by the dominance of the elements that bind the ubiquitous factors over the tissue specific RbFox-1. PMID:22434879

  17. Applying genetic programming to the prediction of alternative mRNA splice variants.

    PubMed

    Vukusic, Ivana; Grellscheid, Sushma Nagaraja; Wiehe, Thomas

    2007-04-01

    Genetic programming (GP) can be used to classify a given gene sequence as either constitutively or alternatively spliced. We describe the principles of GP and apply it to a well-defined data set of alternatively spliced genes. A feature matrix of sequence properties, such as nucleotide composition or exon length, was passed to the GP system "Discipulus." To test its performance we concentrated on cassette exons (SCE) and retained introns (SIR). We analyzed 27,519 constitutively spliced and 9641 cassette exons including their neighboring introns; in addition we analyzed 33,316 constitutively spliced introns compared to 2712 retained introns. We find that the classifier yields highly accurate predictions on the SIR data with a sensitivity of 92.1% and a specificity of 79.2%. Prediction accuracies on the SCE data are lower, 47.3% (sensitivity) and 70.9% (specificity), indicating that alternative splicing of introns can be better captured by sequence properties than that of exons.

  18. Structure of the human myelin/oligodendrocyte glycoprotein gene and multiple alternative spliced isoforms

    SciTech Connect

    Pham-Dinh, D.; Gaspera, D.B.; Dautigny, A.

    1995-09-20

    Myelin/oligodendrocyte glycoprotein (MOG), a special component of the central nervous system localization on the outermost lamellae of mature myelin, is a member of the immunoglobulin superfamily. We report here the organization of the human MOG gene, which spans approximately 17 kb, and the characterization of six MOG mRNA splicing variants. The intron/exon structure of the human MOG gene confirmed the splicing pattern, supporting the hypothesis that mRNA isoforms could arise by alternative splicing of a single gene. In addition to the eight exons coding for the major MOG isoform, the human MOG gene also contains 3` region, a previously unknown alternatively spliced coding exon, VIA. Alternative utilization of two acceptor splicing sites for exon VIII could produce two different C-termini. The nucleotide sequences presented here may be a useful tool to study further possible involvement if the MOG gene in hereditary neurological disorders. 23 refs., 5 figs.

  19. Alternative splicing of breast cancer associated gene BRCA1 from breast cancer cell line.

    PubMed

    Lixia, Miao; Zhijian, Cao; Chao, Shen; Chaojiang, Gu; Congyi, Zheng

    2007-01-31

    Breast cancer is the most common malignancy among women, and mutations in the BRCA1 gene produce increased susceptibility to these malignancies in certain families. In this study, the forward 1-13 exons of breast cancer associated gene BRCA1 were cloned from breast cancer cell line ZR-75-30 by RT-PCR method. Sequence analysis showed that nine BRCA1 splice forms were isolated and characterized, compared with wild-type BRCA1 gene, five splice forms of which were novel. These splice isoforms were produced from the molecular mechanism of 5' and 3' alternative splicing. All these splice forms deleting exon 11b and the locations of alternative splicing were focused on two parts:one was exons 2 and 3, and the other was exons 9 and 10. These splice forms accorded with GT-AG rule. Most these BRCA1 splice variants still kept the original reading frame. Western blot analysis indicated that some BRCA1 splice variants were expressed in ZR-75-30 cell line at the protein level. In addition, we confirmed the presence of these new transcripts of BRCA1 gene in MDA-MB-435S, K562, Hela, HLA, HIC, H9, Jurkat and human fetus samples by RT-PCR analysis. These results suggested that breast cancer associated gene BRCA1 may have unexpectedly a large number of splice variants. We hypothesized that alternative splicing of BRCA1 possibly plays a major role in the tumorigenesis of breast and/or ovarian cancer. Thus, the identification of cancer-specific splice forms will provide a novel source for the discovery of diagnostic or prognostic biomarkers and tumor antigens suitable as targets for therapeutic intervention.

  20. Serine/Arginine-Rich Splicing Factor 3 and Heterogeneous Nuclear Ribonucleoprotein A1 Regulate Alternative RNA Splicing and Gene Expression of Human Papillomavirus 18 through Two Functionally Distinguishable cis Elements

    PubMed Central

    Ajiro, Masahiko; Tang, Shuang; Doorbar, John

    2016-01-01

    ABSTRACT Human papillomavirus 18 (HPV18) is the second most common oncogenic HPV type associated with cervical, anogenital, and oropharyngeal cancers. Like other oncogenic HPVs, HPV18 encodes two major (one early and one late) polycistronic pre-mRNAs that are regulated by alternative RNA splicing to produce a repertoire of viral transcripts for the expression of individual viral genes. However, RNA cis-regulatory elements and trans-acting factors contributing to HPV18 alternative RNA splicing remain unknown. In this study, an exonic splicing enhancer (ESE) in the nucleotide (nt) 3520 to 3550 region in the HPV18 genome was identified and characterized for promotion of HPV18 929^3434 splicing and E1^E4 production through interaction with SRSF3, a host oncogenic splicing factor differentially expressed in epithelial cells and keratinocytes. Introduction of point mutations in the SRSF3-binding site or knockdown of SRSF3 expression in cells reduces 929^3434 splicing and E1^E4 production but activates other, minor 929^3465 and 929^3506 splicing. Knockdown of SRSF3 expression also enhances the expression of E2 and L1 mRNAs. An exonic splicing silencer (ESS) in the HPV18 nt 612 to 639 region was identified as being inhibitory to the 233^416 splicing of HPV18 E6E7 pre-mRNAs via binding to hnRNP A1, a well-characterized, abundantly and ubiquitously expressed RNA-binding protein. Introduction of point mutations into the hnRNP A1-binding site or knockdown of hnRNP A1 expression promoted 233^416 splicing and reduced E6 expression. These data provide the first evidence that the alternative RNA splicing of HPV18 pre-mRNAs is subject to regulation by viral RNA cis elements and host trans-acting splicing factors. IMPORTANCE Expression of HPV18 genes is regulated by alternative RNA splicing of viral polycistronic pre-mRNAs to produce a repertoire of viral early and late transcripts. RNA cis elements and trans-acting factors contributing to HPV18 alternative RNA splicing have been

  1. Serine/Arginine-Rich Splicing Factor 3 and Heterogeneous Nuclear Ribonucleoprotein A1 Regulate Alternative RNA Splicing and Gene Expression of Human Papillomavirus 18 through Two Functionally Distinguishable cis Elements.

    PubMed

    Ajiro, Masahiko; Tang, Shuang; Doorbar, John; Zheng, Zhi-Ming

    2016-10-15

    Human papillomavirus 18 (HPV18) is the second most common oncogenic HPV type associated with cervical, anogenital, and oropharyngeal cancers. Like other oncogenic HPVs, HPV18 encodes two major (one early and one late) polycistronic pre-mRNAs that are regulated by alternative RNA splicing to produce a repertoire of viral transcripts for the expression of individual viral genes. However, RNA cis-regulatory elements and trans-acting factors contributing to HPV18 alternative RNA splicing remain unknown. In this study, an exonic splicing enhancer (ESE) in the nucleotide (nt) 3520 to 3550 region in the HPV18 genome was identified and characterized for promotion of HPV18 929^3434 splicing and E1^E4 production through interaction with SRSF3, a host oncogenic splicing factor differentially expressed in epithelial cells and keratinocytes. Introduction of point mutations in the SRSF3-binding site or knockdown of SRSF3 expression in cells reduces 929^3434 splicing and E1^E4 production but activates other, minor 929^3465 and 929^3506 splicing. Knockdown of SRSF3 expression also enhances the expression of E2 and L1 mRNAs. An exonic splicing silencer (ESS) in the HPV18 nt 612 to 639 region was identified as being inhibitory to the 233^416 splicing of HPV18 E6E7 pre-mRNAs via binding to hnRNP A1, a well-characterized, abundantly and ubiquitously expressed RNA-binding protein. Introduction of point mutations into the hnRNP A1-binding site or knockdown of hnRNP A1 expression promoted 233^416 splicing and reduced E6 expression. These data provide the first evidence that the alternative RNA splicing of HPV18 pre-mRNAs is subject to regulation by viral RNA cis elements and host trans-acting splicing factors. Expression of HPV18 genes is regulated by alternative RNA splicing of viral polycistronic pre-mRNAs to produce a repertoire of viral early and late transcripts. RNA cis elements and trans-acting factors contributing to HPV18 alternative RNA splicing have been discovered in this

  2. Single-Nucleotide Polymorphisms in NAGNAG Acceptors Are Highly Predictive for Variations of Alternative Splicing

    PubMed Central

    Hiller, Michael; Huse, Klaus; Szafranski, Karol; Jahn, Niels; Hampe, Jochen; Schreiber, Stefan; Backofen, Rolf; Platzer, Matthias

    2006-01-01

    Aberrant or modified splicing patterns of genes are causative for many human diseases. Therefore, the identification of genetic variations that cause changes in the splicing pattern of a gene is important. Elsewhere, we described the widespread occurrence of alternative splicing at NAGNAG acceptors. Here, we report a genomewide screen for single-nucleotide polymorphisms (SNPs) that affect such tandem acceptors. From 121 SNPs identified, we extracted 64 SNPs that most likely affect alternative NAGNAG splicing. We demonstrate that the NAGNAG motif is necessary and sufficient for this type of alternative splicing. The evolutionarily young NAGNAG alleles, as determined by the comparison with the chimpanzee genome, exhibit the same biases toward intron phase 1 and single–amino acid insertion/deletions that were already observed for all human NAGNAG acceptors. Since 28% of the NAGNAG SNPs occur in known disease genes, they represent preferable candidates for a more-detailed functional analysis, especially since the splice relevance for some of the coding SNPs is overlooked. Against the background of a general lack of methods for identifying splice-relevant SNPs, the presented approach is highly effective in the prediction of polymorphisms that are causal for variations in alternative splicing. PMID:16400609

  3. Multiple Scenarios of Transition to Chaos in the Alternative Splicing Model

    NASA Astrophysics Data System (ADS)

    Kogai, Vladislav V.; Likhoshvai, Vitaly A.; Fadeev, Stanislav I.; Khlebodarova, Tamara M.

    We have investigated the scenarios of transition to chaos in the mathematical model of a genetic system constituted by a single transcription factor-encoding gene, the expression of which is self-regulated by a feedback loop that involves protein isoforms. Alternative splicing results in the synthesis of protein isoforms providing opposite regulatory outcomes — activation or repression. The model is represented by a differential equation with two delayed arguments. The possibility of transition to chaos dynamics via all classical scenarios: a cascade of period-doubling bifurcations, quasiperiodicity and type-I, type-II and type-III intermittencies, has been numerically demonstrated. The parametric features of each type of transition to chaos have been described.

  4. Alternative splicing in the nervous system: an emerging source of diversity and regulation.

    PubMed

    Lee, Christopher J; Irizarry, Kris

    2003-10-15

    Alternative splicing is emerging as a major mechanism of functional regulation in the human genome. Previously considered to be an unusual event, it has been detected by many genomics studies in 40%-60% of human genes. Moreover, it appears to be of central importance for neuronal genes and other genes involved in "information processing" functions. In this review, we will summarize alternative splicing's effects on mRNA transcripts, protein products, biological function, and human disease, focusing on genes of neuropsychiatric interest. We will also describe the latest experimental methods and database resources that can help neuroscientists make use of alternative splicing in their own research.

  5. Spatio-temporal regulations and functions of neuronal alternative RNA splicing in developing and adult brains.

    PubMed

    Iijima, Takatoshi; Hidaka, Chiharu; Iijima, Yoko

    2016-08-01

    Alternative pre-mRNA splicing is a fundamental mechanism that generates molecular diversity from a single gene. In the central nervous system (CNS), key neural developmental steps are thought to be controlled by alternative splicing decisions, including the molecular diversity underlying synaptic wiring, plasticity, and remodeling. Significant progress has been made in understanding the molecular mechanisms and functions of alternative pre-mRNA splicing in neurons through studies in invertebrate systems; however, recent studies have begun to uncover the potential role of neuronal alternative splicing in the mammalian CNS. This article provides an overview of recent findings regarding the regulation and function of neuronal alternative splicing. In particular, we focus on the spatio-temporal regulation of neurexin, a synaptic adhesion molecule, by neuronal cell type-specific factors and neuronal activity, which are thought to be especially important for characterizing neural development and function within the mammalian CNS. Notably, there is increasing evidence that implicates the dysregulation of neuronal splicing events in several neurological disorders. Therefore, understanding the detailed mechanisms of neuronal alternative splicing in the mammalian CNS may provide plausible treatment strategies for these diseases.

  6. Alternative Splicing Mediates Responses of the Arabidopsis Circadian Clock to Temperature Changes[W

    PubMed Central

    James, Allan B.; Syed, Naeem Hasan; Bordage, Simon; Marshall, Jacqueline; Nimmo, Gillian A.; Jenkins, Gareth I.; Herzyk, Pawel; Brown, John W.S.; Nimmo, Hugh G.

    2012-01-01

    Alternative splicing plays crucial roles by influencing the diversity of the transcriptome and proteome and regulating protein structure/function and gene expression. It is widespread in plants, and alteration of the levels of splicing factors leads to a wide variety of growth and developmental phenotypes. The circadian clock is a complex piece of cellular machinery that can regulate physiology and behavior to anticipate predictable environmental changes on a revolving planet. We have performed a system-wide analysis of alternative splicing in clock components in Arabidopsis thaliana plants acclimated to different steady state temperatures or undergoing temperature transitions. This revealed extensive alternative splicing in clock genes and dynamic changes in alternatively spliced transcripts. Several of these changes, notably those affecting the circadian clock genes LATE ELONGATED HYPOCOTYL (LHY) and PSEUDO RESPONSE REGULATOR7, are temperature-dependent and contribute markedly to functionally important changes in clock gene expression in temperature transitions by producing nonfunctional transcripts and/or inducing nonsense-mediated decay. Temperature effects on alternative splicing contribute to a decline in LHY transcript abundance on cooling, but LHY promoter strength is not affected. We propose that temperature-associated alternative splicing is an additional mechanism involved in the operation and regulation of the plant circadian clock. PMID:22408072

  7. Alternative splicing mediates responses of the Arabidopsis circadian clock to temperature changes.

    PubMed

    James, Allan B; Syed, Naeem Hasan; Bordage, Simon; Marshall, Jacqueline; Nimmo, Gillian A; Jenkins, Gareth I; Herzyk, Pawel; Brown, John W S; Nimmo, Hugh G

    2012-03-01

    Alternative splicing plays crucial roles by influencing the diversity of the transcriptome and proteome and regulating protein structure/function and gene expression. It is widespread in plants, and alteration of the levels of splicing factors leads to a wide variety of growth and developmental phenotypes. The circadian clock is a complex piece of cellular machinery that can regulate physiology and behavior to anticipate predictable environmental changes on a revolving planet. We have performed a system-wide analysis of alternative splicing in clock components in Arabidopsis thaliana plants acclimated to different steady state temperatures or undergoing temperature transitions. This revealed extensive alternative splicing in clock genes and dynamic changes in alternatively spliced transcripts. Several of these changes, notably those affecting the circadian clock genes late elongated hypocotyl (LHY) and pseudo response regulator7, are temperature-dependent and contribute markedly to functionally important changes in clock gene expression in temperature transitions by producing nonfunctional transcripts and/or inducing nonsense-mediated decay. Temperature effects on alternative splicing contribute to a decline in LHY transcript abundance on cooling, but LHY promoter strength is not affected. We propose that temperature-associated alternative splicing is an additional mechanism involved in the operation and regulation of the plant circadian clock.

  8. The evolutionary fate of alternatively spliced homologous exons after gene duplication.

    PubMed

    Abascal, Federico; Tress, Michael L; Valencia, Alfonso

    2015-04-29

    Alternative splicing and gene duplication are the two main processes responsible for expanding protein functional diversity. Although gene duplication can generate new genes and alternative splicing can introduce variation through alternative gene products, the interplay between the two processes is complex and poorly understood. Here, we have carried out a study of the evolution of alternatively spliced exons after gene duplication to better understand the interaction between the two processes. We created a manually curated set of 97 human genes with mutually exclusively spliced homologous exons and analyzed the evolution of these exons across five distantly related vertebrates (lamprey, spotted gar, zebrafish, fugu, and coelacanth). Most of these exons had an ancient origin (more than 400 Ma). We found examples supporting two extreme evolutionary models for the behaviour of homologous axons after gene duplication. We observed 11 events in which gene duplication was accompanied by splice isoform separation, that is, each paralog specifically conserved just one distinct ancestral homologous exon. At other extreme, we identified genes in which the homologous exons were always conserved within paralogs, suggesting that the alternative splicing event cannot easily be separated from the function in these genes. That many homologous exons fall in between these two extremes highlights the diversity of biological systems and suggests that the subtle balance between alternative splicing and gene duplication is adjusted to the specific cellular context of each gene.

  9. Alternative splicing affects the subcellular localization of Drosha

    PubMed Central

    Link, Steffen; Grund, Stefanie E.; Diederichs, Sven

    2016-01-01

    The RNase III enzyme Drosha is a key factor in microRNA (miRNA) biogenesis and as such indispensable for cellular homeostasis and developmental processes. Together with its co-factor DGCR8, it converts the primary transcript (pri-miRNA) into the precursor hairpin (pre-miRNA) in the nucleus. While the middle and the C-terminal domain are crucial for pri-miRNA processing and DGCR8 binding, the function of the N-terminus remains cryptic. Different studies have linked this region to the subcellular localization of Drosha, stabilization and response to stress. In this study, we identify alternatively spliced Drosha transcripts that are devoid of a part of the arginine/serine-rich (RS-rich) domain and expressed in a large set of human cells. In contrast to their expected habitation, we find two isoforms also present in the cytoplasm, while the other two isoforms reside exclusively in the nucleus. Their processing activity for pri-miRNAs and the binding to co-factors remains unaltered. In multiple cell lines, the endogenous mRNA expression of the Drosha isoforms correlates with the localization of endogenous Drosha proteins. The pri-miRNA processing efficiency is not significantly different between groups of cells with or without cytoplasmic Drosha expression. In summary, we discovered novel isoforms of Drosha with differential subcellular localization pointing toward additional layers of complexity in the regulation of its activity. PMID:27185895

  10. Ancient nature of alternative splicing and functions of introns

    SciTech Connect

    Zhou, Kemin; Salamov, Asaf; Kuo, Alan; Aerts, Andrea; Grigoriev, Igor

    2011-03-21

    Using four genomes: Chamydomonas reinhardtii, Agaricus bisporus, Aspergillus carbonarius, and Sporotricum thermophile with EST coverage of 2.9x, 8.9x, 29.5x, and 46.3x respectively, we identified 11 alternative splicing (AS) types that were dominated by intron retention (RI; biased toward short introns) and found 15, 35, 52, and 63percent AS of multiexon genes respectively. Genes with AS were more ancient, and number of AS correlated with number of exons, expression level, and maximum intron length of the gene. Introns with tendency to be retained had either stop codons or length of 3n+1 or 3n+2 presumably triggering nonsense-mediated mRNA decay (NMD), but introns retained in major isoforms (0.2-6percent of all introns) were biased toward 3n length and stop codon free. Stopless introns were biased toward phase 0, but 3n introns favored phase 1 that introduced more flexible and hydrophilic amino acids on both ends of introns which would be less disruptive to protein structure. We proposed a model in which minor RI intron could evolve into major RI that could facilitate intron loss through exonization.

  11. Designing alternative splicing RNA-seq studies. Beyond generic guidelines

    PubMed Central

    Stephan-Otto Attolini, Camille; Peña, Victor; Rossell, David

    2015-01-01

    Motivation: Designing an RNA-seq study depends critically on its specific goals, technology and underlying biology, which renders general guidelines inadequate. We propose a Bayesian framework to customize experiments so that goals can be attained and resources are not wasted, with a focus on alternative splicing. Results: We studied how read length, sequencing depth, library preparation and the number of replicates affects cost-effectiveness of single-sample and group comparison studies. Optimal settings varied strongly according to the target organism or tissue (potential 50–500% cost cuts) and, interestingly, short reads outperformed long reads for standard analyses. Our framework learns key characteristics for study design from the data, and predicts if and how to continue experimentation. These predictions matched several follow-up experimental datasets that were used for validation. We provide default pipelines, but the framework can be combined with other data analysis methods and can help assess their relative merits. Availability and implementation: casper package at www.bioconductor.org/packages/release/bioc/html/casper.html, Supplementary Manual by typing casperDesign() at the R prompt. Contact: rosselldavid@gmail.com Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26220961

  12. Alternatively spliced orcokinin isoforms and their functions in Tribolium castaneum.

    PubMed

    Jiang, Hongbo; Kim, Hong Geun; Park, Yoonseong

    2015-10-01

    Orcokinin and orcomyotropin were originally described as neuropeptides in crustaceans but have now been uncovered in many species of insects in which they are called orcokinin-A (OK-A) and orcokinin-B (OK-B), respectively. The two groups of mature peptides are products of alternatively spliced transcripts of the single copy gene orcokinin in insects. We investigated the expression patterns and the functions of OK-A and OK-B in the red flour beetle Tribolium castaneum. In situ hybridization and immunohistochemistry using isoform-specific probes and antibodies for each OK-A and OK-B suggests that both peptides are co-expressed in 5-7 pairs of brain cells and in the midgut enteroendocrine cells, which contrasts to expression patterns in other insects in which the two peptides are expressed in different cells. We developed a novel behavioral assay to assess the phenotypes of orcokinin RNA interference (RNAi) in T. castaneum. RNAi of ok-a and ok-b alone or in combination resulted in higher frequencies and longer durations of death feigning in response to mechanical stimulation in the adult assay. In the larval behavioral assays, we observed longer recovery times from knockout induced by water submergence in the insects treated with RNAi for ok-a and ok-b alone or in combination. We conclude that both OK-A and OK-B have "awakening" activities and are potentially involved in the control of circadian rhythms.

  13. nagnag: Identification and quantification of NAGNAG alternative splicing using RNA-Seq data.

    PubMed

    Yan, Xiaoyan; Sablok, Gaurav; Feng, Gang; Ma, Jiaxin; Zhao, Hongwei; Sun, Xiaoyong

    2015-07-08

    Regulation of proteome diversity by alternative splicing has been widely demonstrated in plants and animals. NAGNAG splicing, which was recently defined as a tissue specific event, results in the production of two distinct isoforms that are distinguished by three nucleotides (NAG) as a consequence of the intron proximal or distal to the splice site. Since the NAGNAG mechanism is not well characterized, tools for the identification and quantification of NAGNAG splicing events remain under-developed. Here we report nagnag, an R-based NAGNAG splicing detection tool, which accurately identifies and quantifies NAGNAG splicing events using RNA-Seq. Overall, nagnag produces user-friendly visualization reports and highlights differences between the DNA/RNA/protein across the identified isoforms of the reported gene. The package is available on https://sourceforge.net/projects/nagnag/files/; or http://genome.sdau.edu.cn/research/software/nagnag.html.

  14. A genome wide analysis of alternative splicing events during the osteogenic differentiation of human cartilage endplate-derived stem cells.

    PubMed

    Shang, Jin; Wang, Honggang; Fan, Xin; Shangguan, Lei; Liu, Huan

    2016-08-01

    Low back pain is a prevalent disease, which leads to suffering and disabilities in a vast number of individuals. Degenerative disc diseases are usually the underlying causes of low back pain. However, the pathogenesis of degenerative disc diseases is highly complex and difficult to determine. Current therapies for degenerative disc diseases are various. In particular, cell-based therapies have proven to be effective and promising. Our research group has previously isolated and identified the cartilage endplate‑derived stem cells. In addition, alternative splicing is a sophisticated regulatory mechanism, which greatly increases cellular complexity and phenotypic diversity of eukaryotic organisms. The present study continued to investigate alternative splicing events in osteogenic differentiation of cartilage endplate‑derived stem cells. An Affymetrix Human Transcriptome Array 2.0 was used to detect splicing changes between the control and differentiated samples. Additionally, molecular function and pathway analysis were also performed. Following rigorous bioinformatics analysis of the data, 3,802 alternatively spliced genes were identified, and 10 of these were selected for validation by reverse transcription‑polymerase chain reaction. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway analysis also revealed numerous enriched GO terms and signaling pathways. To the best of our knowledge, the present study is the first to investigate alternative splicing mechanisms in osteogenic differentiation of stem cells on a genome‑wide scale. The illumination of molecular mechanisms of stem cell osteogenic differentiation may assist the development novel bioengineered methods to treat degenerative disc diseases.

  15. Regulation of BCL-X splicing reveals a role for the polypyrimidine tract binding protein (PTBP1/hnRNP I) in alternative 5′ splice site selection

    PubMed Central

    Bielli, Pamela; Bordi, Matteo; Biasio, Valentina Di; Sette, Claudio

    2014-01-01

    Alternative splicing (AS) modulates many physiological and pathological processes. For instance, AS of the BCL-X gene balances cell survival and apoptosis in development and cancer. Herein, we identified the polypyrimidine tract binding protein (PTBP1) as a direct regulator of BCL-X AS. Overexpression of PTBP1 promotes selection of the distal 5′ splice site in BCL-X exon 2, generating the pro-apoptotic BCL-Xs splice variant. Conversely, depletion of PTBP1 enhanced splicing of the anti-apoptotic BCL-XL variant. In vivo cross-linking experiments and site-directed mutagenesis restricted the PTBP1 binding site to a polypyrimidine tract located between the two alternative 5′ splice sites. Binding of PTBP1 to this site was required for its effect on splicing. Notably, a similar function of PTBP1 in the selection of alternative 5′ splice sites was confirmed using the USP5 gene as additional model. Mechanistically, PTBP1 displaces SRSF1 binding from the proximal 5′ splice site, thus repressing its selection. Our study provides a novel mechanism of alternative 5′ splice site selection by PTBP1 and indicates that the presence of a PTBP1 binding site between two alternative 5′ splice sites promotes selection of the distal one, while repressing the proximal site by competing for binding of a positive regulator. PMID:25294838

  16. Polypyrimidine tract binding protein homologs from Arabidopsis are key regulators of alternative splicing with implications in fundamental developmental processes.

    PubMed

    Rühl, Christina; Stauffer, Eva; Kahles, André; Wagner, Gabriele; Drechsel, Gabriele; Rätsch, Gunnar; Wachter, Andreas

    2012-11-01

    Alternative splicing (AS) generates transcript variants by variable exon/intron definition and massively expands transcriptome diversity. Changes in AS patterns have been found to be linked to manifold biological processes, yet fundamental aspects, such as the regulation of AS and its functional implications, largely remain to be addressed. In this work, widespread AS regulation by Arabidopsis thaliana Polypyrimidine tract binding protein homologs (PTBs) was revealed. In total, 452 AS events derived from 307 distinct genes were found to be responsive to the levels of the splicing factors PTB1 and PTB2, which predominantly triggered splicing of regulated introns, inclusion of cassette exons, and usage of upstream 5' splice sites. By contrast, no major AS regulatory function of the distantly related PTB3 was found. Dependent on their position within the mRNA, PTB-regulated events can both modify the untranslated regions and give rise to alternative protein products. We find that PTB-mediated AS events are connected to diverse biological processes, and the functional implications of selected instances were further elucidated. Specifically, PTB misexpression changes AS of PHYTOCHROME INTERACTING FACTOR6, coinciding with altered rates of abscisic acid-dependent seed germination. Furthermore, AS patterns as well as the expression of key flowering regulators were massively changed in a PTB1/2 level-dependent manner.

  17. Epithelial splicing regulatory protein 1 and 2 paralogues correlate with splice signatures and favorable outcome in human colorectal cancer

    PubMed Central

    Deloria, Abigail J.; Höflmayer, Doris; Kienzl, Philip; Łopatecka, Justyna; Sampl, Sandra; Klimpfinger, Martin; Braunschmid, Tamara; Bastian, Fabienne; Lu, Lingeng; Marian, Brigitte; Stättner, Stefan; Holzmann, Klaus

    2016-01-01

    ESRPs are master splice regulators implicated in alternative mRNA splicing programs important for epithelial-mesenchymal transition (EMT) and tumor progression. ESRP1 was identified in some tumors as good or worse predictor of outcome, but in colorectal cancer (CRC) the prognostic value of ESRPs and relation with mesenchymal splice variants is not clear. Here, we studied 68 CRC cases, compared tissue expression of ESRPs with clinical data and with EMT gene splice patterns of conditional CRC cells with deficient ESRP1 expression. Around 72% of patients showed global decreased transcript expression of both ESRPs in tumor as compared to matched non-neoplastic colorectal epithelium. Reduction of ESRP1 in tumor cells was evaluated by immunohistochemistry, associated with microsatellite stability and switch to mesenchymal splice signatures of FGFRs, CD44, ENAH and CTNND1(p120-catenin). Expression of ESRPs was significantly associated with favorable overall survival (log-rank test, P=0.0186 and 0.0408), better than prognostic stratification by tumor staging; and for ESRP1 confirmed with second TCGA cohort (log-rank test, P=0.0435). Prognostic value is independent of the pathological stage and microsatellite instability (ESRP1: HR=0.36, 95%CI 0.15–0.91, P=0.032; ESRP2: HR=0.23, 95%CI 0.08–0.65, P=0.006). Our study supports the role of ESRP1 as tumor suppressor and strongly suggests that ESRPs are candidate markers for early detection, diagnosis, and prognosis of CRC. PMID:27650542

  18. Effects of alternative splicing on function of Bestrophin-1 calcium-activated chloride channels

    PubMed Central

    Kuo, Yu-Hung; Abdullaev, Iskandar F.; Hyzinski-García, María C.; Mongin, Alexander A.

    2014-01-01

    Synopsis The proposed Ca2+-activated Cl− channel protein Bestrophin 1 (Best1) is expressed and functionally important in the retina and in the brain. Human BEST1 has two known splice variants, Best1V1 and Best1V2, which arise from alternative splicing of two exons: exon 2 splicing results in a unique N-terminal domain, whereas alternative splicing of exon 11 produces two mutually exclusive C-termini. Prior studies were limited to Best1V1 and its clinically relevant mutations. In the present work, we cloned a novel splice variant of Best1V1 missing exon 2 (Best1V1Δex2) and differing from each of the two previously identified isoforms by one alternatively spliced domain. This finding allowed us to determine the role for alternative splicing of the Best1 N- and C-termini. We heteroexpressed Best1V1Δex2 in HEK293 cells, and compared its properties to Best1V1 and Best1V2. Western blot analysis confirmed protein expression from all three splice variants. Both Best1V1 and Best1V1Δex2 successfully formed Ca2+-activated Cl− channels, demonstrating that the N-terminus encoded by exon 2 is not essential for channel function. In contrast, Best1V2 expressing cells had no detectable Ca2+-activated Cl− currents, pointing to a critical role for splicing of the C-terminus. Surface protein biotinylation demonstrated that Best1V1 and Best1V1Δex2 are trafficked to the plasma membrane, whereas Best1V2 is not. These results define the impact of alternative splicing on Best1 function, and should be taken into consideration in future modeling of the Best1 protein structure. PMID:24341532

  19. Alternative splicing and genomic structure of the Wilms tumor gene WT1

    SciTech Connect

    Haber, D.A. Massachusetts General Hospital Cancer Center, Charlestown ); Sohn, R.L.; Buckler, A.J.; Pelletier, J.; Call, K.M.; Housman, D.E. )

    1991-11-01

    The chromosome 11p13 Wilms tumor susceptibility gene WT1 appears to play a crucial role in regulating the proliferation and differentiation of nephroblasts and gonadal tissue. The WT1 gene consists of 10 exons, encoding a complex pattern of mRNA species: four distinct transcripts are expressed, reflecting the presence or absence of two alternative splices. Splice I consists of a separate exon, encoding 17 amino acids, which is inserted between the proline-rich amino terminus and the zinc finger domains. Splice II arises from the use of an alternative 5{prime} splice junction and results in the insertion of 3 amino acids between zinc fingers 3 and 4. RNase protection analysis demonstrates that the most prevalent splice variant in both human and mouse is that which contains both alternative splices, whereas the least common is the transcript missing both splices. The relative distribution of splice variants is highly conserved between normal fetal kidney tissue and Wilms tumors that have intact WT1 transcripts. The ratio of these different WT1 mRNA species is also maintained as a function of development in the mouse kidney and in various mouse tissues expressing WT1. The conservation in structure and relative levels of each of the four WT1 mRNA species suggest that each encoded polypeptide makes a significant contribution to normal gene function. The control of cellular proliferation and differentiation exerted by the WT1 gene products may involve interactions between four polypeptides with distinct targets and functions.

  20. Cancer-Associated Perturbations in Alternative Pre-messenger RNA Splicing.

    PubMed

    Shkreta, Lulzim; Bell, Brendan; Revil, Timothée; Venables, Julian P; Prinos, Panagiotis; Elela, Sherif Abou; Chabot, Benoit

    2013-01-01

    For most of our 25,000 genes, the removal of introns by pre-messenger RNA (pre-mRNA) splicing represents an essential step toward the production of functional messenger RNAs (mRNAs). Alternative splicing of a single pre-mRNA results in the production of different mRNAs. Although complex organisms use alternative splicing to expand protein function and phenotypic diversity, patterns of alternative splicing are often altered in cancer cells. Alternative splicing contributes to tumorigenesis by producing splice isoforms that can stimulate cell proliferation and cell migration or induce resistance to apoptosis and anticancer agents. Cancer-specific changes in splicing profiles can occur through mutations that are affecting splice sites and splicing control elements, and also by alterations in the expression of proteins that control splicing decisions. Recent progress in global approaches that interrogate splicing diversity should help to obtain specific splicing signatures for cancer types. The development of innovative approaches for annotating and reprogramming splicing events will more fully establish the essential contribution of alternative splicing to the biology of cancer and will hopefully provide novel targets and anticancer strategies. Metazoan genes are usually made up of several exons interrupted by introns. The introns are removed from the pre-mRNA by RNA splicing. In conjunction with other maturation steps, such as capping and polyadenylation, the spliced mRNA is then transported to the cytoplasm to be translated into a functional protein. The basic mechanism of splicing requires accurate recognition of each extremity of each intron by the spliceosome. Introns are identified by the binding of U1 snRNP to the 5' splice site and the U2AF65/U2AF35 complex to the 3' splice site. Following these interactions, other proteins and snRNPs are recruited to generate the complete spliceosomal complex needed to excise the intron. While many introns are constitutively

  1. The Role of Alternative Splicing in the Control of Immune Homeostasis and Cellular Differentiation.

    PubMed

    Yabas, Mehmet; Elliott, Hannah; Hoyne, Gerard F

    2015-12-22

    Alternative splicing of pre-mRNA helps to enhance the genetic diversity within mammalian cells by increasing the number of protein isoforms that can be generated from one gene product. This provides a great deal of flexibility to the host cell to alter protein function, but when dysregulation in splicing occurs this can have important impact on health and disease. Alternative splicing is widely used in the mammalian immune system to control the development and function of antigen specific lymphocytes. In this review we will examine the splicing of pre-mRNAs yielding key proteins in the immune system that regulate apoptosis, lymphocyte differentiation, activation and homeostasis, and discuss how defects in splicing can contribute to diseases. We will describe how disruption to trans-acting factors, such as heterogeneous nuclear ribonucleoproteins (hnRNPs), can impact on cell survival and differentiation in the immune system.

  2. Regulation of the beta-hydroxyacyl ACP dehydratase gene of Picea mariana by alternative splicing.

    PubMed

    Tai, Helen H; Williams, Martin; Iyengar, Abhinav; Yeates, Jessica; Beardmore, Tannis

    2007-01-01

    The gene for beta-hydroxyacyl ACP dehydratase, a de novo fatty acid biosynthetic enzyme, was cloned from Picea mariana (black spruce) and consists of five exons and four introns. The first intron of the beta-hydroxyacyl ACP dehydratase mRNA is alternatively spliced. Retention of intron 1 in splice variants results in truncation of the beta-hydroxyacyl ACP dehydratase ORF at a premature termination codon. In addition, splicing of intron 1 was found to be associated with cold temperature. mRNAs retaining intron 1 increase with seed imbibition at 22 degrees C but not 4 degrees C, whereas, splicing of intron 1 increases in winter weeks with temperatures below freezing. These results provide evidence that alternative splicing may also contribute to regulation of lipid biosynthesis in Picea mariana.

  3. The Role of Alternative Splicing in the Control of Immune Homeostasis and Cellular Differentiation

    PubMed Central

    Yabas, Mehmet; Elliott, Hannah; Hoyne, Gerard F.

    2015-01-01

    Alternative splicing of pre-mRNA helps to enhance the genetic diversity within mammalian cells by increasing the number of protein isoforms that can be generated from one gene product. This provides a great deal of flexibility to the host cell to alter protein function, but when dysregulation in splicing occurs this can have important impact on health and disease. Alternative splicing is widely used in the mammalian immune system to control the development and function of antigen specific lymphocytes. In this review we will examine the splicing of pre-mRNAs yielding key proteins in the immune system that regulate apoptosis, lymphocyte differentiation, activation and homeostasis, and discuss how defects in splicing can contribute to diseases. We will describe how disruption to trans-acting factors, such as heterogeneous nuclear ribonucleoproteins (hnRNPs), can impact on cell survival and differentiation in the immune system. PMID:26703587

  4. Bovine Herpesvirus 4 Modulates Its β-1,6-N-Acetylglucosaminyltransferase Activity through Alternative Splicing

    PubMed Central

    Lété, Céline; Markine-Goriaynoff, Nicolas; Machiels, Bénédicte; Pang, Poh-Choo; Xiao, Xue; Canis, Kevin; Suzuki, Masami; Fukuda, Minoru; Dell, Anne; Haslam, Stuart M.; Vanderplasschen, Alain

    2015-01-01

    enzyme which is a key enzyme for the synthesis of complex O-glycans. In this study, we show that, in contrast to cellular homologues, this virus has evolved to alternatively express two proteins from this gene. While the first one is enzymatically active, the second results from the alternative splicing of the region encoding the catalytic site of the enzyme. We postulate that this regulatory mechanism could allow the virus to modulate the synthesis of some particular glycans for function at the location and/or the moment of infection. PMID:26656682

  5. Regulation of Translation Factor EEF1D Gene Function by Alternative Splicing

    PubMed Central

    Kaitsuka, Taku; Matsushita, Masayuki

    2015-01-01

    Alternative splicing is an exquisite mechanism that allows one coding gene to have multiple functions. The alternative splicing machinery is necessary for proper development, differentiation and stress responses in a variety of organisms, and disruption of this machinery is often implicated in human diseases. Previously, we discovered a long form of eukaryotic elongation factor 1Bδ (eEF1Bδ; this long-form eEF1Bδ results from alternative splicing of EEF1D transcripts and regulates the cellular stress response by transcriptional activation, not translational enhancement, of heat-shock responsive genes. In this review, we discuss the molecular function of EEF1D alternative splicing products and the estimated implication of human diseases. PMID:25686034

  6. Corticotropin (ACTH) regulates alternative RNA splicing in Y1 mouse adrenocortical tumor cells.

    PubMed

    Schimmer, Bernard P; Cordova, Martha

    2015-06-15

    The stimulatory effect of ACTH on gene expression is well documented and is thought to be a major mechanism by which ACTH maintains the functional and structural integrity of the gland. Previously, we showed that ACTH regulates the accumulation of over 1200 transcripts in Y1 adrenal cells, including a cluster with functions in alternative splicing of RNA. On this basis, we postulated that some of the effects of ACTH on the transcription landscape of Y1 cells are mediated by alternative splicing. In this study, we demonstrate that ACTH regulates the alternative splicing of four transcripts - Gnas, Cd151, Dab2 and Tia1. Inasmuch as alternative splicing potentially affects transcripts from more than two-thirds of the mouse genome, we suggest that these findings are representative of a genome-wide effect of ACTH that impacts on the mRNA and protein composition of the adrenal cortex.

  7. Differential gene expression and alternative splicing between diploid and tetraploid watermelon lines

    USDA-ARS?s Scientific Manuscript database

    Synthetic tetraploid plants have been used for production of seedless triploid watermelon lines being pollinated with diploid plants. When compared to their diploid or triploid counterparts, the tetraploid exhibit wide phenotypic differences. Though many factors, including alternative splicing (AS),...

  8. Identification by high-throughput imaging of the histone methyltransferase EHMT2 as an epigenetic regulator of VEGFA alternative splicing

    PubMed Central

    Salton, Maayan; Voss, Ty C.; Misteli, Tom

    2014-01-01

    Recent evidence points to a role of chromatin in regulation of alternative pre-mRNA splicing (AS). In order to identify novel chromatin regulators of AS, we screened an RNAi library of chromatin proteins using a cell-based high-throughput in vivo assay. We identified a set of chromatin proteins that regulate AS. Using simultaneous genome-wide expression and AS analysis, we demonstrate distinct and non-overlapping functions of these chromatin modifiers on transcription and AS. Detailed mechanistic characterization of one dual function chromatin modifier, the H3K9 methyltransferase EHMT2 (G9a), identified VEGFA as a major chromatin-mediated AS target. Silencing of EHMT2, or its heterodimer partner EHMT1, affects AS by promoting exclusion of VEGFA exon 6a, but does not alter total VEGFA mRNA levels. The epigenetic regulatory mechanism of AS by EHMT2 involves an adaptor system consisting of the chromatin modulator HP1γ, which binds methylated H3K9 and recruits splicing regulator SRSF1. The epigenetic regulation of VEGFA is physiologically relevant since EHMT2 is transcriptionally induced in response to hypoxia and triggers concomitant changes in AS of VEGFA. These results characterize a novel epigenetic regulatory mechanism of AS and they demonstrate separate roles of epigenetic modifiers in transcription and alternative splicing. PMID:25414343

  9. Alternative splicing and nonsense-mediated decay of circadian clock genes under environmental stress conditions in Arabidopsis.

    PubMed

    Kwon, Young-Ju; Park, Mi-Jeong; Kim, Sang-Gyu; Baldwin, Ian T; Park, Chung-Mo

    2014-05-19

    The circadian clock enables living organisms to anticipate recurring daily and seasonal fluctuations in their growth habitats and synchronize their biology to the environmental cycle. The plant circadian clock consists of multiple transcription-translation feedback loops that are entrained by environmental signals, such as light and temperature. In recent years, alternative splicing emerges as an important molecular mechanism that modulates the clock function in plants. Several clock genes are known to undergo alternative splicing in response to changes in environmental conditions, suggesting that the clock function is intimately associated with environmental responses via the alternative splicing of the clock genes. However, the alternative splicing events of the clock genes have not been studied at the molecular level. We systematically examined whether major clock genes undergo alternative splicing under various environmental conditions in Arabidopsis. We also investigated the fates of the RNA splice variants of the clock genes. It was found that the clock genes, including EARLY FLOWERING 3 (ELF3) and ZEITLUPE (ZTL) that have not been studied in terms of alternative splicing, undergo extensive alternative splicing through diverse modes of splicing events, such as intron retention, exon skipping, and selection of alternative 5' splice site. Their alternative splicing patterns were differentially influenced by changes in photoperiod, temperature extremes, and salt stress. Notably, the RNA splice variants of TIMING OF CAB EXPRESSION 1 (TOC1) and ELF3 were degraded through the nonsense-mediated decay (NMD) pathway, whereas those of other clock genes were insensitive to NMD. Taken together, our observations demonstrate that the major clock genes examined undergo extensive alternative splicing under various environmental conditions, suggesting that alternative splicing is a molecular scheme that underlies the linkage between the clock and environmental stress

  10. Alternative splicing and nonsense-mediated decay of circadian clock genes under environmental stress conditions in Arabidopsis

    PubMed Central

    2014-01-01

    Background The circadian clock enables living organisms to anticipate recurring daily and seasonal fluctuations in their growth habitats and synchronize their biology to the environmental cycle. The plant circadian clock consists of multiple transcription-translation feedback loops that are entrained by environmental signals, such as light and temperature. In recent years, alternative splicing emerges as an important molecular mechanism that modulates the clock function in plants. Several clock genes are known to undergo alternative splicing in response to changes in environmental conditions, suggesting that the clock function is intimately associated with environmental responses via the alternative splicing of the clock genes. However, the alternative splicing events of the clock genes have not been studied at the molecular level. Results We systematically examined whether major clock genes undergo alternative splicing under various environmental conditions in Arabidopsis. We also investigated the fates of the RNA splice variants of the clock genes. It was found that the clock genes, including EARLY FLOWERING 3 (ELF3) and ZEITLUPE (ZTL) that have not been studied in terms of alternative splicing, undergo extensive alternative splicing through diverse modes of splicing events, such as intron retention, exon skipping, and selection of alternative 5′ splice site. Their alternative splicing patterns were differentially influenced by changes in photoperiod, temperature extremes, and salt stress. Notably, the RNA splice variants of TIMING OF CAB EXPRESSION 1 (TOC1) and ELF3 were degraded through the nonsense-mediated decay (NMD) pathway, whereas those of other clock genes were insensitive to NMD. Conclusion Taken together, our observations demonstrate that the major clock genes examined undergo extensive alternative splicing under various environmental conditions, suggesting that alternative splicing is a molecular scheme that underlies the linkage between the clock

  11. Physiological state co-regulates thousands of mammalian mRNA splicing events at tandem splice sites and alternative exons.

    PubMed

    Szafranski, Karol; Fritsch, Claudia; Schumann, Frank; Siebel, Lisa; Sinha, Rileen; Hampe, Jochen; Hiller, Michael; Englert, Christoph; Huse, Klaus; Platzer, Matthias

    2014-08-01

    Thousands of tandem alternative splice sites (TASS) give rise to mRNA insertion/deletion variants with small size differences. Recent work has concentrated on the question of biological relevance in general, and the physiological regulation of TASS in particular. We have quantitatively studied 11 representative TASS cases in comparison to one mutually exclusive exon case and two cassette exons (CEs) using a panel of human and mouse tissues, as well as cultured cell lines. Tissues show small but significant differences in TASS isoform ratios, with a variance 4- to 20-fold lower than seen for CEs. Remarkably, in cultured cells, all studied alternative splicing (AS) cases showed a cell-density-dependent shift of isoform ratios with similar time series profiles. A respective genome-wide co-regulation of TASS splicing was shown by next-generation mRNA sequencing data. Moreover, data from human and mouse organs indicate that this co-regulation of TASS occurs in vivo, with brain showing the strongest difference to other organs. Together, the results indicate a physiological AS regulation mechanism that functions almost independently from the splice site context and sequence. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. HITS-CLIP and integrative modeling define the Rbfox splicing-regulatory network linked to brain development and autism.

    PubMed

    Weyn-Vanhentenryck, Sebastien M; Mele, Aldo; Yan, Qinghong; Sun, Shuying; Farny, Natalie; Zhang, Zuo; Xue, Chenghai; Herre, Margaret; Silver, Pamela A; Zhang, Michael Q; Krainer, Adrian R; Darnell, Robert B; Zhang, Chaolin

    2014-03-27

    The RNA binding proteins Rbfox1/2/3 regulate alternative splicing in the nervous system, and disruption of Rbfox1 has been implicated in autism. However, comprehensive identification of functional Rbfox targets has been challenging. Here, we perform HITS-CLIP for all three Rbfox family members in order to globally map, at a single-nucleotide resolution, their in vivo RNA interaction sites in the mouse brain. We find that the two guanines in the Rbfox binding motif UGCAUG are critical for protein-RNA interactions and crosslinking. Using integrative modeling, these interaction sites, combined with additional datasets, define 1,059 direct Rbfox target alternative splicing events. Over half of the quantifiable targets show dynamic changes during brain development. Of particular interest are 111 events from 48 candidate autism-susceptibility genes, including syndromic autism genes Shank3, Cacna1c, and Tsc2. Alteration of Rbfox targets in some autistic brains is correlated with downregulation of all three Rbfox proteins, supporting the potential clinical relevance of the splicing-regulatory network.

  13. Widespread JNK-dependent alternative splicing induces a positive feedback loop through CELF2-mediated regulation of MKK7 during T-cell activation.

    PubMed

    Martinez, Nicole M; Agosto, Laura; Qiu, Jinsong; Mallory, Michael J; Gazzara, Matthew R; Barash, Yoseph; Fu, Xiang-Dong; Lynch, Kristen W

    2015-10-01

    Alternative splicing is prevalent among genes encoding signaling molecules; however, the functional consequence of differential isoform expression remains largely unknown. Here we demonstrate that, in response to T-cell activation, the Jun kinase (JNK) kinase MAP kinase kinase 7 (MKK7) is alternatively spliced to favor an isoform that lacks exon 2. This isoform restores a JNK-docking site within MKK7 that is disrupted in the larger isoform. Consistently, we show that skipping of MKK7 exon 2 enhances JNK pathway activity, as indicated by c-Jun phosphorylation and up-regulation of TNF-α. Moreover, this splicing event is itself dependent on JNK signaling. Thus, MKK7 alternative splicing represents a positive feedback loop through which JNK promotes its own signaling. We further show that repression of MKK7 exon 2 is dependent on the presence of flanking sequences and the JNK-induced expression of the RNA-binding protein CELF2, which binds to these regulatory elements. Finally, we found that ∼25% of T-cell receptor-mediated alternative splicing events are dependent on JNK signaling. Strikingly, these JNK-dependent events are also significantly enriched for responsiveness to CELF2. Together, our data demonstrate a widespread role for the JNK-CELF2 axis in controlling splicing during T-cell activation, including a specific role in propagating JNK signaling. © 2015 Martinez et al.; Published by Cold Spring Harbor Laboratory Press.

  14. Changes in the expression of splicing factor transcripts and variations in alternative splicing are associated with lifespan in mice and humans.

    PubMed

    Lee, Benjamin P; Pilling, Luke C; Emond, Florence; Flurkey, Kevin; Harrison, David E; Yuan, Rong; Peters, Luanne L; Kuchel, George A; Ferrucci, Luigi; Melzer, David; Harries, Lorna W

    2016-10-01

    Dysregulation of splicing factor expression and altered alternative splicing are associated with aging in humans and other species, and also with replicative senescence in cultured cells. Here, we assess whether expression changes of key splicing regulator genes and consequent effects on alternative splicing are also associated with strain longevity in old and young mice, across 6 different mouse strains with varying lifespan (A/J, NOD.B10Sn-H2(b) /J, PWD.Phj, 129S1/SvlmJ, C57BL/6J and WSB/EiJ). Splicing factor expression and changes to alternative splicing were associated with strain lifespan in spleen and to a lesser extent in muscle. These changes mainly involved hnRNP splicing inhibitor transcripts with most changes more marked in spleens of young animals from long-lived strains. Changes in spleen isoform expression were suggestive of reduced cellular senescence and retained cellular proliferative capacity in long-lived strains. Changes in muscle isoform expression were consistent with reduced pro-inflammatory signalling in longer-lived strains. Two splicing regulators, HNRNPA1 and HNRNPA2B1, were also associated with parental longevity in humans, in the InCHIANTI aging study. Splicing factors may represent a driver, mediator or early marker of lifespan in mouse, as expression differences were present in the young animals of long-lived strains. Changes to alternative splicing patterns of key senescence genes in spleen and key remodelling genes in muscle suggest that correct regulation of alternative splicing may enhance lifespan in mice. Expression of some splicing factors in humans was also associated with parental longevity, suggesting that splicing regulation may also influence lifespan in humans. © 2016 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  15. Integrating Omics and Alternative Splicing Reveals Insights into Grape Response to High Temperature1[OPEN

    PubMed Central

    Jiang, Jianfu; Liu, Xinna; Liu, Guotian; Li, Shaohua

    2017-01-01

    Heat stress is one of the primary abiotic stresses that limit crop production. Grape (Vitis vinifera) is a cultivated fruit with high economic value throughout the world, with its growth and development often influenced by high temperature. Alternative splicing (AS) is a widespread phenomenon increasing transcriptome and proteome diversity. We conducted high-temperature treatments (35°C, 40°C, and 45°C) on grapevines and assessed transcriptomic (especially AS) and proteomic changes in leaves. We found that nearly 70% of the genes were alternatively spliced under high temperature. Intron retention (IR), exon skipping, and alternative donor/acceptor sites were markedly induced under different high temperatures. Among all differential AS events, IR was the most abundant up- and down-regulated event. Moreover, the occurrence frequency of IR events at 40°C and 45°C was far higher than at 35°C. These results indicated that AS, especially IR, is an important posttranscriptional regulatory event during grape leaf responses to high temperature. Proteomic analysis showed that protein levels of the RNA-binding proteins SR45, SR30, and SR34 and the nuclear ribonucleic protein U1A gradually rose as ambient temperature increased, which revealed a reason why AS events occurred more frequently under high temperature. After integrating transcriptomic and proteomic data, we found that heat shock proteins and some important transcription factors such as MULTIPROTEIN BRIDGING FACTOR1c and HEAT SHOCK TRANSCRIPTION FACTOR A2 were involved mainly in heat tolerance in grape through up-regulating transcriptional (especially modulated by AS) and translational levels. To our knowledge, these results provide the first evidence for grape leaf responses to high temperature at simultaneous transcriptional, posttranscriptional, and translational levels. PMID:28049741

  16. Integrating Omics and Alternative Splicing Reveals Insights into Grape Response to High Temperature.

    PubMed

    Jiang, Jianfu; Liu, Xinna; Liu, Chonghuai; Liu, Guotian; Li, Shaohua; Wang, Lijun

    2017-02-01

    Heat stress is one of the primary abiotic stresses that limit crop production. Grape (Vitis vinifera) is a cultivated fruit with high economic value throughout the world, with its growth and development often influenced by high temperature. Alternative splicing (AS) is a widespread phenomenon increasing transcriptome and proteome diversity. We conducted high-temperature treatments (35°C, 40°C, and 45°C) on grapevines and assessed transcriptomic (especially AS) and proteomic changes in leaves. We found that nearly 70% of the genes were alternatively spliced under high temperature. Intron retention (IR), exon skipping, and alternative donor/acceptor sites were markedly induced under different high temperatures. Among all differential AS events, IR was the most abundant up- and down-regulated event. Moreover, the occurrence frequency of IR events at 40°C and 45°C was far higher than at 35°C. These results indicated that AS, especially IR, is an important posttranscriptional regulatory event during grape leaf responses to high temperature. Proteomic analysis showed that protein levels of the RNA-binding proteins SR45, SR30, and SR34 and the nuclear ribonucleic protein U1A gradually rose as ambient temperature increased, which revealed a reason why AS events occurred more frequently under high temperature. After integrating transcriptomic and proteomic data, we found that heat shock proteins and some important transcription factors such as MULTIPROTEIN BRIDGING FACTOR1c and HEAT SHOCK TRANSCRIPTION FACTOR A2 were involved mainly in heat tolerance in grape through up-regulating transcriptional (especially modulated by AS) and translational levels. To our knowledge, these results provide the first evidence for grape leaf responses to high temperature at simultaneous transcriptional, posttranscriptional, and translational levels.

  17. An alternative splicing switch shapes neurexin repertoires in principal neurons versus interneurons in the mouse hippocampus

    PubMed Central

    Nguyen, Thi-Minh; Schreiner, Dietmar; Xiao, Le; Traunmüller, Lisa; Bornmann, Caroline; Scheiffele, Peter

    2016-01-01

    The unique anatomical and functional features of principal and interneuron populations are critical for the appropriate function of neuronal circuits. Cell type-specific properties are encoded by selective gene expression programs that shape molecular repertoires and synaptic protein complexes. However, the nature of such programs, particularly for post-transcriptional regulation at the level of alternative splicing is only beginning to emerge. We here demonstrate that transcripts encoding the synaptic adhesion molecules neurexin-1,2,3 are commonly expressed in principal cells and interneurons of the mouse hippocampus but undergo highly differential, cell type-specific alternative splicing. Principal cell-specific neurexin splice isoforms depend on the RNA-binding protein Slm2. By contrast, most parvalbumin-positive (PV+) interneurons lack Slm2, express a different neurexin splice isoform and co-express the corresponding splice isoform-specific neurexin ligand Cbln4. Conditional ablation of Nrxn alternative splice insertions selectively in PV+ cells results in elevated hippocampal network activity and impairment in a learning task. Thus, PV-cell-specific alternative splicing of neurexins is critical for neuronal circuit function DOI: http://dx.doi.org/10.7554/eLife.22757.001 PMID:27960072

  18. Alternative Splicing of G-protein Coupled Receptors: Relevance to Pain Management

    PubMed Central

    Oladosu, Folabomi A.; Maixner, William; Nackley, Andrea G.

    2015-01-01

    Drugs that target G-protein coupled receptors (GPCRs) represent the primary treatment strategy for patients with acute and chronic pain; however, there is substantial individual variability in both the efficacy and adverse side effects associated with these drugs. Variability in drug responses is, in part, due to individuals’ diversity in alternative splicing of pain-relevant GPCRs. GPCR alternative splice variants often exhibit distinct tissue distribution patterns, drug binding properties, and signaling characteristics that may impact disease pathology as well as the size and direction of analgesic effects. Here, we review the importance of GPCRs and their known splice variants to the management of pain. PMID:26250730

  19. New way of regulating alternative splicing in retroviruses: the promoter makes a difference.

    PubMed

    Bohne, Jens; Schambach, Axel; Zychlinski, Daniela

    2007-04-01

    Alternative splicing has been recognized as a major mechanism for creating proteomic diversity from a limited number of genes. However, not all determinants regulating this process have been characterized. Using subviral human immunodeficiency virus (HIV) env constructs we observed an enhanced splicing of the RNA when expression was under control of the cytomegalovirus (CMV) promoter instead of the HIV long terminal repeat (LTR). We extended these observations to LTR- or CMV-driven murine leukemia proviruses, suggesting that retroviral LTRs are adapted to inefficient alternative splicing at most sites in order to maintain balanced gene expression.

  20. Alternative Splicing of G Protein-Coupled Receptors: Relevance to Pain Management.

    PubMed

    Oladosu, Folabomi A; Maixner, William; Nackley, Andrea G

    2015-08-01

    Drugs that target G protein-coupled receptors (GPCRs) represent the primary treatment strategy for patients with acute and chronic pain; however, there is substantial individual variability in both the efficacy and adverse effects associated with these drugs. Variability in drug responses is due, in part, to individuals' diversity in alternative splicing of pain-relevant GPCRs. G protein-coupled receptor alternative splice variants often exhibit distinct tissue distribution patterns, drug-binding properties, and signaling characteristics that may impact disease pathology as well as the extent and direction of analgesic effects. We review the importance of GPCRs and their known splice variants to the management of pain.

  1. Detecting tissue-specific alternative splicing and disease-associated aberrant splicing of the PTCH gene with exon junction microarrays.

    PubMed

    Nagao, Kazuaki; Togawa, Naoyuki; Fujii, Katsunori; Uchikawa, Hideki; Kohno, Yoichi; Yamada, Masao; Miyashita, Toshiyuki

    2005-11-15

    Mutations in the human ortholog of Drosophila patched (PTCH) have been identified in patients with autosomal dominant nevoid basal cell carcinoma syndrome (NBCCS), characterized by minor developmental anomalies and an increased incidence of cancers such as medulloblastoma and basal cell carcinoma. We identified many isoforms of PTCH mRNA involving exons 1-5, exon 10 and a novel exon, 12b, generated by alternative splicing (AS), most of which have not been deposited in GenBank nor discussed earlier. To monitor splicing events of the PTCH gene, we designed oligonucleotide arrays on which exon probes and exon-exon junction probes as well as a couple of intron probes for the PTCH gene were placed in duplicate. Probe intensities were normalized on the basis of the total expression of PTCH and probe sensitivity. Tissue-specific regulation of AS identified with the microarrays closely correlated with the results obtained by RT-PCR. Of note, the novel exon, exon 12b, was specifically expressed in the brain and heart, especially in the cerebellum. Additionally, using these microarrays, we were able to detect disease-associated aberrant splicings of the PTCH gene in two patients with NBCCS. In both cases, cryptic splice donor sites located either in an exon or in an intron were activated because of the partial disruption of the consensus sequence for the authentic splice donor sites due to point mutations. Taken together, oligonucleotide microarrays containing exon junction probes are demonstrated to be a powerful tool to investigate tissue-specific regulation of AS and aberrant splicing taking place in genetic disorders.

  2. Small Molecule Amiloride Modulates Oncogenic RNA Alternative Splicing to Devitalize Human Cancer Cells

    PubMed Central

    Chang, Jan-Gowth; Chang, Wen-Hsin; Chow, Lu-Ping; Chan, Wen-Ling; Lin, Hui-Hua; Huang, Hsien-Da; Chang, Ya-Sian; Hung, Cheng-Hao; Yang, Wen-Kuang

    2011-01-01

    Alternative splicing involves differential exon selection of a gene transcript to generate mRNA and protein isoforms with structural and functional diversity. Abnormal alternative splicing has been shown to be associated with malignant phenotypes of cancer cells, such as chemo-resistance and invasive activity. Screening small molecules and drugs for modulating RNA splicing in human hepatocellular carcinoma cell line Huh-7, we discovered that amiloride, distinct from four pH-affecting amiloride analogues, could “normalize” the splicing of BCL-X, HIPK3 and RON/MISTR1 transcripts. Our proteomic analyses of amiloride-treated cells detected hypo-phosphorylation of splicing factor SF2/ASF, and decreased levels of SRp20 and two un-identified SR proteins. We further observed decreased phosphorylation of AKT, ERK1/2 and PP1, and increased phosphorylation of p38 and JNK, suggesting that amiloride treatment down-regulates kinases and up-regulates phosphatases in the signal pathways known to affect splicing factor protein phosphorylation. These amiloride effects of “normalized” oncogenic RNA splicing and splicing factor hypo-phosphorylation were both abrogated by pre-treatment with a PP1 inhibitor. Global exon array of amiloride-treated Huh-7 cells detected splicing pattern changes involving 584 exons in 551 gene transcripts, many of which encode proteins playing key roles in ion transport, cellular matrix formation, cytoskeleton remodeling, and genome maintenance. Cellular functional analyses revealed subsequent invasion and migration defects, cell cycle disruption, cytokinesis impairment, and lethal DNA degradation in amiloride-treated Huh-7 cells. Other human solid tumor and leukemic cells, but not a few normal cells, showed similar amiloride-altered RNA splicing with devitalized consequence. This study thus provides mechanistic underpinnings for exploiting small molecule modulation of RNA splicing for cancer therapeutics. PMID:21694768

  3. Alternative Splicing in Breast Cancer and the Potential Development of Therapeutic Tools.

    PubMed

    Martínez-Montiel, Nancy; Anaya-Ruiz, Maricruz; Pérez-Santos, Martín; Martínez-Contreras, Rebeca D

    2017-10-05

    Alternative splicing is a key molecular mechanism now considered as a hallmark of cancer that has been associated with the expression of distinct isoforms during the onset and progression of the disease. The leading cause of cancer-related deaths in women worldwide is breast cancer, and even when the role of alternative splicing in this type of cancer has been established, the function of this mechanism in breast cancer biology is not completely decoded. In order to gain a comprehensive view of the role of alternative splicing in breast cancer biology and development, we summarize here recent findings regarding alternative splicing events that have been well documented for breast cancer evolution, considering its prognostic and therapeutic value. Moreover, we analyze how the response to endocrine and chemical therapies could be affected due to alternative splicing and differential expression of variant isoforms. With all this knowledge, it becomes clear that targeting alternative splicing represents an innovative approach for breast cancer therapeutics and the information derived from current studies could guide clinical decisions with a direct impact in the clinical advances for breast cancer patients nowadays.

  4. SRSF10 regulates alternative splicing and is required for adipocyte differentiation.

    PubMed

    Li, Huang; Cheng, Yuanming; Wu, Wenwu; Liu, Yuguo; Wei, Ning; Feng, Xiaoyan; Xie, Zhiqin; Feng, Ying

    2014-06-01

    During adipocyte differentiation, significant alternative splicing changes occur in association with the adipogenic process. However, little is known about roles played by splicing factors in this process. We observed that mice deficient for the splicing factor SRSF10 exhibit severely impaired development of subcutaneous white adipose tissue (WAT) as a result of defects in adipogenic differentiation. To identify splicing events responsible for this, transcriptome sequencing (RNA-seq) analysis was performed using embryonic fibroblast cells. Several SRSF10-affected splicing events that are implicated in adipogenesis have been identified. Notably, lipin1, known as an important regulator during adipogenesis, was further investigated. While lipin1β is mainly involved in lipogenesis, its alternatively spliced isoform lipin1α, generated through the skipping of exon 7, is primarily required for initial adipocyte differentiation. Skipping of exon 7 is controlled by an SRSF10-regulated cis element located in the constitutive exon 8. The activity of this element depends on the binding of SRSF10 and correlates with the relative abundance of lipin1α mRNA. A series of experiments demonstrated that SRSF10 controls the production of lipin1α and thus promotes adipocyte differentiation. Indeed, lipin1α expression could rescue SRSF10-mediated adipogenic defects. Taken together, our results identify SRSF10 as an essential regulator for adipocyte differentiation and also provide new insights into splicing control by SRSF10 in lipin1 pre-mRNA splicing. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  5. Alternative splicing: functional diversity among voltage-gated calcium channels and behavioral consequences.

    PubMed

    Lipscombe, Diane; Andrade, Arturo; Allen, Summer E

    2013-07-01

    Neuronal voltage-gated calcium channels generate rapid, transient intracellular calcium signals in response to membrane depolarization. Neuronal Ca(V) channels regulate a range of cellular functions and are implicated in a variety of neurological and psychiatric diseases including epilepsy, Parkinson's disease, chronic pain, schizophrenia, and bipolar disorder. Each mammalian Cacna1 gene has the potential to generate tens to thousands of Ca(V) channels by alternative pre-mRNA splicing, a process that adds fine granulation to the pool of Ca(V) channel structures and functions. The precise composition of Ca(V) channel splice isoform mRNAs expressed in each cell are controlled by cell-specific splicing factors. The activity of splicing factors are in turn regulated by molecules that encode various cellular features, including cell-type, activity, metabolic states, developmental state, and other factors. The cellular and behavioral consequences of individual sites of Ca(V) splice isoforms are being elucidated, as are the cell-specific splicing factors that control splice isoform selection. Altered patterns of alternative splicing of Ca(V) pre-mRNAs can alter behavior in subtle but measurable ways, with the potential to influence drug efficacy and disease severity. This article is part of a Special Issue entitled: Calcium channels.

  6. Alternative splicing, a new target to block cellular gene expression by poliovirus 2A protease

    SciTech Connect

    Alvarez, Enrique; Castello, Alfredo; Carrasco, Luis; Izquierdo, Jose M.

    2011-10-14

    Highlights: {yields} Novel role for poliovirus 2A protease as splicing modulator. {yields} Poliovirus 2A protease inhibits the alternative splicing of pre-mRNAs. {yields} Poliovirus 2A protease blocks the second catalytic step of splicing. -- Abstract: Viruses have developed multiple strategies to interfere with the gene expression of host cells at different stages to ensure their own survival. Here we report a new role for poliovirus 2A{sup pro} modulating the alternative splicing of pre-mRNAs. Expression of 2A{sup pro} potently inhibits splicing of reporter genes in HeLa cells. Low amounts of 2A{sup pro} abrogate Fas exon 6 skipping, whereas higher levels of protease fully abolish Fas and FGFR2 splicing. In vitro splicing of MINX mRNA using nuclear extracts is also strongly inhibited by 2A{sup pro}, leading to accumulation of the first exon and the lariat product containing the unspliced second exon. These findings reveal that the mechanism of action of 2A{sup pro} on splicing is to selectively block the second catalytic step.

  7. Context-dependent regulatory mechanism of the splicing factor hnRNP L

    PubMed Central

    Motta-Mena, Laura B.; Heyd, Florian; Lynch, Kristen W.

    2010-01-01

    Splicing regulatory proteins often have distinct activities when bound to exons versus introns. However, less clear is whether variables besides location can influence activity. HnRNP L binds to a motif present in both CD45 variable exons 4 and 5 to affect their coordinate repression. Here we show that, in contrast to its direct repression of exon 4, hnRNP L represses exon 5 by countering the activity of a neighboring splicing enhancer. In the absence of the enhancer hnRNP L unexpectedly activates exon inclusion. As the splice sites flanking exon 4 and 5 are distinct, we directly examined the effect of varying splice site strength on the mechanism of hnRNP L function. Remarkably, binding of hnRNP L to an exon represses strong splice sites but enhances weak splice sites. A model in which hnRNP L stabilizes snRNP binding can explain both effects in a manner determined by the inherent snRNP-substrate affinity. PMID:20122404

  8. Mutational bias is the driving force for shaping the synonymous codon usage pattern of alternatively spliced genes in rice (Oryza sativa L.).

    PubMed

    Liu, Qingpo; Hu, Haichao; Wang, Hong

    2015-04-01

    Alternative splicing plays important roles in diverse aspects of plant development, metabolism, and stress responses. However, the regulatory mechanisms of alternative splicing of genes still remain incompletely elucidated, especially in plants. In this study, the synonymous codon usage pattern of alternatively spliced (AS) genes in rice was firstly explored using the combination of correspondence analysis (CA), internal CA, correlation and ANOVA analyses. The results show that alternatively and non-alternatively spliced (non-AS) genes have similar tendency for overall codon usage, but exhibit significant difference in 58 out of 64 codons. AS and non-AS genes are both under strong purifying selection, but the former ones have significant lower mutation rate and are prone to be enriched towards the chromosomal ends. In the group of AS genes, the variability in synonymous codon usage between genes is mainly due to the variations in GC content, CDS length, as well as gene functions. Mutational bias that accounts for 25.85 % of the total codon usage variability plays a major role in shaping the codon usage pattern of AS genes. In contrast, no obvious evidence is found for the contributions of translational selection, AS types, the conservation of AS events, and numbers of AS variants to the codon usage divergence between AS genes. These findings may be useful for further understanding the mechanisms of origination, differentiation and regulation of alternatively spliced genes in plants.

  9. The evolution of alternative splicing in the Pax family: the view from the Basal chordate amphioxus.

    PubMed

    Short, Stephen; Holland, Linda Z

    2008-06-01

    Pax genes encode transcription factors critical for metazoan development. Large-scale gene duplication with subsequent gene losses during vertebrate evolution has resulted in two human genes for each of the Pax1/9, Pax3/7, and Pax4/6 subfamilies and three for the Pax2/5/8 subfamily, compared to one each in the cephalochordate amphioxus. In addition, alternative splicing occurs in vertebrate Pax transcripts from all four subfamilies, and many splice forms are known to have functional importance. To better understand the evolution of alternative splicing within the Pax family, we systematically surveyed transcripts of the four amphioxus Pax genes. We have found alternative splicing in every gene. Comparisons with vertebrates suggest that the number of alternative splicing events per gene has not decreased following duplication; there are comparable levels in the four amphioxus Pax genes as in each gene of the equivalent vertebrate families. Thus, the total number of isoforms for the nine vertebrate genes is considerably higher than for the four amphioxus genes. Most alternative splicing events appear to have arisen since the divergence of amphioxus and vertebrate lineages, suggesting that differences in alternative splicing could account for divergent functions of the highly conserved Pax genes in both lineages. However, several events predicted to dramatically alter known functional domains are conserved between amphioxus and vertebrates, suggestive of a common chordate function. Our results, together with previous studies of vertebrate Pax genes, support the theory that alternative splicing impacts functional motifs more than gene duplication followed by divergence.

  10. RBM25 is a global splicing factor promoting inclusion of alternatively spliced exons and is itself regulated by lysine mono-methylation.

    PubMed

    Carlson, Scott M; Soulette, Cameron M; Yang, Ze; Elias, Joshua E; Brooks, Angela N; Gozani, Or

    2017-08-11

    In eukaryotes, precursor mRNA (pre-mRNA) splicing removes non-coding intron sequences to produce mature mRNA. This removal is controlled in part by RNA-binding proteins that regulate alternative splicing decisions through interactions with the splicing machinery. RNA binding motif protein 25 (RBM25) is a putative splicing factor strongly conserved across eukaryotic lineages. However, the role of RBM25 in global splicing regulation and its cellular functions are unknown. Here we show that RBM25 is required for the viability of multiple human cell lines, suggesting that it could play a key role in pre-mRNA splicing. Indeed, transcriptome-wide analysis of splicing events demonstrated that RBM25 regulates a large fraction of alternatively spliced exons throughout the human genome. Moreover, proteomic analysis indicated that RBM25 interacts with components of the early spliceosome and regulators of alternative splicing. Previously, we identified an RBM25 species that is mono-methylated at lysine 77 (RBM25K77me1), and here we used quantitative mass spectrometry to show that RBM25K77me1 is abundant in multiple human cell lines. We also identified a region of RBM25 spanning Lys-77 that binds with high affinity to serine- and arginine-rich splicing factor 2 (SRSF2), a crucial protein in exon definition, but only when Lys-77 is unmethylated. Together, our findings uncover a pivotal role for RBM25 as an essential regulator of alternative splicing and reveal a new potential mechanism for regulation of pre-mRNA splicing by lysine methylation of a splicing factor. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. SRSF10 Plays a Role in Myoblast Differentiation and Glucose Production via Regulation of Alternative Splicing.

    PubMed

    Wei, Ning; Cheng, Yuanming; Wang, Zhijia; Liu, Yuguo; Luo, Chunling; Liu, Lina; Chen, Linlin; Xie, Zhiqin; Lu, Yun; Feng, Ying

    2015-11-24

    Alternative splicing is a major mechanism of controlling gene expression and protein diversity in higher eukaryotes. We report that the splicing factor SRSF10 functions during striated muscle development, myoblast differentiation, and glucose production both in cells and in mice. A combination of RNA-sequencing and molecular analysis allowed us to identify muscle-specific splicing events controlled by SRSF10 that are critically involved in striated muscle development. Inclusion of alternative exons 16 and 17 of Lrrfip1 is a muscle-specific event that is activated by SRSF10 and essential for myoblast differentiation. On the other hand, in mouse primary hepatocytes, PGC1α is a key target of SRSF10 that regulates glucose production by fasting. SRSF10 represses inclusion of PGC1α exon 7a and facilitates the production of functional protein. The results highlight the biological significance of SRSF10 and regulated alternative splicing in vivo.

  12. Fluorescence-based alternative splicing reporters for the study of epithelial plasticity in vivo.

    PubMed

    Somarelli, Jason A; Schaeffer, Daneen; Bosma, Reggie; Bonano, Vivian I; Sohn, Jang Wook; Kemeny, Gabor; Ettyreddy, Abhinav; Garcia-Blanco, Mariano A

    2013-01-01

    Alternative splicing generates a vast diversity of protein isoforms from a limited number of protein-coding genes, with many of the isoforms possessing unique, and even contrasting, functions. Fluorescence-based splicing reporters have the potential to facilitate studies of alternative splicing at the single-cell level and can provide valuable information on phenotypic transitions in almost real time. Fibroblast growth factor receptor 2 (FGFR2) pre-mRNA is alternatively spliced to form the epithelial-specific and mesenchymal-specific IIIb and IIIc isoforms, respectively, which are useful markers of epithelial-mesenchymal transitions (EMT). We have used our knowledge of FGFR2 splicing regulation to develop a fluorescence-based reporter system to visualize exon IIIc regulation in vitro and in vivo. Here we show the application of this reporter system to the study of EMT in vitro in cell culture and in vivo in transgenic mice harboring these splicing constructs. In explant studies, the reporters revealed that FGFR2 isoform switching is not required for keratinocyte migration during cutaneous wound closure. Our results demonstrate the value of the splicing reporters as tools to study phenotypic transitions and cell fates at single cell resolution. Moreover, our data suggest that keratinocytes migrate efficiently in the absence of a complete EMT.

  13. Antagonistic regulation of α-actinin alternative splicing by CELF proteins and polypyrimidine tract binding protein

    PubMed Central

    GROMAK, NATALIA; MATLIN, ARIANNE J.; COOPER, THOMAS A.; SMITH, CHRISTOPHER W.J.

    2003-01-01

    The α-actinin gene has a pair of alternatively spliced exons. The smooth muscle (SM) exon is repressed in most cell types by polypyrimidine tract binding protein (PTB). CELF (CUG-BP and ETR3-like factors) family proteins, splicing regulators whose activities are altered in myotonic dystrophy, were found to coordinately regulate selection of the two α-actinin exons. CUG-BP and ETR3 activated the SM exon, and along with CELF4 they were also able to repress splicing of the NM (nonmuscle) exon both in vivo and in vitro. Activation of SM exon splicing was associated with displacement of PTB from the polypyrimidine tract by binding of CUG-BP at adjacent sites. Our data provides direct evidence for the activity of CELF proteins as both activators and repressors of splicing within a single-model system of alternative splicing, and suggests a model whereby α-actinin alternative splicing is regulated by synergistic and antagonistic interactions between members of the CELF and PTB families. PMID:12649496

  14. ROS and p53 in regulation of UVB-induced HDM2 alternative splicing.

    PubMed

    Tong, Lingying; Wu, Shiyong

    2015-01-01

    Alternative splicing plays an important role in proteasome diversity and gene expression regulation in eukaryotic cells. Hdm2, the human homolog of mdm2 (murine double minute oncogene 2), is known to be an oncogene as its role in suppression of p53. Hdm2 alternative splicing, occurs in both tumor and normal tissues, is believed to be a response of cells for cellular stress, and thus modulate p53 activity. Therefore, understanding the regulation of hdm2 splicing is critical in elucidating the mechanisms of tumor development and progression. In this study, we determined the effect of ultraviolet B light (UVB) on alternative splicing of hdm2. Our data indicated that UVB (50 mJ cm(-2)) alone is not a good inducer of alternative splicing of hdm2. The less effectiveness could be due to the induction of ROS and p53 by UVB because removing ROS by L-NAC (10 mm) in p53 null cells could lead to alternative splicing of hdm2 upon UVB irradiation.

  15. Human telomerase reverse transcriptase regulation by DNA methylation, transcription factor binding and alternative splicing (Review).

    PubMed

    Avin, Brittany A; Umbricht, Christopher B; Zeiger, Martha A

    2016-12-01

    The catalytic subunit of telomerase, human telomerase reverse transcriptase (hTERT), plays an essential role in telomere maintenance to oppose cellular senescence and, is highly regulated in normal and cancerous cells. Regulation of hTERT occurs through multiple avenues, including a unique pattern of CpG promoter methylation and alternative splicing. Promoter methylation affects the binding of transcription factors, resulting in changes in expression of the gene. In addition to expression level changes, changes in promoter binding can affect alternative splicing in a cotranscriptional manner. The alternative splicing of hTERT results in either the full length transcript which can form the active telomerase complex with hTR, or numerous inactive isoforms. Both regulation strategies are exploited in cancer to activate telomerase, however, the exact mechanism is unknown. Therefore, unraveling the link between promoter methylation status and alternative splicing for hTERT could expose yet another level of hTERT regulation. In an attempt to provide insight into the cellular control of active telomerase in cancer, this review will discuss our current perspective on CpG methylation of the hTERT promoter region, summarize the different forms of alternatively spliced variants, and examine examples of transcription factor binding that affects splicing.

  16. Regulation of alternative splicing by local histone modifications: potential roles for RNA-guided mechanisms

    PubMed Central

    Zhou, Hua-Lin; Luo, Guangbin; Wise, Jo Ann; Lou, Hua

    2014-01-01

    The molecular mechanisms through which alternative splicing and histone modifications regulate gene expression are now understood in considerable detail. Here, we discuss recent studies that connect these two previously separate avenues of investigation, beginning with the unexpected discoveries that nucleosomes are preferentially positioned over exons and DNA methylation and certain histone modifications also show exonic enrichment. These findings have profound implications linking chromatin structure, histone modification and splicing regulation. Complementary single gene studies provided insight into the mechanisms through which DNA methylation and histones modifications modulate alternative splicing patterns. Here, we review an emerging theme resulting from these studies: RNA-guided mechanisms integrating chromatin modification and splicing. Several groundbreaking papers reported that small noncoding RNAs affect alternative exon usage by targeting histone methyltransferase complexes to form localized facultative heterochromatin. More recent studies provided evidence that pre-messenger RNA itself can serve as a guide to enable precise alternative splicing regulation via local recruitment of histone-modifying enzymes, and emerging evidence points to a similar role for long noncoding RNAs. An exciting challenge for the future is to understand the impact of local modulation of transcription elongation rates on the dynamic interplay between histone modifications, alternative splicing and other processes occurring on chromatin. PMID:24081581

  17. Isolation and characterization of alternatively spliced variants of the mouse sigma1 receptor gene, Sigmar1

    PubMed Central

    Pan, Ling; Pasternak, David A.; Xu, Jin; Xu, Mingming; Lu, Zhigang; Pasternak, Gavril W.

    2017-01-01

    The sigma1 receptor acts as a chaperone at the endoplasmic reticulum, associates with multiple proteins in various cellular systems, and involves in a number of diseases, such as addiction, pain, cancer and psychiatric disorders. The sigma1 receptor is encoded by the single copy SIGMAR1 gene. The current study identifies five alternatively spliced variants of the mouse sigma1 receptor gene using a polymerase chain reaction cloning approach. All the splice variants are generated by exon skipping or alternative 3’ or 5’ splicing, producing the truncated sigma1 receptor. Similar alternative splicing has been observed in the human SIGMAR1 gene based on the molecular cloning or genome sequence prediction, suggesting conservation of alternative splicing of SIGMAR1 gene. Using quantitative polymerase chain reactions, we demonstrate differential expression of several splice variants in mouse tissues and brain regions. When expressed in HEK293 cells, all the splice variants fail to bind sigma ligands, implicating that each truncated region in these splice variants is important for ligand binding. However, co-immunoprecipitation (Co-IP) study in HEK293 cells co-transfected with tagged constructs reveals that all the splice variants maintain their ability to physically associate with a mu opioid receptor (mMOR-1), providing useful information to correlate the motifs/sequences necessary for their physical association. Furthermore, a competition Co-IP study showed that all the variants can disrupt in a dose-dependent manner the dimerization of the original sigma1 receptor with mMOR-1, suggesting a potential dominant negative function and providing significant insights into their function. PMID:28350844

  18. Alternatives to animal experimentation: The regulatory background

    SciTech Connect

    Garthoff, Bernward . E-mail: bernward.garthoff@bayercropscience.com

    2005-09-01

    The framework, in which alternatives to animal experiments can be developed, standardized, respectively formally validated, has to be seen in a global context. The ever increasing demand of testing for hazard and risk assessment in health and environment, exemplified by the EU REACH program, subsequently triggers laboratory animal testing. This holds especially true, if no valid alternative methods agreed to by the regulatory authorities and the scientific community are available. At least for regulatory toxicity testing, the global frame and network are given by institutions such as OECD, ICH, and alike. However, due to the necessity of global consent of states, organizations, and stakeholders, the time gap between availability of a novel alternative test method and its final acceptance by authorities and implementation thereafter is widening. The lack of new technologies or opportunities for alternative method application such as, for example, the broad use of transgenic animals for refinement of existing tests, adds to the problem. The bare existence of certain in vivo tests increases also the gap between public demands for testing versus availability of alternative tests. Industries operating on a worldwide basis support the alternative test development in their respective area of research and operational business. However, a more coordinating approach such as that of the ecopa-organization (European Consensus Platform on Alternatives) is needed to exploit the existing possibilities within the current regulatory framework. This will speed up the process of acceptance and challenge the political worldto feel responsible for the sequels of their demanding more testing, that is, by funding alternative method development in academia and industry.

  19. Alternatives to animal experimentation: the regulatory background.

    PubMed

    Garthoff, Bernward

    2005-09-01

    The framework, in which alternatives to animal experiments can be developed, standardized, respectively formally validated, has to be seen in a global context. The ever increasing demand of testing for hazard and risk assessment in health and environment, exemplified by the EU REACH program, subsequently triggers laboratory animal testing. This holds especially true, if no valid alternative methods agreed to by the regulatory authorities and the scientific community are available. At least for regulatory toxicity testing, the global frame and network are given by institutions such as OECD, ICH, and alike. However, due to the necessity of global consent of states, organizations, and stakeholders, the time gap between availability of a novel alternative test method and its final acceptance by authorities and implementation thereafter is widening. The lack of new technologies or opportunities for alternative method application such as, for example, the broad use of transgenic animals for refinement of existing tests, adds to the problem. The bare existence of certain in vivo tests increases also the gap between public demands for testing versus availability of alternative tests. Industries operating on a worldwide basis support the alternative test development in their respective area of research and operational business. However, a more coordinating approach such as that of the ecopa-organization (European Consensus Platform on Alternatives) is needed to exploit the existing possibilities within the current regulatory framework. This will speed up the process of acceptance and challenge the political world to feel responsible for the sequels of their demanding more testing, that is, by funding alternative method development in academia and industry.

  20. Toll-Like Receptor 9 Alternatively Spliced Isoform Negatively Regulates TLR9 Signaling in Teleost Fish

    PubMed Central

    Chen, Nai-Yu; Nagarajan, Govindarajulu; Chiou, Pinwen Peter

    2015-01-01

    Toll-like receptor 9 (TLR9) recognizes and binds unmethylated CpG motifs in DNA, which are found in the genomes of bacteria and DNA viruses. In fish, Tlr9 is highly diverse, with the number of introns ranging from 0 to 4. A fish Tlr9 gene containing two introns has been reported to express two alternatively spliced isoforms, namely gTLR9A (full-length) and gTLR9B (with a truncated Cʹ-terminal signal transducing domain), whose regulation and function remain unclear. Here, we report a unique regulatory mechanism of gTLR9 signaling in orange-spotted grouper (Epinephelus coioides), whose gTlr9 sequence also contains two introns. We demonstrated that the grouper gTlr9 gene indeed has the capacity to produce two gTLR9 isoforms via alternative RNA splicing. We found that gTLR9B could function as a negative regulator to suppress gTLR9 signaling as demonstrated by the suppression of downstream gene expression. Following stimulation with CpG oligodeoxynucleotide (ODN), gTLR9A and gTLR9B were observed to translocate into endosomes and co-localize with ODN and the adaptor protein gMyD88. Both gTLR9A and gTLR9B could interact with gMyD88; however, gTLR9B could not interact with downstream IRAK4 and TRAF6. Further analysis of the expression profile of gTlr9A and gTlr9B upon immune-stimulation revealed that the two isoforms were differentially regulated in a time-dependent manner. Overall, these data suggest that fish TLR9B functions as a negative regulator, and that its temporal expression is mediated by alternative RNA splicing. This has not been observed in mammalian TLR9s and might have been acquired relatively recently in the evolution of fish. PMID:25955250

  1. The Conserved Splicing Factor SUA Controls Alternative Splicing of the Developmental Regulator ABI3 in Arabidopsis[W][OA

    PubMed Central

    Sugliani, Matteo; Brambilla, Vittoria; Clerkx, Emile J.M.; Koornneef, Maarten; Soppe, Wim J.J.

    2010-01-01

    ABSCISIC ACID INSENSITIVE3 (ABI3) is a major regulator of seed maturation in Arabidopsis thaliana. We detected two ABI3 transcripts, ABI3-α and ABI3-β, which encode full-length and truncated proteins, respectively. Alternative splicing of ABI3 is developmentally regulated, and the ABI3-β transcript accumulates at the end of seed maturation. The two ABI3 transcripts differ by the presence of a cryptic intron in ABI3-α, which is spliced out in ABI3-β. The suppressor of abi3-5 (sua) mutant consistently restores wild-type seed features in the frameshift mutant abi3-5 but does not suppress other abi3 mutant alleles. SUA is a conserved splicing factor, homologous to the human protein RBM5, and reduces splicing of the cryptic ABI3 intron, leading to a decrease in ABI3-β transcript. In the abi3-5 mutant, ABI3-β codes for a functional ABI3 protein due to frameshift restoration. PMID:20525852

  2. Genome-wide cataloging and analysis of alternatively spliced genes in cereal crops.

    PubMed

    Min, Xiang Jia; Powell, Brian; Braessler, Jonathan; Meinken, John; Yu, Feng; Sablok, Gaurav

    2015-09-21

    Protein functional diversity at the post-transcriptional level is regulated through spliceosome mediated pre-mRNA alternative splicing (AS) events and that has been widely demonstrated to be a key player in regulating the functional diversity in plants. Identification and analysis of AS genes in cereal crop plants are critical for crop improvement and understanding regulatory mechanisms. We carried out the comparative analyses of the functional landscapes of the AS using the consensus assembly of expressed sequence tags and available mRNA sequences in four cereal plants. We identified a total of 8,734 in Oryza sativa subspecies (ssp) japonica, 2,657 in O. sativa ssp indica, 3,971 in Sorghum bicolor, and 10,687 in Zea mays AS genes. Among the identified AS events, intron retention remains to be the dominant type accounting for 23.5 % in S. bicolor, and up to 55.8 % in O. sativa ssp indica. We identified a total of 887 AS genes that were conserved among Z. mays, S. bicolor, and O. sativa ssp japonica; and 248 AS genes were found to be conserved among all four studied species or ssp. Furthermore, we identified 53 AS genes conserved with Brachypodium distachyon. Gene Ontology classification of AS genes revealed functional assignment of these genes in many biological processes with diverse molecular functions. AS is common in cereal plants. The AS genes identified in four cereal crops in this work provide the foundation for further studying the roles of AS in regulation of cereal plant growth and development. The data can be accessed at Plant Alternative Splicing Database (http://proteomics.ysu.edu/altsplice/).

  3. Negative feedback control of jasmonate signaling by an alternative splice variant of JAZ10.

    PubMed

    Moreno, Javier E; Shyu, Christine; Campos, Marcelo L; Patel, Lalita C; Chung, Hoo Sun; Yao, Jian; He, Sheng Yang; Howe, Gregg A

    2013-06-01

    The plant hormone jasmonate (JA) activates gene expression by promoting ubiquitin-dependent degradation of jasmonate ZIM domain (JAZ) transcriptional repressor proteins. A key feature of all JAZ proteins is the highly conserved Jas motif, which mediates both JAZ degradation and JAZ binding to the transcription factor MYC2. Rapid expression of JAZ genes in response to JA is thought to attenuate JA responses, but little is known about the mechanisms by which newly synthesized JAZ proteins exert repression in the presence of the hormone. Here, we show in Arabidopsis (Arabidopsis thaliana) that desensitization to JA is mediated by an alternative splice variant (JAZ10.4) of JAZ10 that lacks the Jas motif. Unbiased protein-protein interaction screens identified three related basic helix-loop-helix transcription factors (MYC2, MYC3, and MYC4) and the corepressor NINJA as JAZ10.4-binding partners. We show that the amino-terminal region of JAZ10.4 contains a cryptic MYC2-binding site that resembles the Jas motif and that the ZIM motif of JAZ10.4 functions as a transferable repressor domain whose activity is associated with the recruitment of NINJA. Functional studies showed that the expression of JAZ10.4 from the native JAZ10 promoter complemented the JA-hypersensitive phenotype of a jaz10 mutant. Moreover, treatment of these complemented lines with JA resulted in the rapid accumulation of JAZ10.4 protein. Our results provide an explanation for how the unique domain architecture of JAZ10.4 links transcription factors to a corepressor complex and suggest how JA-induced transcription and alternative splicing of JAZ10 premessenger RNA creates a regulatory circuit to attenuate JA responses.

  4. Potential control of human immunodeficiency virus type 1 asp expression by alternative splicing in the upstream untranslated region.

    PubMed

    Barbagallo, Michael S; Birch, Katherine E; Deacon, Nicholas J; Mosse, Jennifer A

    2012-07-01

    The negative-sense asp open reading frame (ORF) positioned opposite to the human immunodeficiency virus type 1 (HIV-1) env gene encodes the 189 amino acid, membrane-associated ASP protein. Negative-sense transcription, regulated by long terminal repeat sequences, has been observed early in HIV-1 infection in vitro. All subtypes of HIV-1 were scanned to detect the negative-sense asp ORF and to identify potential regulatory sequences. A series of highly conserved upstream short open reading frames (sORFs) was identified. This potential control region from HIV-1(NL4-3), containing six sORFs, was cloned upstream of the reporter gene EGFP. Expression by transfection of HEK293 cells indicated that the introduction of this sORF region inhibits EGFP reporter expression; analysis of transcripts revealed no significant changes in levels of EGFP mRNA. Reverse transcriptase-polymerase chain reaction analysis (RT-PCR) further demonstrated that the upstream sORF region undergoes alternative splicing in vitro. The most abundant product is spliced to remove sORFs I to V, leaving only the in-frame sORF VI upstream of asp. Sequence analysis revealed the presence of typical splice donor- and acceptor-site motifs. Mutation of the highly conserved splice donor and acceptor sites modulates, but does not fully relieve, inhibition of EGFP production. The strong conservation of asp and its sORFs across all HIV-1 subtypes suggests that the asp gene product may have a role in the pathogenesis of HIV-1. Alternative splicing of the upstream sORF region provides a potential mechanism for controlling expression of the asp gene.

  5. A computational and experimental approach toward a priori identification of alternatively spliced exons

    PubMed Central

    PHILIPPS, DANA L.; PARK, JUNG W.; GRAVELEY, BRENTON R.

    2004-01-01

    Alternative splicing is a powerful means of regulating gene expression and enhancing protein diversity. In fact, the majority of metazoan genes encode pre-mRNAs that are alternatively spliced to produce anywhere from two to tens of thousands of mRNA isoforms. Thus, an important part of determining the complete proteome of an organism is developing a catalog of all mRNA isoforms. Alternatively spliced exons are typically identified by aligning EST clusters to reference mRNAs or genomic DNA. However, this approach is not useful for genomes that lack robust EST coverage, and tools that enable accurate prediction of alternatively spliced exons would be extraordinarily useful. Here, we use comparative genomics to identify, and experimentally verify, potential alternative exons based solely on their high degree of conservation between Drosophila melanogaster and D. pseudoobscura. At least 40% of the exons that fit our prediction criteria are in fact alternatively spliced. Thus, comparative genomics can be used to accurately predict certain classes of alternative exons without relying on EST data. PMID:15525709

  6. A methyl transferase links the circadian clock to the regulation of alternative splicing.

    PubMed

    Sanchez, Sabrina E; Petrillo, Ezequiel; Beckwith, Esteban J; Zhang, Xu; Rugnone, Matias L; Hernando, C Esteban; Cuevas, Juan C; Godoy Herz, Micaela A; Depetris-Chauvin, Ana; Simpson, Craig G; Brown, John W S; Cerdán, Pablo D; Borevitz, Justin O; Mas, Paloma; Ceriani, M Fernanda; Kornblihtt, Alberto R; Yanovsky, Marcelo J

    2010-11-04

    Circadian rhythms allow organisms to time biological processes to the most appropriate phases of the day-night cycle. Post-transcriptional regulation is emerging as an important component of circadian networks, but the molecular mechanisms linking the circadian clock to the control of RNA processing are largely unknown. Here we show that PROTEIN ARGININE METHYL TRANSFERASE 5 (PRMT5), which transfers methyl groups to arginine residues present in histones and Sm spliceosomal proteins, links the circadian clock to the control of alternative splicing in plants. Mutations in PRMT5 impair several circadian rhythms in Arabidopsis thaliana and this phenotype is caused, at least in part, by a strong alteration in alternative splicing of the core-clock gene PSEUDO RESPONSE REGULATOR 9 (PRR9). Furthermore, genome-wide studies show that PRMT5 contributes to the regulation of many pre-messenger-RNA splicing events, probably by modulating 5'-splice-site recognition. PRMT5 expression shows daily and circadian oscillations, and this contributes to the mediation of the circadian regulation of expression and alternative splicing of a subset of genes. Circadian rhythms in locomotor activity are also disrupted in dart5-1, a mutant affected in the Drosophila melanogaster PRMT5 homologue, and this is associated with alterations in splicing of the core-clock gene period and several clock-associated genes. Our results demonstrate a key role for PRMT5 in the regulation of alternative splicing and indicate that the interplay between the circadian clock and the regulation of alternative splicing by PRMT5 constitutes a common mechanism that helps organisms to synchronize physiological processes with daily changes in environmental conditions.

  7. Modulation of alternative splicing with chemical compounds in new therapeutics for human diseases.

    PubMed

    Ohe, Kenji; Hagiwara, Masatoshi

    2015-04-17

    Alternative splicing is a critical step where a limited number of human genes generate a complex and diverse proteome. Various diseases, including inherited diseases with abnormalities in the "genome code," have been found to result in an aberrant mis-spliced "transcript code" with correlation to the resulting phenotype. Chemical compound-based and nucleic acid-based strategies are trying to target this mis-spliced "transcript code". We will briefly mention about how to obtain splicing-modifying-compounds by high-throughput screening and overview of what is known about compounds that modify splicing pathways. The main focus will be on RNA-binding protein kinase inhibitors. In the main text, we will refer to diseases where splicing-modifying-compounds have been intensively investigated, with comparison to nucleic acid-based strategies. The information on their involvement in mis-splicing as well as nonsplicing events will be helpful in finding better compounds with less off-target effects for future implications in mis-splicing therapy.

  8. The Orthologue of the Fruitfly Sex Behaviour Gene Fruitless in the Mosquito Aedes aegypti: Evolution of Genomic Organisation and Alternative Splicing

    PubMed Central

    Salvemini, Marco; D'Amato, Rocco; Petrella, Valeria; Aceto, Serena; Nimmo, Derric; Neira, Marco; Alphey, Luke; Polito, Lino C.; Saccone, Giuseppe

    2013-01-01

    In Drosophila melanogaster the doublesex (dsx) and fruitless (fru) regulatory genes act at the bottom of the somatic sex determination pathway. Both are regulated via alternative splicing by an upstream female-specific TRA/TRA-2 complex, recognizing a common cis element. dsx controls somatic sexual differentiation of non-neural as well as of neural tissues. fru, on the other hand, expresses male-specific functions only in neural system where it is required to built the neural circuits underlying proper courtship behaviour. In the mosquito Aedes aegypti sex determination is different from Drosophila. The key male determiner M, which is located on one of a pair of homomorphic sex chromosomes, controls sex-specific splicing of the mosquito dsx orthologue. In this study we report the genomic organization and expression of the fru homologue in Ae. aegypti (Aeafru). We found that it is sex-specifically spliced suggesting that it is also under the control of the sex determination pathway. Comparative analyses between the Aeafru and Anopheles gambiae fru (Angfru) genomic loci revealed partial conservation of exon organization and extensive divergence of intron lengths. We find that Aeadsx and Aeafru share novel cis splicing regulatory elements conserved in the alternatively spliced regions. We propose that in Aedes aegypti sex-specific splicing of dsx and fru is most likely under the control of splicing regulatory factors which are different from TRA and TRA-2 found in other dipteran insects and discuss the potential use of fru and dsx for developing new genetic strategies in vector control. PMID:23418412

  9. The orthologue of the fruitfly sex behaviour gene fruitless in the mosquito Aedes aegypti: evolution of genomic organisation and alternative splicing.

    PubMed

    Salvemini, Marco; D'Amato, Rocco; Petrella, Valeria; Aceto, Serena; Nimmo, Derric; Neira, Marco; Alphey, Luke; Polito, Lino C; Saccone, Giuseppe

    2013-01-01

    In Drosophila melanogaster the doublesex (dsx) and fruitless (fru) regulatory genes act at the bottom of the somatic sex determination pathway. Both are regulated via alternative splicing by an upstream female-specific TRA/TRA-2 complex, recognizing a common cis element. dsx controls somatic sexual differentiation of non-neural as well as of neural tissues. fru, on the other hand, expresses male-specific functions only in neural system where it is required to built the neural circuits underlying proper courtship behaviour. In the mosquito Aedes aegypti sex determination is different from Drosophila. The key male determiner M, which is located on one of a pair of homomorphic sex chromosomes, controls sex-specific splicing of the mosquito dsx orthologue. In this study we report the genomic organization and expression of the fru homologue in Ae. aegypti (Aeafru). We found that it is sex-specifically spliced suggesting that it is also under the control of the sex determination pathway. Comparative analyses between the Aeafru and Anopheles gambiae fru (Angfru) genomic loci revealed partial conservation of exon organization and extensive divergence of intron lengths. We find that Aeadsx and Aeafru share novel cis splicing regulatory elements conserved in the alternatively spliced regions. We propose that in Aedes aegypti sex-specific splicing of dsx and fru is most likely under the control of splicing regulatory factors which are different from TRA and TRA-2 found in other dipteran insects and discuss the potential use of fru and dsx for developing new genetic strategies in vector control.

  10. Development and media regulate alternative splicing of a methyltransferase pre-mRNA in Monascus pilosus.

    PubMed

    Zhang, Ming-Yong; Miyake, Tsuyoshi

    2009-05-27

    Two alternatively spliced mRNAs (d- and l-MpLaeA) of a methyltransferase gene (MpLaeA) were identified from Monascus pilosus IFO4520 and its mutant MK-1. Alternative splicing of the MpLaeA pre-mRNA occurred in the 5'-untranslated region (5'-UTR). The alternative splicing patterns of MpLaeA were regulated by the fungal growth stage and the principal nutrients: that is, the short l-MpLaeA mRNA was a constitutive transcript at all growth stages and different carbon or nitrogen sources, but the glutamate and NaNO(3) as main nitrogen source could up-regulate the long d-MpLaeA mRNA form. The long spliced 5'-UTR of d-MpLaeA blocked GFP expression in Escherichia coli , suggesting that d-MpLaeA mRNA was an ineffective spliced mRNA. Down-regulation of MpLaeA by transgenic antisense d-MpLaeA cDNA resulted in decreasing synthesis of monacolin K in M. pilosus. This suggested that the alternative splicing of MpLaeA mRNA might regulate the synthesis of monacolin K.

  11. In Vitro and In Vivo Modulation of Alternative Splicing by the Biguanide Metformin

    PubMed Central

    Laustriat, Delphine; Gide, Jacqueline; Barrault, Laetitia; Chautard, Emilie; Benoit, Clara; Auboeuf, Didier; Boland, Anne; Battail, Christophe; Artiguenave, François; Deleuze, Jean-François; Bénit, Paule; Rustin, Pierre; Franc, Sylvia; Charpentier, Guillaume; Furling, Denis; Bassez, Guillaume; Nissan, Xavier; Martinat, Cécile; Peschanski, Marc; Baghdoyan, Sandrine

    2015-01-01

    Major physiological changes are governed by alternative splicing of RNA, and its misregulation may lead to specific diseases. With the use of a genome-wide approach, we show here that this splicing step can be modified by medication and demonstrate the effects of the biguanide metformin, on alternative splicing. The mechanism of action involves AMPK activation and downregulation of the RBM3 RNA-binding protein. The effects of metformin treatment were tested on myotonic dystrophy type I (DM1), a multisystemic disease considered to be a spliceopathy. We show that this drug promotes a corrective effect on several splicing defects associated with DM1 in derivatives of human embryonic stem cells carrying the causal mutation of DM1 as well as in primary myoblasts derived from patients. The biological effects of metformin were shown to be compatible with typical therapeutic dosages in a clinical investigation involving diabetic patients. The drug appears to act as a modifier of alternative splicing of a subset of genes and may therefore have novel therapeutic potential for many more diseases besides those directly linked to defective alternative splicing. PMID:26528939

  12. MBNL proteins repress embryonic stem cell-specific alternative splicing and reprogramming

    PubMed Central

    Han, Hong; Irimia, Manuel; Ross, P. Joel; Sung, Hoon-Ki; Alipanahi, Babak; David, Laurent; Golipour, Azadeh; Gabut, Mathieu; Michael, Iacovos P.; Nachman, Emil N.; Wang, Eric; Trcka, Dan; Thompson, Tadeo; O’Hanlon, Dave; Slobodeniuc, Valentina; Barbosa-Morais, Nuno L.; Burge, Christopher B.; Moffat, Jason; Frey, Brendan J.; Nagy, Andras; Ellis, James; Wrana, Jeffrey L.; Blencowe, Benjamin J.

    2014-01-01

    Previous investigations of the core gene regulatory circuitry that controls embryonic stem cell (ESC) pluripotency have largely focused on the roles of transcription, chromatin and non-coding RNA regulators1–3. Alternative splicing (AS) represents a widely acting mode of gene regulation4–8, yet its role in regulating ESC pluripotency and differentiation is poorly understood. Here, we identify the Muscleblind-like RNA binding proteins, MBNL1 and MBNL2, as conserved and direct negative regulators of a large program of cassette exon AS events that are differentially regulated between ESCs and other cell types. Knockdown of MBNL proteins in differentiated cells causes switching to an ESC-like AS pattern for approximately half of these events, whereas over-expression of MBNL proteins in ESCs promotes differentiated cell-like AS patterns. Among the MBNL-regulated events is an ESC-specific AS switch in the forkhead family transcription factor FOXP1 that controls pluripotency9. Consistent with a central and negative regulatory role for MBNL proteins in pluripotency, their knockdown significantly enhances the expression of key pluripotency genes and the formation of induced pluripotent stem cells (iPSCs) during somatic cell reprogramming. PMID:23739326

  13. Role of six single nucleotide polymorphisms, risk factors in coronary disease, in OLR1 alternative splicing.

    PubMed

    Tejedor, J Ramón; Tilgner, Hagen; Iannone, Camilla; Guigó, Roderic; Valcárcel, Juan

    2015-06-01

    The OLR1 gene encodes the oxidized low-density lipoprotein receptor (LOX-1), which is responsible for the cellular uptake of oxidized LDL (Ox-LDL), foam cell formation in atheroma plaques and atherosclerotic plaque rupture. Alternative splicing (AS) of OLR1 exon 5 generates two protein isoforms with antagonistic functions in Ox-LDL uptake. Previous work identified six single nucleotide polymorphisms (SNPs) in linkage disequilibrium that influence the inclusion levels of OLR1 exon 5 and correlate with the risk of cardiovascular disease. Here we use minigenes to recapitulate the effects of two allelic series (Low- and High-Risk) on OLR1 AS and identify one SNP in intron 4 (rs3736234) as the main contributor to the differences in exon 5 inclusion, while the other SNPs in the allelic series attenuate the drastic effects of this key SNP. Bioinformatic, proteomic, mutational and functional high-throughput analyses allowed us to define regulatory sequence motifs and identify SR protein family members (SRSF1, SRSF2) and HMGA1 as factors involved in the regulation of OLR1 AS. Our results suggest that antagonism between SRSF1 and SRSF2/HMGA1, and differential recognition of their regulatory motifs depending on the identity of the rs3736234 polymorphism, influence OLR1 exon 5 inclusion and the efficiency of Ox-LDL uptake, with potential implications for atherosclerosis and coronary disease.

  14. [Deregulation of pre-messenger RNA splicing and rare diseases].

    PubMed

    de la Grange, Pierre

    2016-12-01

    Most of protein-coding human genes are subjected to alternative pre-mRNA splicing. This mechanism is highly regulated to precisely modulate detection of specific splice sites. This regulation is under control of the spliceosome and several splicing factors are also required to modulate the alternative usage of splice sites. Splicing factors and spliceosome components recognize splicing signals and regulatory sequences of the pre-mRNAs. These splicing sequences make splicing susceptible to polymorphisms and mutations. Examples of associations between human rare diseases and defects in pre-messenger RNA splicing are accumulating. Although many alterations are caused by mutations in splicing sequence (i.e., cis acting mutations), recent studies described the disruptive impact of mutations within spliceosome components or splicing factors (i.e., trans acting mutations). Following growing of knowledge regarding splicing regulation, several approaches have been developed to compensate for the effect of deleterious mutations and to restore sufficient amounts of functional protein.

  15. The Role of Alternative Splicing in Breast Cancer Progression

    DTIC Science & Technology

    2007-09-01

    tumorigenesis Reportable Outcomes: -portions of the work have been chosen for an oral presentation at this year’s Cold Spring Harbor “Eukaryotic mRNA...splicing alterations. References: N/A Appendices: Copy of abstract of the work presented during the Cold Spring Harbor “Eukaryotic mRNA Processing” meeting, August 22-26, 2007

  16. In Vivo Analysis of Alternative Pre-mRNA Splicing.

    DTIC Science & Technology

    1996-10-01

    SF2 influences 5’ splice site selection by activating proximal sites. Cell 62: 35-42 Koehorst SG, Jacobs HM, Thijssen JH, Blankenstein MA (1993) Wild...S, Frati L and Gulino A (1995) Estrogen receptors: new perspectives in breast cancer management. J. Ster. Bioch . Mol. Biol. 49:327-331. Pfeffer U

  17. Alternative splicing in chronic myeloid leukemia (CML): a novel therapeutic target?

    PubMed

    Adamia, Sophia; Pilarski, Patrick M; Bar-Natan, Michal; Stone, Richard M; Griffin, James D

    2013-09-01

    Although the imatinib based therapy of chronic myeloid leukemia (CML) represents a triumph of medicine, not all patients with CML benefit from this drug due to the development of resistance and intolerance. The interruption of imatinib treatment is often followed by clinical relapse, suggesting a failure in the killing of residual leukaemic stem cells. There is need to identify alternative selective molecular targets for this disease and develop more effective therapeutic approaches. Alternative pre-mRNA splicing (AS) is an epigenetic process that greatly diversifies the repertoire of the transcriptome. AS orchestrates interactions between various types of proteins and between proteins and nucleic acids. Changes caused by individual splicing events in the cells are small, however, "splicing programs" typically react to these individual changes with considerable effects in cell proliferation, cell survival, and apoptosis. Current evidence suggests a pivotal role of AS in leukemias, particularly in myelodisplastic syndrome (MDS) and chronic lymphocyte leukemia (CLL). From these studies and studies in other malignances, it is clear that splicing abnormalities play a significant role in malignant transformation. Evaluation of AS events in CML can be used to identify novel disease markers and drugsensitive targets to overcome the limits of the small molecule inhibitors currently used for treating patients with CML. The use of aberrant splice variants as disease markers has been reported, however, little is known about the use of splicing abnormalities as drug targets in CML. Herein we discuss potential therapeutic approaches that can be used to target splicing abnormalities in CML.

  18. Alternative splicing of the maize Ac transposase transcript in transgenic sugar beet (Beta vulgaris L.).

    PubMed

    Lisson, Ralph; Hellert, Jan; Ringleb, Malte; Machens, Fabian; Kraus, Josef; Hehl, Reinhard

    2010-09-01

    The maize Activator/Dissociation (Ac/Ds) transposable element system was introduced into sugar beet. The autonomous Ac and non-autonomous Ds element excise from the T-DNA vector and integrate at novel positions in the sugar beet genome. Ac and Ds excisions generate footprints in the donor T-DNA that support the hairpin model for transposon excision. Two complete integration events into genomic sugar beet DNA were obtained by IPCR. Integration of Ac leads to an eight bp duplication, while integration of Ds in a homologue of a sugar beet flowering locus gene did not induce a duplication. The molecular structure of the target site indicates Ds integration into a double strand break. Analyses of transposase transcription using RT-PCR revealed low amounts of alternatively spliced mRNAs. The fourth intron of the transposase was found to be partially misspliced. Four different splice products were identified. In addition, the second and third exon were found to harbour two and three novel introns, respectively. These utilize each the same splice donor but several alternative splice acceptor sites. Using the SplicePredictor online tool, one of the two introns within exon two is predicted to be efficiently spliced in maize. Most interestingly, splicing of this intron together with the four major introns of Ac would generate a transposase that lacks the DNA binding domain and two of its three nuclear localization signals, but still harbours the dimerization domain.

  19. Hydrogen peroxide triggers a novel alternative splicing of arsenic (+3 oxidation state) methyltransferase gene.

    PubMed

    Sumi, Daigo; Takeda, Chieri; Yasuoka, Daiki; Himeno, Seiichiro

    2016-11-04

    We previously reported that two splicing variants of human AS3MT mRNA, exon-3 skipping form (Δ3) and exons-4 and -5 skipping form (Δ4,5), were detected in HepG2 cells and that both variants lacked arsenic methylation activity (Sumi et al., 2011). Here we studied whether hydrogen peroxide (H2O2) triggers alternative splicing of AS3MT mRNA. The results showed that exposure of HepG2 cells to H2O2 resulted in increased levels of a novel spliced form skipping exon-3 to exon-10 (Δ3-10) in an H2O2-concentration-dependent manner, although no change was detected in the mRNA levels of Δ3 AS3MT. We found decreased protein levels of serine/arginine-rich 40 (SRp40), which we determined to be a candidate splice factor for controlling the splicing of AS3MT mRNA. We next compared the amounts of methylated arsenic metabolites between control and H2O2-exposed HepG2 cells after the addition of arsenite as a substance. The results showed lower levels of methylated arsenic metabolites in HepG2 cells exposed to H2O2. These data suggest that the splicing of AS3MT pre-mRNA was disconcerted by oxidative stress and that abnormal alternative splicing of AS3MT mRNA may affect arsenic methylation ability.

  20. Hypoxia Regulates Alternative Splicing of HIF and non-HIF Target Genes

    PubMed Central

    Sena, Johnny A.; Wang, Liyi; Heasley, Lynn E.; Hu, Cheng-Jun

    2014-01-01

    Hypoxia is a common characteristic of many solid tumors. The hypoxic microenvironment stabilizes hypoxia-inducible transcription factor 1α (HIF1A) and 2α (HIF2α/EPAS1) to activate gene transcription, which promotes tumor cell survival. The majority of human genes are alternatively spliced, producing RNA isoforms that code for functionally distinct proteins. Thus, an effective hypoxia response requires increased HIF target gene expression as well as proper RNA splicing of these HIF-dependent transcripts. However, it is unclear if and how hypoxia regulates RNA splicing of HIF targets. This study determined the effects of hypoxia on alternative splicing (AS) of HIF and non-HIF target genes in hepatocellular carcinoma (HCC) cells and characterized the role of HIF in regulating AS of HIF induced genes. The results indicate that hypoxia generally promotes exon inclusion for hypoxia-induced, but reduces exon inclusion for hypoxia reduced genes. Mechanistically, HIF activity, but not hypoxia per se is found to be necessary and sufficient to increase exon inclusion of several HIF targets including pyruvate dehydrogenase kinase 1 (PDK1). PDK1 splicing reporters confirm that transcriptional activation by HIF is sufficient to increase exon inclusion of PDK1 splicing reporter. In contrast, transcriptional activation of a PDK1 minigene by other transcription factors in the absence of endogenous HIF target gene activation fails to alter PDK1 RNA splicing. PMID:24850901

  1. Alternative splicing of the maize Ac transposase transcript in transgenic sugar beet (Beta vulgaris L.)

    PubMed Central

    Lisson, Ralph; Hellert, Jan; Ringleb, Malte; Machens, Fabian; Kraus, Josef

    2010-01-01

    The maize Activator/Dissociation (Ac/Ds) transposable element system was introduced into sugar beet. The autonomous Ac and non-autonomous Ds element excise from the T-DNA vector and integrate at novel positions in the sugar beet genome. Ac and Ds excisions generate footprints in the donor T-DNA that support the hairpin model for transposon excision. Two complete integration events into genomic sugar beet DNA were obtained by IPCR. Integration of Ac leads to an eight bp duplication, while integration of Ds in a homologue of a sugar beet flowering locus gene did not induce a duplication. The molecular structure of the target site indicates Ds integration into a double strand break. Analyses of transposase transcription using RT–PCR revealed low amounts of alternatively spliced mRNAs. The fourth intron of the transposase was found to be partially misspliced. Four different splice products were identified. In addition, the second and third exon were found to harbour two and three novel introns, respectively. These utilize each the same splice donor but several alternative splice acceptor sites. Using the SplicePredictor online tool, one of the two introns within exon two is predicted to be efficiently spliced in maize. Most interestingly, splicing of this intron together with the four major introns of Ac would generate a transposase that lacks the DNA binding domain and two of its three nuclear localization signals, but still harbours the dimerization domain. PMID:20512402

  2. Dose-Dependent Regulation of Alternative Splicing by MBNL Proteins Reveals Biomarkers for Myotonic Dystrophy

    PubMed Central

    Struck, Adam J.; Gupta, Riti; Farnsworth, Dylan R.; Mahady, Amy E.; Eichinger, Katy; Thornton, Charles A.; Wang, Eric T.; Berglund, J. Andrew

    2016-01-01

    Alternative splicing is a regulated process that results in expression of specific mRNA and protein isoforms. Alternative splicing factors determine the relative abundance of each isoform. Here we focus on MBNL1, a splicing factor misregulated in the disease myotonic dystrophy. By altering the concentration of MBNL1 in cells across a broad dynamic range, we show that different splicing events require different amounts of MBNL1 for half-maximal response, and respond more or less steeply to MBNL1. Motifs around MBNL1 exon 5 were studied to assess how cis-elements mediate the MBNL1 dose-dependent splicing response. A framework was developed to estimate MBNL concentration using splicing responses alone, validated in the cell-based model, and applied to myotonic dystrophy patient muscle. Using this framework, we evaluated the ability of individual and combinations of splicing events to predict functional MBNL concentration in human biopsies, as well as their performance as biomarkers to assay mild, moderate, and severe cases of DM. PMID:27681373

  3. Alternative Splicing of the Amelogenin Gene in a Caudate Amphibian, Plethodoncinereus

    PubMed Central

    Wang, Xinping; Xing, Zeli; Zhang, Xichen; Zhu, Lisai; Diekwisch, Thomas G. H.

    2013-01-01

    As the major enamel matrix protein contributing to tooth development, amelogenin has been demonstrated to play a crucial role in tooth enamel formation. Previous studies have revealed amelogenin alternative splicing as a mechanism for amelogenin heterogeneous expression in mammals. While amelogenin and its splicing forms in mammalian vertebrates have been characterized, splicing variants of amelogenin gene still remains largely unknown in non-mammalian species. Here, using PCR and sequence analysis we discovered two novel amelogenin transcript variants in tooth organ extracts from a caudate amphibian, the salamander Plethodoncinereus. The one was shorter -S- (416 nucleotides including untranslated regions, 5 exons) and the other larger -L- (851 nt, 7 exons) than the previously published “normal” gene in this species -M- (812 nucleotides, 6 exons). This is the first report demonstrating the amelogenin alternative splicing in amphibian, revealing a unique exon 2b and two novel amelogenin gene transcripts in Plethodoncinereus. PMID:23840861

  4. A Novel Subgenomic Murine Leukemia Virus RNA Transcript Results from Alternative Splicing

    PubMed Central

    Déjardin, Jérôme; Bompard-Maréchal, Guillaume; Audit, Muriel; Hope, Thomas J.; Sitbon, Marc; Mougel, Marylène

    2000-01-01

    Here we show the existence of a novel subgenomic 4.4-kb RNA in cells infected with the prototypic replication-competent Friend or Moloney murine leukemia viruses (MuLV). This RNA derives by splicing from an alternative donor site (SD′) within the capsid-coding region to the canonical envelope splice acceptor site. The position and the sequence of SD′ was highly conserved among mammalian type C and D oncoviruses. Point mutations used to inactivate SD′ without changing the capsid-coding ability affected viral RNA splicing and reduced viral replication in infected cells. PMID:10729146

  5. SPACE: an algorithm to predict and quantify alternatively spliced isoforms using microarrays

    PubMed Central

    Anton, Miguel A; Gorostiaga, Dorleta; Guruceaga, Elizabeth; Segura, Victor; Carmona-Saez, Pedro; Pascual-Montano, Alberto; Pio, Ruben; Montuenga, Luis M; Rubio, Angel

    2008-01-01

    Exon and exon+junction microarrays are promising tools for studying alternative splicing. Current analytical tools applied to these arrays lack two relevant features: the ability to predict unknown spliced forms and the ability to quantify the concentration of known and unknown isoforms. SPACE is an algorithm that has been developed to (1) estimate the number of different transcripts expressed under several conditions, (2) predict the precursor mRNA splicing structure and (3) quantify the transcript concentrations including unknown forms. The results presented here show its robustness and accuracy for real and simulated data. PMID:18312629

  6. Genome-wide profiling of chemoradiation‑induced changes in alternative splicing in colon cancer cells.

    PubMed

    Xiong, Wei; Gao, Depei; Li, Yunfeng; Liu, Xin; Dai, Peiling; Qin, Jiyong; Wang, Guanshun; Li, Kangming; Bai, Han; Li, Wenhui

    2016-10-01

    Alternative splicing is a key mechanism that regulates protein diversity and has been found to be associated with colon cancer progression and metastasis. However, the function of alternative splicing in chemoradiation‑resistant colon cancer remains elusive. In this study, we constructed a chemoradiation‑resistant colon cancer cell line. Through RNA-sequencing of normal and chemoradiation‑resistant colon cancer cells (HCT116), we found 818 genes that were highly expressed in the normal HCT116 cells, whereas 285 genes were highly expressed in the chemoradiation-resistant HCT116 (RCR-HCT116) cells. Gene ontology (GO) analysis showed that genes that were highly expressed in the HCT116 cells were enriched in GO categories related to cell cycle and cell division, whereas genes that were highly expressed in the RCR-HCT116 cells were associated with regulation of system processes and response to wounding. Analysis of alternative splicing events revealed that exon skipping was significantly increased in the chemoradiation‑resistant colon cancer cells. Moreover, we identified 323 alternative splicing events in 293 genes that were significantly different between the two different HCT116 cell types. These alternative splicing‑related genes were clustered functionally into several groups related with DNA replication, such as deoxyribonucleotide metabolic/catabolic processes, response to DNA damage stimulus and helicase activity. These findings enriched our knowledge by elucidating the function of alternative splicing in chemoradiation-resistant colon cancer.

  7. Alternatively spliced insertions in the paired domain restrict the DNA sequence specificity of Pax6 and Pax8.

    PubMed Central

    Kozmik, Z; Czerny, T; Busslinger, M

    1997-01-01

    Transcription factors of the Pax family bind to their target genes via the paired domain which is known to be composed of two subdomains each recognizing distinct half-sites in adjacent major grooves of the DNA helix. We now demonstrate that the mammalian Pax8 gene gives rise, by alternative mRNA splicing, to a protein isoform containing an extra serine residue in the recognition alpha-helix 3 of the paired domain. This Pax8(S) protein does not interact with bipartite paired domain-binding sites, indicating that inactivation of the N-terminal DNA-binding motif severely restricts the sequence specificity of the paired domain. However, the Pax8(S) protein binds in vitro and in vivo to the 5aCON sequence which was previously identified as a high-affinity binding site for the Pax6(5a) splice variant carrying a 14-amino-acid insertion in the paired domain. The 5aCON sequence is shown to consist of four interdigitated 5' half-sites of the bipartite consensus sequence and is thus bound by four Pax8(S) molecules via the intact C-terminal DNA-binding motif of the paired domain. Together these data suggest that inactivation of the N-terminal region of the paired domain by alternative splicing is used in vivo to selectively target Pax transcription factors to gene regulatory regions containing highly specialized 5aCON-like sequences. PMID:9362493

  8. Regulation of corepressor alternative mRNA splicing by hormonal and metabolic signaling

    PubMed Central

    Snyder, Chelsea A.; Goodson, Michael L.; Schroeder, Amy C.; Privalsky, Martin L.

    2015-01-01

    Alternative mRNA splicing diversifies the products encoded by the NCoR and SMRT corepressor loci. There is a programmed alteration in NCoR mRNA splicing during adipocyte differentiation from an NCoRδ isoform, which contains three nuclear receptor interaction domains, to an NCoRδ isoform that contains two nuclear receptor interaction domains. This alternative mRNA splicing of NCoR has profound effects on adiposity and on diabetes in mouse models. We report here that dexamethasone, a powerful regulator of metabolism and of adipocyte differentiation, confers this change in NCoR mRNA splicing in cultured adipocytes. We also demonstrate that changes in dietary components can consistently, if moderately, modulate the total transcript levels and the mRNA splicing of NCoR and SMRT in both cultured cells and intact mice. This ability of alternative corepressor mRNA splicing to respond to nutritional changes confirms its importance in regulating glucose and lipid metabolism, and its promise as a therapeutic candidate for metabolic disorders such as type 2 diabetes. PMID:26166430

  9. EMT and stemness: flexible processes tuned by alternative splicing in development and cancer progression.

    PubMed

    Pradella, Davide; Naro, Chiara; Sette, Claudio; Ghigna, Claudia

    2017-01-30

    Epithelial-to-mesenchymal transition (EMT) is associated with metastasis formation as well as with generation and maintenance of cancer stem cells. In this way, EMT contributes to tumor invasion, heterogeneity and chemoresistance. Morphological and functional changes involved in these processes require robust reprogramming of gene expression, which is only partially accomplished at the transcriptional level. Alternative splicing is another essential layer of gene expression regulation that expands the cell proteome. This step in post-transcriptional regulation of gene expression tightly controls cell identity between epithelial and mesenchymal states and during stem cell differentiation. Importantly, dysregulation of splicing factor function and cancer-specific splicing isoform expression frequently occurs in human tumors, suggesting the importance of alternative splicing regulation for cancer biology.In this review, we briefly discuss the role of EMT programs in development, stem cell differentiation and cancer progression. Next, we focus on selected examples of key factors involved in EMT and stem cell differentiation that are regulated post-transcriptionally through alternative splicing mechanisms. Lastly, we describe relevant oncogenic splice-variants that directly orchestrate cancer stem cell biology and tumor EMT, which may be envisioned as novel targets for therapeutic intervention.

  10. Regulation of corepressor alternative mRNA splicing by hormonal and metabolic signaling.

    PubMed

    Snyder, Chelsea A; Goodson, Michael L; Schroeder, Amy C; Privalsky, Martin L

    2015-09-15

    Alternative mRNA splicing diversifies the products encoded by the NCoR and SMRT corepressor loci. There is a programmed alteration in NCoR mRNA splicing during adipocyte differentiation from an NCoRω isoform, which contains three nuclear receptor interaction domains, to an NCoRδ isoform that contains two nuclear receptor interaction domains. This alternative mRNA splicing of NCoR has profound effects on adiposity and on diabetes in mouse models. We report here that dexamethasone, a powerful regulator of metabolism and of adipocyte differentiation, confers this change in NCoR mRNA splicing in cultured adipocytes. We also demonstrate that changes in dietary components can consistently, if moderately, modulate the total transcript levels and the mRNA splicing of NCoR and SMRT in both cultured cells and intact mice. This ability of alternative corepressor mRNA splicing to respond to nutritional changes confirms its importance in regulating glucose and lipid metabolism, and its promise as a therapeutic candidate for metabolic disorders such as type 2 diabetes.

  11. Global Profiling and Molecular Characterization of Alternative Splicing Events Misregulated in Lung Cancer ▿ †

    PubMed Central

    Misquitta-Ali, Christine M.; Cheng, Edith; O'Hanlon, Dave; Liu, Ni; McGlade, C. Jane; Tsao, Ming Sound; Blencowe, Benjamin J.

    2011-01-01

    Alternative splicing (AS) is a widespread mechanism underlying the generation of proteomic and regulatory complexity. However, which of the myriad of human AS events play important roles in disease is largely unknown. To identify frequently occurring AS events in lung cancer, we used AS microarray profiling and reverse transcription-PCR (RT-PCR) assays to survey patient-matched normal and adenocarcinoma tumor tissues from the lungs of 29 individuals diagnosed with non-small cell lung cancer (NSCLC). Of 5,183 profiled alternative exons, four displayed tumor-associated changes in the majority of the patients. These events affected transcripts from the VEGFA, MACF1, APP, and NUMB genes. Similar AS changes were detected in NUMB and APP transcripts in primary breast and colon tumors. Tumor-associated increases in NUMB exon 9 inclusion correlated with reduced levels of NUMB protein expression and activation of the Notch signaling pathway, an event that has been linked to tumorigenesis. Moreover, short hairpin RNA (shRNA) knockdown of NUMB followed by isoform-specific rescue revealed that expression of the exon 9-skipped (nontumor) isoform represses Notch target gene activation whereas expression of the exon 9-included (tumor) isoform lacks this activity and is capable of promoting cell proliferation. The results thus reveal widespread AS changes in NSCLC that impact cell signaling in a manner that likely contributes to tumorigenesis. PMID:21041478

  12. Changes in alternative splicing of human and mouse genes are accompanied by faster evolution of constitutive exons.

    PubMed

    Cusack, Brian P; Wolfe, Kenneth H

    2005-11-01

    Alternative splicing is known to be an important source of protein sequence variation, but its evolutionary impact has not been explored in detail. Studying alternative splicing requires extensive sampling of the transcriptome, but new data sets based on expressed sequence tags aligned to chromosomes make it possible to study alternative splicing on a genome-wide scale. Although genes showing alternative splicing by exon skipping are conserved as compared to the genome as a whole, we find that genes where structural differences between human and mouse result in genome-specific alternatively spliced exons in one species show almost 60% greater nonsynonymous divergence in constitutive exons than genes where exon skipping is conserved. This effect is also seen for genes showing species-specific patterns of alternative splicing where gene structure is conserved. Our observations are not attributable to an inherent difference in rate of evolution between these two sets of proteins or to differences with respect to predictors of evolutionary rate such as expression level, tissue specificity, or genetic redundancy. Where genome-specific alternatively spliced exons are seen in mammals, the vast majority of skipped exons appear to be recent additions to gene structures. Furthermore, among genes with genome-specific alternatively spliced exons, the degree of nonsynonymous divergence in constitutive sequence is a function of the frequency of incorporation of these alternative exons into transcripts. These results suggest that alterations in alternative splicing pattern can have knock-on effects in terms of accelerated sequence evolution in constant regions of the protein.

  13. Domestication reduces alternative splicing expression variations in sorghum.

    PubMed

    Ranwez, Vincent; Serra, Audrey; Pot, David; Chantret, Nathalie

    2017-01-01

    Domestication is known to strongly reduce genomic diversity through population bottlenecks. The resulting loss of polymorphism has been thoroughly documented in numerous cultivated species. Here we investigate the impact of domestication on the diversity of alternative transcript expressions using RNAseq data obtained on cultivated and wild sorghum accessions (ten accessions for each pool). In that aim, we focus on genes expressing two isoforms in sorghum and estimate the ratio between expression levels of those isoforms in each accession. Noticeably, for a given gene, one isoform can either be overexpressed or underexpressed in some wild accessions, whereas in the cultivated accessions, the balance between the two isoforms of the same gene appears to be much more homogenous. Indeed, we observe in sorghum significantly more variation in isoform expression balance among wild accessions than among domesticated accessions. The possibility exists that the loss of nucleotide diversity due to domestication could affect regulatory elements, controlling transcription or degradation of these isoforms. Impact on the isoform expression balance is discussed. As far as we know, this is the first time that the impact of domestication on transcript isoform balance has been studied at the genomic scale. This could pave the way towards the identification of key domestication genes with finely tuned isoform expressions in domesticated accessions while being highly variable in their wild relatives.

  14. Domestication reduces alternative splicing expression variations in sorghum

    PubMed Central

    Ranwez, Vincent; Serra, Audrey; Pot, David

    2017-01-01

    Domestication is known to strongly reduce genomic diversity through population bottlenecks. The resulting loss of polymorphism has been thoroughly documented in numerous cultivated species. Here we investigate the impact of domestication on the diversity of alternative transcript expressions using RNAseq data obtained on cultivated and wild sorghum accessions (ten accessions for each pool). In that aim, we focus on genes expressing two isoforms in sorghum and estimate the ratio between expression levels of those isoforms in each accession. Noticeably, for a given gene, one isoform can either be overexpressed or underexpressed in some wild accessions, whereas in the cultivated accessions, the balance between the two isoforms of the same gene appears to be much more homogenous. Indeed, we observe in sorghum significantly more variation in isoform expression balance among wild accessions than among domesticated accessions. The possibility exists that the loss of nucleotide diversity due to domestication could affect regulatory elements, controlling transcription or degradation of these isoforms. Impact on the isoform expression balance is discussed. As far as we know, this is the first time that the impact of domestication on transcript isoform balance has been studied at the genomic scale. This could pave the way towards the identification of key domestication genes with finely tuned isoform expressions in domesticated accessions while being highly variable in their wild relatives. PMID:28886042

  15. Analysis of genetic interaction networks shows that alternatively spliced genes are highly versatile.

    PubMed

    Talavera, David; Sheoran, Ritika; Lovell, Simon C

    2013-01-01

    Alternative splicing has the potential to increase the diversity of the transcriptome and proteome. Where more than one transcript arises from a gene they are often so different that they are quite unlikely to have the same function. However, it remains unclear if alternative splicing generally leads to a gene being involved in multiple biological processes or whether it alters the function within a single process. Knowing that genetic interactions occur between functionally related genes, we have used them as a proxy for functional versatility, and have analysed the sets of genes of two well-characterised model organisms: Caenorhabditis elegans and Drosophila melanogaster. Using network analyses we find that few genes are functionally homogenous (only involved in a few functionally-related biological processes). Moreover, there are differences between alternatively spliced genes and genes with a single transcript; specifically, genes with alternatively splicing are, on average, involved in more biological processes. Finally, we suggest that factors other than specific functional classes determine whether a gene is alternatively spliced.

  16. Changes in cell wall polysaccharide composition, gene transcription and alternative splicing in germinating barley embryos.

    PubMed

    Zhang, Qisen; Zhang, Xiaoqi; Pettolino, Filomena; Zhou, Gaofeng; Li, Chengdao

    2016-02-01

    Barley (Hordeum vulgare L.) seed germination initiates many important biological processes such as DNA, membrane and mitochondrial repairs. However, little is known on cell wall modifications in germinating embryos. We have investigated cell wall polysaccharide composition change, gene transcription and alternative splicing events in four barley varieties at 24h and 48 h germination. Cell wall components in germinating barley embryos changed rapidly, with increases in cellulose and (1,3)(1,4)-β-D-glucan (20-100%) within 24h, but decreases in heteroxylan and arabinan (3-50%). There were also significant changes in the levels of type I arabinogalactans and heteromannans. Alternative splicing played very important roles in cell wall modifications. At least 22 cell wall transcripts were detected to undergo either alternative 3' splicing, alternative 5' splicing or intron retention type of alternative splicing. These genes coded enzymes catalyzing synthesis and degradation of cellulose, heteroxylan, (1,3)(1,4)-β-D-glucan and other cell wall polymers. Furthermore, transcriptional regulation also played very important roles in cell wall modifications. Transcript levels of primary wall cellulase synthase, heteroxylan synthesizing and nucleotide sugar inter-conversion genes were very high in germinating embryos. At least 50 cell wall genes changed transcript levels significantly. Expression patterns of many cell wall genes coincided with changes in polysaccharide composition. Our data showed that cell wall polysaccharide metabolism was very active in germinating barley embryos, which was regulated at both transcriptional and post-transcriptional levels.

  17. Caffeine induces tumor cytotoxicity via the regulation of alternative splicing in subsets of cancer-associated genes.

    PubMed

    Lu, Guan-Yu; Huang, Shih-Ming; Liu, Shu-Ting; Liu, Pei-Yao; Chou, Wei-Yuan; Lin, Wei-Shiang

    2014-02-01

    Caffeine causes a diverse range of pharmacological effects that are time- and concentration-dependent and reversible. The detailed mechanisms of caffeine in tumor suppression via tumor suppressor protein p53 remain unclear. The isoforms of p53 are physiological proteins that are expressed in normal cells and generated via alternative promoters, splicing sites and/or translational initiation sites. In this study, we investigated how caffeine modulated cell cycle arrest and apoptosis via the expression of various alternatively spliced p53 isoforms. Caffeine reduced p53α expression and induced the expression of p53β, which contains an alternatively spliced p53 C-terminus. In HeLa cells, the expression levels of many serine/arginine-rich splicing factors, including serine/arginine-rich splicing factors 2 and 3, were altered by caffeine. Serine/arginine-rich splicing factor 3 was a promising candidate for the serine/arginine-rich splicing factors responsible for the alternative splicing of p53 in response to caffeine treatment. In addition to p53-dependent functions, multiple target genes of serine/arginine-rich splicing factor 3 suggest that caffeine can regulate epithelial-mesenchymal-transition and hypoxic conditions to inhibit the survival of tumor cells. In summary, our data provide a new pathway of caffeine-modulated tumor suppression via the alternative splicing of the target genes of serine/arginine-rich splicing factor 3.

  18. Alternate splicing of transcripts shape macrophage response to Mycobacterium tuberculosis infection

    PubMed Central

    Kalam, Haroon; Fontana, Mary F.

    2017-01-01

    Transcriptional reprogramming of macrophages upon Mycobacterium tuberculosis (Mtb) infection is widely studied; however, the significance of alternate splicing (AS) in shaping cellular responses to mycobacterial infections is not yet appreciated. Alternate splicing can influence transcript stability or structure, function and localization of corresponding proteins thereby altering protein stoichiometry and physiological consequences. Using comprehensive analysis of a time-series RNA-seq data obtained from human macrophages infected with virulent or avirulent strains of Mtb, we show extensive remodeling of alternate splicing in macrophage transcriptome. The global nature of this regulation was evident since genes belonging to functional classes like trafficking, immune response, autophagy, redox and metabolism showed marked departure in the pattern of splicing in the infected macrophages. The systemic perturbation of splicing machinery in the infected macrophages was apparent as genes involved at different stages of spliceosome assembly were also regulated at the splicing level. Curiously there was a considerable increase in the expression of truncated/non-translatable variants of several genes, specifically upon virulent infections. Increased expression of truncated transcripts correlated with a decline in the corresponding protein levels. We verified the physiological relevance for one such candidate gene RAB8B; whose truncated variant gets enriched in H37Rv infected cells. Upon tweaking relative abundance of longer or shorter variants of RAB8B transcripts by specialized transduction, mycobacterial targeting to lysosomes could be promoted or blocked respectively, which also resulted in corresponding changes in the bacterial survival. Our results show RAB8B recruitment to the mycobacterial phagosomes is required for phagosome maturation. Thus the abundance of truncated RAB8B variant helps virulent Mtb survival by limiting the RAB8B levels in the cells, a mechanism

  19. Control of alternative splicing by forskolin through hnRNP K during neuronal differentiation.

    PubMed

    Cao, Wenguang; Razanau, Aleh; Feng, Dairong; Lobo, Vincent G; Xie, Jiuyong

    2012-09-01

    The molecular basis of cell signal-regulated alternative splicing at the 3' splice site remains largely unknown. We isolated a protein kinase A-responsive ribonucleic acid (RNA) element from a 3' splice site of the synaptosomal-associated protein 25 (Snap25) gene for forskolin-inhibited splicing during neuronal differentiation of rat pheochromocytoma PC12 cells. The element binds specifically to heterogeneous nuclear ribonucleo protein (hnRNP) K in a phosphatase-sensitive way, which directly competes with the U2 auxiliary factor U2AF65, an essential component of early spliceosomes. Transcripts with similarly localized hnRNP K target motifs upstream of alternative exons are enriched in genes often associated with neurological diseases. We show that such motifs upstream of the Runx1 exon 6 also bind hnRNP K, and importantly, hnRNP K is required for forskolin-induced repression of the exon. Interestingly, this exon encodes the peptide domain that determines the switch of the transcriptional repressor/activator activity of Runx1, a change known to be critical in specifying neuron lineages. Consistent with an important role of the target genes in neurons, knocking down hnRNP K severely disrupts forskolin-induced neurite growth. Thus, through hnRNP K, the neuronal differentiation stimulus forskolin targets a critical 3' splice site component of the splicing machinery to control alternative splicing of crucial genes. This also provides a regulated direct competitor of U2AF65 for cell signal control of 3' splice site usage.

  20. Meta-Analysis of Multiple Sclerosis Microarray Data Reveals Dysregulation in RNA Splicing Regulatory Genes.

    PubMed

    Paraboschi, Elvezia Maria; Cardamone, Giulia; Rimoldi, Valeria; Gemmati, Donato; Spreafico, Marta; Duga, Stefano; Soldà, Giulia; Asselta, Rosanna

    2015-09-30

    Abnormalities in RNA metabolism and alternative splicing (AS) are emerging as important players in complex disease phenotypes. In particular, accumulating evidence suggests the existence of pathogenic links between multiple sclerosis (MS) and altered AS, including functional studies showing that an imbalance in alternatively-spliced isoforms may contribute to disease etiology. Here, we tested whether the altered expression of AS-related genes represents a MS-specific signature. A comprehensive comparative analysis of gene expression profiles of publicly-available microarray datasets (190 MS cases, 182 controls), followed by gene-ontology enrichment analysis, highlighted a significant enrichment for differentially-expressed genes involved in RNA metabolism/AS. In detail, a total of 17 genes were found to be differentially expressed in MS in multiple datasets, with CELF1 being dysregulated in five out of seven studies. We confirmed CELF1 downregulation in MS (p=0.0015) by real-time RT-PCRs on RNA extracted from blood cells of 30 cases and 30 controls. As a proof of concept, we experimentally verified the unbalance in alternatively-spliced isoforms in MS of the NFAT5 gene, a putative CELF1 target. In conclusion, for the first time we provide evidence of a consistent dysregulation of splicing-related genes in MS and we discuss its possible implications in modulating specific AS events in MS susceptibility genes.

  1. Regulation of transcription of the RNA splicing factor hSlu7 by Elk-1 and Sp1 affects alternative splicing.

    PubMed

    Alberstein, Moti; Amit, Maayan; Vaknin, Keren; O'Donnell, Amanda; Farhy, Chen; Lerenthal, Yaniv; Shomron, Noam; Shaham, Ohad; Sharrocks, Andrew D; Ashery-Padan, Ruth; Ast, Gil

    2007-11-01

    Alternative splicing plays a major role in transcriptome diversity and plasticity, but it is largely unknown how tissue-specific and embryogenesis-specific alternative splicing is regulated. The highly conserved splicing factor Slu7 is involved in 3' splice site selection and also regulates alternative splicing. We show that Slu7 has a unique spatial pattern of expression among human and mouse embryonic and adult tissues. We identified several functional Ets binding sites and GC-boxes in the human Slu7 (hSlu7) promoter region. The Ets and GC-box binding transcription factors, Elk-1 and Sp1, respectively, exerted opposite effects on hSlu7 transcription: Sp1 protein enhances and Elk-1 protein represses transcription in a dose-dependent manner. Sp1 protein bound to the hSlu7 promoter in vivo, and depletion of Sp1 by RNA interference (RNAi) repressed hSlu7 expression. Elk-1 protein bound to the hSlu7 promoter in vivo, and depletion of Elk-1 by RNAi caused an increase in the endogenous level of hSlu7 mRNA. Further, depletion of either Sp1 or Elk-1 affected alternative splicing. Our results provide indications of a complex transcription regulation mechanism that controls the spatial and temporal expression of Slu7, presumably allowing regulation of tissue-specific alternative splicing events.

  2. Chemotherapy induces alternative transcription and splicing: Facts and hopes for cancer treatment.

    PubMed

    Lambert, Charles A; Garbacki, Nancy; Colige, Alain C

    2017-04-20

    Alternative promoter usage, alternative splicing and alternative cleavage/polyadenylation (referred here as to alternative transcription and splicing) are main instruments to diversify the transcriptome from a limited set of genes. There is a good deal of evidence that chemotherapeutic drugs affect these processes, but the therapeutic incidence of these effects is poorly documented. The scope of this study is to review the impact of chemotherapy on alternative transcription and splicing and to discuss potential implications in cancer therapy. A literature survey identified >2200 events induced by chemotherapeutic drugs. The molecular pathways involved in these regulations are briefly discussed. The GO terms associated with the alternative transcripts are mainly related to cell cycle/division, mRNA processing, DNA repair, macromolecules catabolism and chromatin. A large fraction (43%) of transcripts are also related to the new hallmarks of cancer, mostly genetic instability and replicative immortality. Finally, we ask the question of the impact of alternative transcription and splicing on drug efficacy and of the possible curative benefit of combining chemotherapy and pharmaceutical regulation of this process. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Alternative splicing of a group II intron in a surface layer protein gene in Clostridium tetani.

    PubMed

    McNeil, Bonnie A; Simon, Dawn M; Zimmerly, Steven

    2014-02-01

    Group II introns are ribozymes and retroelements found in bacteria, and are thought to have been the ancestors of nuclear pre-mRNA introns. Whereas nuclear introns undergo prolific alternative splicing in some species, group II introns are not known to carry out equivalent reactions. Here we report a group II intron in the human pathogen Clostridium tetani, which undergoes four alternative splicing reactions in vivo. Together with unspliced transcript, five mRNAs are produced, each encoding a distinct surface layer protein isoform. Correct fusion of exon reading frames requires a shifted 5' splice site located 8 nt upstream of the canonical boundary motif. The shifted junction is accomplished by an altered IBS1-EBS1 pairing between the intron and 5' exon. Growth of C. tetani under a variety of conditions did not result in large changes in alternative splicing levels, raising the possibility that alternative splicing is constitutive. This work demonstrates a novel type of gene organization and regulation in bacteria, and provides an additional parallel between group II and nuclear pre-mRNA introns.

  4. Rbfox3-regulated alternative splicing of Numb promotes neuronal differentiation during development

    PubMed Central

    Kim, Kee K.; Nam, Joseph

    2013-01-01

    Alternative premRNA splicing is a major mechanism to generate diversity of gene products. However, the biological roles of alternative splicing during development remain elusive. Here, we focus on a neuron-specific RNA-binding protein, Rbfox3, recently identified as the antigen of the widely used anti-NeuN antibody. siRNA-mediated loss-of-function studies using the developing chicken spinal cord revealed that Rbfox3 is required to promote neuronal differentiation of postmitotic neurons. Numb premRNA encoding a signaling adaptor protein was found to be a target of Rbfox3 action, and Rbfox3 repressed the inclusion of an alternative exon via binding to the conserved UGCAUG element in the upstream intron. Depleting a specific Numb splice isoform reproduced similar neuronal differentiation defects. Forced expression of the relevant Numb splice isoform was sufficient to rescue, in an isoform-specific manner, postmitotic neurons from defects in differentiation caused by Rbfox3 depletion. Thus, Rbfox3-dependent Numb alternative splicing plays an important role in the progression of neuronal differentiation during vertebrate development. PMID:23420872

  5. Alternative splicing of a group II intron in a surface layer protein gene in Clostridium tetani

    PubMed Central

    McNeil, Bonnie A.; Simon, Dawn M.; Zimmerly, Steven

    2014-01-01

    Group II introns are ribozymes and retroelements found in bacteria, and are thought to have been the ancestors of nuclear pre-mRNA introns. Whereas nuclear introns undergo prolific alternative splicing in some species, group II introns are not known to carry out equivalent reactions. Here we report a group II intron in the human pathogen Clostridium tetani, which undergoes four alternative splicing reactions in vivo. Together with unspliced transcript, five mRNAs are produced, each encoding a distinct surface layer protein isoform. Correct fusion of exon reading frames requires a shifted 5′ splice site located 8 nt upstream of the canonical boundary motif. The shifted junction is accomplished by an altered IBS1-EBS1 pairing between the intron and 5′ exon. Growth of C. tetani under a variety of conditions did not result in large changes in alternative splicing levels, raising the possibility that alternative splicing is constitutive. This work demonstrates a novel type of gene organization and regulation in bacteria, and provides an additional parallel between group II and nuclear pre-mRNA introns. PMID:24214997

  6. Regulation of Dscam exon 17 alternative splicing by steric hindrance in combination with RNA secondary structures.

    PubMed

    Yue, Yuan; Li, Guoli; Yang, Yun; Zhang, Wenjing; Pan, Huawei; Chen, Ran; Shi, Feng; Jin, Yongfeng

    2013-12-01

    The gene Down syndrome cell adhesion molecule (Dscam) potentially encodes 38 016 distinct isoforms in Drosophila melanogaster via mutually exclusive splicing. Here we reveal a combinatorial mechanism of regulation of Dscam exon 17 mutually exclusive splicing through steric hindrance in combination with RNA secondary structure. This mutually exclusive behavior is enforced by steric hindrance, due to the close proximity of the exon 17.2 branch point to exon 17.1 in Diptera, and the interval size constraint in non-Dipteran species. Moreover, intron-exon RNA structures are evolutionarily conserved in 36 non-Drosophila species of six distantly related orders (Diptera, Lepidoptera, Coleoptera, Hymenoptera, Hemiptera, and Phthiraptera), which regulates the selection of exon 17 variants via masking the splice site. By contrast, a previously uncharacterized RNA structure specifically activated exon 17.1 by bringing splice sites closer together in Drosophila, while the other moderately suppressed exon 17.1 selection by hindering the accessibility of polypyrimidine sequences. Taken together, these data suggest a phylogeny of increased complexity in regulating alternative splicing of Dscam exon 17 spanning more than 300 million years of insect evolution. These results also provide models of the regulation of alternative splicing through steric hindrance in combination with dynamic structural codes.

  7. Regulation of Dscam exon 17 alternative splicing by steric hindrance in combination with RNA secondary structures

    PubMed Central

    Yue, Yuan; Li, Guoli; Yang, Yun; Zhang, Wenjing; Pan, Huawei; Chen, Ran; Shi, Feng; Jin, Yongfeng

    2013-01-01

    The gene Down syndrome cell adhesion molecule (Dscam) potentially encodes 38 016 distinct isoforms in Drosophila melanogaster via mutually exclusive splicing. Here we reveal a combinatorial mechanism of regulation of Dscam exon 17 mutually exclusive splicing through steric hindrance in combination with RNA secondary structure. This mutually exclusive behavior is enforced by steric hindrance, due to the close proximity of the exon 17.2 branch point to exon 17.1 in Diptera, and the interval size constraint in non-Dipteran species. Moreover, intron-exon RNA structures are evolutionarily conserved in 36 non-Drosophila species of six distantly related orders (Diptera, Lepidoptera, Coleoptera, Hymenoptera, Hemiptera, and Phthiraptera), which regulates the selection of exon 17 variants via masking the splice site. By contrast, a previously uncharacterized RNA structure specifically activated exon 17.1 by bringing splice sites closer together in Drosophila, while the other moderately suppressed exon 17.1 selection by hindering the accessibility of polypyrimidine sequences. Taken together, these data suggest a phylogeny of increased complexity in regulating alternative splicing of Dscam exon 17 spanning more than 300 million years of insect evolution. These results also provide models of the regulation of alternative splicing through steric hindrance in combination with dynamic structural codes. PMID:24448213

  8. Identification of interleukin-26 in the dromedary camel (Camelus dromedarius): Evidence of alternative splicing and isolation of novel splice variants.

    PubMed

    Premraj, Avinash; Nautiyal, Binita; Aleyas, Abi G; Rasool, Thaha Jamal

    2015-10-01

    Interleukin-26 (IL-26) is a member of the IL-10 family of cytokines. Though conserved across vertebrates, the IL-26 gene is functionally inactivated in a few mammals like rat, mouse and horse. We report here the identification, isolation and cloning of the cDNA of IL-26 from the dromedary camel. The camel cDNA contains a 516 bp open reading frame encoding a 171 amino acid precursor protein, including a 21 amino acid signal peptide. Sequence analysis revealed high similarity with other mammalian IL-26 homologs and the conservation of IL-10 cytokine family domain structure including key amino acid residues. We also report the identification and cloning of four novel transcript variants produced by alternative splicing at the Exon 3-Exon 4 regions of the gene. Three of the alternative splice variants had premature termination codons and are predicted to code for truncated proteins. The transcript variant 4 (Tv4) having an insertion of an extra 120 bp nucleotides in the ORF was predicted to encode a full length protein product with 40 extra amino acid residues. The mRNA transcripts of all the variants were identified in lymph node, where as fewer variants were observed in other tissues like blood, liver and kidney. The expression of Tv2 and Tv3 were found to be up regulated in mitogen induced camel peripheral blood mononuclear cells. IL-26-Tv2 expression was also induced in camel fibroblast cells infected with Camel pox virus in-vitro. The identification of the transcript variants of IL-26 from the dromedary camel is the first report of alternative splicing for IL-26 in a species in which the gene has not been inactivated. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Dynamic regulation of alternative splicing and chromatin structure in Drosophila gonads revealed by RNA-seq

    PubMed Central

    Gan, Qiang; Chepelev, Iouri; Wei, Gang; Tarayrah, Lama; Cui, Kairong; Zhao, Keji; Chen, Xin

    2010-01-01

    Both transcription and post-transcriptional processes, such as alternative splicing, play crucial roles in controlling developmental programs in metazoans. Recently emerged RNA-seq method has brought our understandings of eukaryotic transcriptomes to a new level, because it can resolve both gene expression level and alternative splicing events simultaneously. To gain a better understanding of cellular differentiation in gonads, we analyzed mRNA profiles from Drosophila testes and ovaries using RNA-seq. We identified a set of genes that have sex-specific isoforms in wild-type (wt) gonads, including several transcription factors. We found that differentiation of sperms from undifferentiated germ cells induced a dramatic down-regulation of RNA splicing factors. Our data confirmed that RNA splicing events are significantly more frequent in the undifferentiated-cell enriched bag of marbles (bam) mutant testis, but down-regulated upon differentiation in wt testis. Consistent with this, we showed that genes required for meiosis and terminal differentiation in wt testis were mainly regulated at the transcriptional level, but not by alternative splicing. Unexpectedly, we observed an increase in expression of all families of chromatin remodeling factors and histone modifying enzymes in the undifferentiated cell-enriched bam testis. More interestingly, chromatin regulators and histone modifying enzymes with opposite enzymatic activities are co-enriched in undifferentiated cells in testis, suggesting these cells may possess dynamic chromatin architecture. Finally, our data revealed many new features of the Drosophila gonadal transcriptomes, and will lead to a more comprehensive understanding of how differential gene expression and splicing regulate gametogenesis in Drosophila. Our data provided a foundation for the systematic study of gene expression and alternative splicing in many interesting areas of germ cell biology in Drosophila, such as the molecular basis for sexual

  10. Distinct roles of two alternative splice variants of matrilin-2 in protein oligomerization and proteolysis.

    PubMed

    Li, Longxuan; Zhang, Liangqing; Shao, Yiming; Wang, Guirong; Gong, Rujun; Wang, Zhengke; Peng, Jinwu; Wang, Shuhua; Genochio, David; Zhao, Bin; Luo, Junming

    2012-11-01

    Matrilin-2 (matn2) contains a unique domain, between the second von Willebrand factor A (vWFA) domain and the C-terminal coiled-coil domain, with no sequence homology with other family members. Complementary DNA (cDNA) sequence analysis of matn2 expression in both mice and humans revealed an alternative splice site in the region of the unique domain, which forms a short and a long splicing variant (containing an additional 19 amino acids). However, the expression heterogeneity of the alternative spliced variants, and the roles of the unique domain in oligomerization and proteolysis of matn2 are unknown. In this study, we examined the expression of the two alternative splice variants of matn2 in several skeletal and non-skeletal tissues by reverse transcription-polymerase chain reaction. Both splice variants of matn2 were detected at the mRNA level in all tissues studied. To explore the biochemical significance, several minigene constructs containing the second vWFA domain, the unique domain (with either a long or short form) and the coiled-coil domain of mouse mini matn2 were generated. Ectopic expression of these constructs demonstrated that the long form of matn2 is capable of self-assembling into several oligomeric forms, including a tetramer, trimer, pentamer or multimer; but the short form is only capable of forming a tetramer, trimer or dimer. Moreover, we observed that the splice variants of matn2 are important in modulating matn2 cleavage when co-expressed with matrilin-1 or matrilin-3. These results indicate that the two alternative splice variants have distinct roles in the processes of post-translational modification of matn2, which may have an impact on the homeostasis of the matrilin filamentous network of the extracellular matrix.

  11. Alternative RNA splicing of KSHV ORF57 produces two different RNA isoforms.

    PubMed

    Majerciak, Vladimir; Zheng, Zhi-Ming

    2016-01-15

    In lytically infected B cells Kaposi sarcoma-associated herpesvirus (KSHV) ORF57 gene encodes two RNA isoforms by alternative splicing of its pre-mRNA, which contains a small, constitutive intron in its 5' half and a large, suboptimal intron in its 3's half. The RNA1 isoform encodes full-length ORF57 and is a major isoform derived from splicing of the constitutive small intron, but retaining the suboptimal large intron as the coding region. A small fraction (<5%) of ORF57 RNA undergoes double splicing to produce a smaller non-coding RNA2 due to lack of a translational termination codon. Both RNAs are cleaved and polyadenylated at the same cleavage site CS83636. The insertion of ORF57 RNA1 into a restriction cutting site in certain mammalian expression vectors activates splicing of the subopitmal intron and produces a truncated ORF57 protein.

  12. Alternative transcription exceeds alternative splicing in generating the transcriptome diversity of cerebellar development.

    PubMed

    Pal, Sharmistha; Gupta, Ravi; Kim, Hyunsoo; Wickramasinghe, Priyankara; Baubet, Valérie; Showe, Louise C; Dahmane, Nadia; Davuluri, Ramana V

    2011-08-01

    Despite our growing knowledge that many mammalian genes generate multiple transcript variants that may encode functionally distinct protein isoforms, the transcriptomes of various tissues and their developmental stages are poorly defined. Identifying the transcriptome and its regulation in a cell/tissue is the key to deciphering the cell/tissue-specific functions of a gene. We built a genome-wide inventory of noncoding and protein-coding transcripts (transcriptomes), their promoters (promoteromes) and histone modification states (epigenomes) for developing, and adult cerebella using integrative massive-parallel sequencing and bioinformatics approach. The data consists of 61,525 (12,796 novel) distinct mRNAs transcribed by 29,589 (4792 novel) promoters corresponding to 15,669 protein-coding and 7624 noncoding genes. Importantly, our results show that the transcript variants from a gene are predominantly generated using alternative transcriptional rather than splicing mechanisms, highlighting alternative promoters and transcriptional terminations as major sources of transcriptome diversity. Moreover, H3K4me3, and not H3K27me3, defined the use of alternative promoters, and we identified a combinatorial role of H3K4me3 and H3K27me3 in regulating the expression of transcripts, including transcript variants of a gene during development. We observed a strong bias of both H3K4me3 and H3K27me3 for CpG-rich promoters and an exponential relationship between their enrichment and corresponding transcript expression. Furthermore, the majority of genes associated with neurological diseases expressed multiple transcripts through alternative promoters, and we demonstrated aberrant use of alternative promoters in medulloblastoma, cancer arising in the cerebellum. The transcriptomes of developing and adult cerebella presented in this study emphasize the importance of analyzing gene regulation and function at the isoform level.

  13. Identification of a cis element for tissue-specific alternative splicing of chloroplast ascorbate peroxidase pre-mRNA in higher plants.

    PubMed

    Yoshimura, Kazuya; Yabuta, Yukinori; Ishikawa, Takahiro; Shigeoka, Shigeru

    2002-10-25

    Alternative splicing events in the 3'-terminal region of chloroplast ascorbate peroxidase (chlAPX) pre-mRNA in spinach and tobacco, which produced four types of mRNA variants, one form (tAPX-I) encoding thylakoid-bound APX (tAPX) and three forms (sAPX-I, -II, and -III) encoding stromal APX (sAPX), were regulated in a tissue-specific manner. The ratio of the level of sAPX mRNAs (sAPX-I, -II, and -III) to tAPX-I mRNA was close to 1 in leaf, whereas the ratio in root was greatly elevated due to an increase in sAPX-III and a decrease in tAPX-I resulting from the alternative excision of intron 11 and intron 12, respectively. A putative splicing regulatory cis element (SRE), which is highly conserved in the sequences of chlAPX genes of higher plants, was identified upstream of the acceptor site in intron 12. The deletion of the SRE sequence diminished the splicing efficiency of intron 12 in tobacco leaf in vivo. Gel-shift analysis showed that SRE interacts strongly with a nuclear protein from leaves but not those from the roots of spinach and tobacco. These results indicate that the tissue-specific alternative splicing of chlAPX pre-mRNA is regulated by the splicing enhancer SRE.

  14. Identification of alternative splicing events regulated by the oncogenic factor SRSF1 in lung cancer.

    PubMed

    de Miguel, Fernando J; Sharma, Ravi D; Pajares, María J; Montuenga, Luis M; Rubio, Angel; Pio, Ruben

    2014-02-15

    Abnormal alternative splicing has been associated with cancer. Genome-wide microarrays can be used to detect differential splicing events. In this study, we have developed ExonPointer, an algorithm that uses data from exon and junction probes to identify annotated cassette exons. We used the algorithm to profile differential splicing events in lung adenocarcinoma A549 cells after downregulation of the oncogenic serine/arginine-rich splicing factor 1 (SRSF1). Data were generated using two different microarray platforms. The PCR-based validation rate of the top 20 ranked genes was 60% and 100%. Functional enrichment analyses found a substantial number of splicing events in genes related to RNA metabolism. These analyses also identified genes associated with cancer and developmental and hereditary disorders, as well as biologic processes such as cell division, apoptosis, and proliferation. Most of the top 20 ranked genes were validated in other adenocarcinoma and squamous cell lung cancer cells, with validation rates of 80% to 95% and 70% to 75%, respectively. Moreover, the analysis allowed us to identify four genes, ATP11C, IQCB1, TUBD1, and proline-rich coiled-coil 2C (PRRC2C), with a significantly different pattern of alternative splicing in primary non-small cell lung tumors compared with normal lung tissue. In the case of PRRC2C, SRSF1 downregulation led to the skipping of an exon overexpressed in primary lung tumors. Specific siRNA downregulation of the exon-containing variant significantly reduced cell growth. In conclusion, using a novel analytical tool, we have identified new splicing events regulated by the oncogenic splicing factor SRSF1 in lung cancer.

  15. Hypoxia regulates alternative splicing of HIF and non-HIF target genes.

    PubMed

    Sena, Johnny A; Wang, Liyi; Heasley, Lynn E; Hu, Cheng-Jun

    2014-09-01

    Hypoxia is a common characteristic of many solid tumors. The hypoxic microenvironment stabilizes hypoxia-inducible transcription factor 1α (HIF1α) and 2α (HIF2α/EPAS1) to activate gene transcription, which promotes tumor cell survival. The majority of human genes are alternatively spliced, producing RNA isoforms that code for functionally distinct proteins. Thus, an effective hypoxia response requires increased HIF target gene expression as well as proper RNA splicing of these HIF-dependent transcripts. However, it is unclear if and how hypoxia regulates RNA splicing of HIF targets. This study determined the effects of hypoxia on alternative splicing (AS) of HIF and non-HIF target genes in hepatocellular carcinoma cells and characterized the role of HIF in regulating AS of HIF-induced genes. The results indicate that hypoxia generally promotes exon inclusion for hypoxia-induced, but reduces exon inclusion for hypoxia-reduced genes. Mechanistically, HIF activity, but not hypoxia per se is found to be necessary and sufficient to increase exon inclusion of several HIF targets, including pyruvate dehydrogenase kinase 1 (PDK1). PDK1 splicing reporters confirm that transcriptional activation by HIF is sufficient to increase exon inclusion of PDK1 splicing reporter. In contrast, transcriptional activation of a PDK1 minigene by other transcription factors in the absence of endogenous HIF target gene activation fails to alter PDK1 RNA splicing. This study demonstrates a novel function of HIF in regulating RNA splicing of HIF target genes. ©2014 American Association for Cancer Research.

  16. RNA splicing during terminal erythropoiesis.

    PubMed

    Conboy, John G

    2017-05-01

    Erythroid progenitors must accurately and efficiently splice thousands of pre-mRNAs as the cells undergo extensive changes in gene expression and cellular remodeling during terminal erythropoiesis. Alternative splicing choices are governed by interactions between RNA binding proteins and cis-regulatory binding motifs in the RNA. This review will focus on recent studies that define the genome-wide scope of splicing in erythroblasts and discuss what is known about its regulation. RNA-seq analysis of highly purified erythroblast populations has revealed an extensive program of alternative splicing of both exons and introns. During normal erythropoiesis, stage-specific splicing transitions alter the structure and abundance of protein isoforms required for optimized red cell production. Mutation or deficiency of splicing regulators underlies hematopoietic disease in myelopdysplasia syndrome patients via disrupting the splicing program. Erythroid progenitors execute an elaborate alternative splicing program that modulates gene expression posttranscriptionally, ultimately regulating the structure and function of the proteome in a differentiation stage-specific manner during terminal erythropoiesis. This program helps drive differentiation and ensure synthesis of the proper protein isoforms required to produce mechanically stable red cells. Mutation or deficiency of key splicing regulatory proteins disrupts the splicing program to cause disease.

  17. The Ski2-family helicase Obelus regulates Crumbs alternative splicing and cell polarity

    PubMed Central

    Vichas, Athea; Laurie, Matthew T.

    2015-01-01

    Alternative splicing can have profound consequences for protein activity, but the functions of most alternative splicing regulators are not known. We show that Obelus, a conserved Ski2-family helicase, is required for cell polarity and adherens junction organization in the Drosophila melanogaster embryo. In obelus mutants, epithelial cells display an expanded apical domain, aggregation of adherens junctions at the cell membrane, and microtubule-dependent defects in centrosome positioning. Through whole-genome transcriptome analysis, we found that Obelus is required for the alternative splicing of a small number of transcripts in the early embryo, including the pre-mRNA that encodes the apical polarity protein Crumbs. In obelus mutants, inclusion of an alternative exon results in increased expression of a Crumbs isoform that contains an additional epidermal growth factor–like repeat in the extracellular domain. Overexpression of this alternative Crumbs isoform recapitulates the junctional aggregation and centrosome positioning defects of obelus mutants. These results indicate that regulation of Crumbs alternative splicing by the Obelus helicase modulates epithelial polarity during development. PMID:26644515

  18. Optimization of oligonucleotide arrays and RNA amplification protocols for analysis of transcript structure and alternative splicing

    PubMed Central

    Castle, John; Garrett-Engele, Phil; Armour, Christopher D; Duenwald, Sven J; Loerch, Patrick M; Meyer, Michael R; Schadt, Eric E; Stoughton, Roland; Parrish, Mark L; Shoemaker, Daniel D; Johnson, Jason M

    2003-01-01

    Microarrays offer a high-resolution means for monitoring pre-mRNA splicing on a genomic scale. We have developed a novel, unbiased amplification protocol that permits labeling of entire transcripts. Also, hybridization conditions, probe characteristics, and analysis algorithms were optimized for detection of exons, exon-intron edges, and exon junctions. These optimized protocols can be used to detect small variations and isoform mixtures, map the tissue specificity of known human alternative isoforms, and provide a robust, scalable platform for high-throughput discovery of alternative splicing. PMID:14519201

  19. RNA Polymerase II Elongation at the Crossroads of Transcription and Alternative Splicing

    PubMed Central

    de la Mata, Manuel; Muñoz, Manuel J.; Alló, Mariano; Fededa, Juan Pablo; Schor, Ignacio E.; Kornblihtt, Alberto R.

    2011-01-01

    The elongation phase of transcription lies at the core of several simultaneous and coupled events leading to alternative splicing regulation. Although underestimated in the past, it is at this phase of the transcription cycle where complexes affecting the transcription machinery itself, chromatin structure, posttranscriptional gene regulation and pre-mRNA processing converge to regulate each other or simply to consolidate higher-order complexes and functions. This paper focuses on the multiple processes that take place during transcription elongation which ultimately regulate the outcome of alternative splicing decisions. PMID:22567350

  20. General splicing factors SF2 and SC35 have equivalent activities in vitro, and both affect alternative 5' and 3' splice site selection.

    PubMed Central

    Fu, X D; Mayeda, A; Maniatis, T; Krainer, A R

    1992-01-01

    The human pre-mRNA splicing factors SF2 and SC35 have similar electrophoretic mobilities, and both of them contain an N-terminal ribonucleoprotein (RNP)-type RNA-recognition motif and a C-terminal arginine/serine-rich domain. However, the two proteins are encoded by different genes and display only 31% amino acid sequence identity. Here we report a systematic comparison of the splicing activities of recombinant SF2 and SC35. We find that either protein can reconstitute the splicing activity of S100 extracts and of SC35-immunodepleted nuclear extracts. Previous studies revealed that SF2 influences alternative 5' splice site selection in vitro, by favoring proximal over distal 5' splice sites, and that the A1 protein of heterogeneous nuclear RNP counteracts this effect. We now show that SC35 has a similar effect on competing 5' splice sites and is also antagonized by A1 protein. In addition, we report that both SF2 and SC35 also favor the proximal site in a pre-mRNA containing duplicated 3' splice sites, but this effect is not modulated by A1. We conclude that SF2 and SC35 are distinct splicing factors, but they display indistinguishable splicing activities in vitro. Images PMID:1454802

  1. Cell Type-specific Alternative Splicing Governs Cell Fate in the Developing Cerebral Cortex

    PubMed Central

    Zhang, Xiaochang; Chen, Ming Hui; Wu, Xuebing; Kodani, Andrew; Fan, Jean; Doan, Ryan; Ozawa, Manabu; Ma, Jacqueline; Yoshida, Nobuaki; Reiter, Jeremy F.; Black, Douglas L.; Kharchenko, Peter V.; Sharp, Phillip A.; Walsh, Christopher A.

    2017-01-01

    SUMMARY Alternative splicing is prevalent in the mammalian brain. To interrogate the functional role of alternative splicing in neural development, we analyzed purified neural progenitor cells (NPCs) and neurons from developing cerebral cortices, revealing hundreds of differentially spliced exons that preferentially alter key protein domains—especially in cytoskeletal proteins—and can harbor disease-causing mutations. We show that Ptbp1 and Rbfox proteins antagonistically govern the NPC-to-neuron transition by regulating neuron-specific exons. While Ptbp1 maintains apical progenitors partly through suppressing a poison exon of Flna in NPCs, Rbfox proteins promote neuronal differentiation by switching Ninein from a centrosomal splice form in NPCs to a non-centrosomal isoform in neurons. We further uncover an intronic human mutation within a PTBP1 binding site that disrupts normal skipping of the FLNA poison exon in NPCs and causes a brain-specific malformation. Our study indicates that dynamic control of alternative splicing governs cell fate in cerebral cortical development. PMID:27565344

  2. TET-catalyzed oxidation of intragenic 5-methylcytosine regulates CTCF-dependent alternative splicing.

    PubMed

    Marina, Ryan J; Sturgill, David; Bailly, Marc A; Thenoz, Morgan; Varma, Garima; Prigge, Maria F; Nanan, Kyster K; Shukla, Sanjeev; Haque, Nazmul; Oberdoerffer, Shalini

    2016-02-01

    Intragenic 5-methylcytosine and CTCF mediate opposing effects on pre-mRNA splicing: CTCF promotes inclusion of weak upstream exons through RNA polymerase II pausing, whereas 5-methylcytosine evicts CTCF, leading to exon exclusion. However, the mechanisms governing dynamic DNA methylation at CTCF-binding sites were unclear. Here, we reveal the methylcytosine dioxygenases TET1 and TET2 as active regulators of CTCF-mediated alternative splicing through conversion of 5-methylcytosine to its oxidation derivatives. 5-hydroxymethylcytosine and 5-carboxylcytosine are enriched at an intragenic CTCF-binding sites in the CD45 model gene and are associated with alternative exon inclusion. Reduced TET levels culminate in increased 5-methylcytosine, resulting in CTCF eviction and exon exclusion. In vitro analyses establish the oxidation derivatives are not sufficient to stimulate splicing, but efficiently promote CTCF association. We further show genomewide that reciprocal exchange of 5-hydroxymethylcytosine and 5-methylcytosine at downstream CTCF-binding sites is a general feature of alternative splicing in naïve and activated CD4(+) T cells. These findings significantly expand our current concept of the pre-mRNA "splicing code" to include dynamic intragenic DNA methylation catalyzed by the TET proteins.

  3. Alternative Splicing within and between Drosophila Species, Sexes, Tissues, and Developmental Stages

    PubMed Central

    Gibilisco, Lauren; Zhou, Qi; Mahajan, Shivani; Bachtrog, Doris

    2016-01-01

    Alternative pre-mRNA splicing (“AS”) greatly expands proteome diversity, but little is known about the evolutionary landscape of AS in Drosophila and how it differs between embryonic and adult stages or males and females. Here we study the transcriptomes from several tissues and developmental stages in males and females from four species across the Drosophila genus. We find that 20–37% of multi-exon genes are alternatively spliced. While males generally express a larger number of genes, AS is more prevalent in females, suggesting that the sexes adopt different expression strategies for their specialized function. While the number of total genes expressed increases during early embryonic development, the proportion of expressed genes that are alternatively spliced is highest in the very early embryo, before the onset of zygotic transcription. This indicates that females deposit a diversity of isoforms into the egg, consistent with abundant AS found in ovary. Cluster analysis by gene expression (“GE”) levels shows mostly stage-specific clustering in embryonic samples, and tissue-specific clustering in adult tissues. Clustering embryonic stages and adult tissues based on AS profiles results in stronger species-specific clustering, suggesting that diversification of splicing contributes to lineage-specific evolution in Drosophila. Most sex-biased AS found in flies is due to AS in gonads, with little sex-specific splicing in somatic tissues. PMID:27935948

  4. Alternative Splicing within and between Drosophila Species, Sexes, Tissues, and Developmental Stages.

    PubMed

    Gibilisco, Lauren; Zhou, Qi; Mahajan, Shivani; Bachtrog, Doris

    2016-12-01

    Alternative pre-mRNA splicing ("AS") greatly expands proteome diversity, but little is known about the evolutionary landscape of AS in Drosophila and how it differs between embryonic and adult stages or males and females. Here we study the transcriptomes from several tissues and developmental stages in males and females from four species across the Drosophila genus. We find that 20-37% of multi-exon genes are alternatively spliced. While males generally express a larger number of genes, AS is more prevalent in females, suggesting that the sexes adopt different expression strategies for their specialized function. While the number of total genes expressed increases during early embryonic development, the proportion of expressed genes that are alternatively spliced is highest in the very early embryo, before the onset of zygotic transcription. This indicates that females deposit a diversity of isoforms into the egg, consistent with abundant AS found in ovary. Cluster analysis by gene expression ("GE") levels shows mostly stage-specific clustering in embryonic samples, and tissue-specific clustering in adult tissues. Clustering embryonic stages and adult tissues based on AS profiles results in stronger species-specific clustering, suggesting that diversification of splicing contributes to lineage-specific evolution in Drosophila. Most sex-biased AS found in flies is due to AS in gonads, with little sex-specific splicing in somatic tissues.

  5. Altered motor activity of alternative splice variants of the mammalian kinesin-3 protein KIF1B.

    PubMed

    Matsushita, Masafumi; Yamamoto, Ruri; Mitsui, Keiji; Kanazawa, Hiroshi

    2009-11-01

    Several mammalian kinesin motor proteins exist as multiple isoforms that arise from alternative splicing of a single gene. However, the roles of many motor protein splice variants remain unclear. The kinesin-3 motor protein KIF1B has alternatively spliced isoforms distinguished by the presence or absence of insertion sequences in the conserved amino-terminal region of the protein. The insertions are located in the loop region containing the lysine-rich cluster, also known as the K-loop, and in the hinge region adjacent to the motor domain. To clarify the functions of these alternative splice variants of KIF1B, we examined the biochemical properties of recombinant KIF1B with and without insertion sequences. In a microtubule-dependent ATPase assay, KIF1B variants that contained both insertions had higher activity and affinity for microtubules than KIF1B variants that contained no insertions. Mutational analysis of the K-loop insertion revealed that variants with a longer insertion sequence at this site had higher activity. However, the velocity of movement in motility assays was similar between KIF1B with and without insertion sequences. Our results indicate that splicing isoforms of KIF1B that vary in their insertion sequences have different motor activities.

  6. Alternative splicing and gene duplication differentially shaped the regulation of isochorismate synthase in Populus and Arabidopsis

    PubMed Central

    Yuan, Yinan; Chung, Jeng-Der; Fu, Xueyan; Johnson, Virgil E.; Ranjan, Priya; Booth, Sarah L.; Harding, Scott A.; Tsai, Chung-Jui

    2009-01-01

    Isochorismate synthase (ICS) converts chorismate to isochorismate for the biosynthesis of phylloquinone, an essential cofactor for photosynthetic electron transport. ICS is also required for salicylic acid (SA) synthesis during Arabidopsis defense. In several other species, including Populus, SA is derived primarily from the phenylpropanoid pathway. We therefore sought to investigate ICS regulation in Populus to learn the extent of ICS involvement in SA synthesis and defense. Arabidopsis harbors duplicated AtICS genes that differ in their exon-intron structure, basal expression, and stress inducibility. In contrast, we found a single ICS gene in Populus and six other sequenced plant genomes, pointing to the AtICS duplication as a lineage-specific event. The Populus ICS encodes a functional plastidic enzyme, and was not responsive to stresses that stimulated phenylpropanoid accumulation. Populus ICS underwent extensive alternative splicing that was rare for the duplicated AtICSs. Sequencing of 184 RT-PCR Populus clones revealed 37 alternative splice variants, with normal transcripts representing ≈50% of the population. When expressed in Arabidopsis, Populus ICS again underwent alternative splicing, but did not produce normal transcripts to complement AtICS1 function. The splice-site sequences of Populus ICS are unusual, suggesting a causal link between junction sequence, alternative splicing, and ICS function. We propose that gene duplication and alternative splicing of ICS evolved independently in Arabidopsis and Populus in accordance with their distinct defense strategies. AtICS1 represents a divergent isoform for inducible SA synthesis during defense. Populus ICS primarily functions in phylloquinone biosynthesis, a process that can be sustained at low ICS transcript levels. PMID:19996170

  7. A Recently Evolved Alternative Splice Site in the BRANCHED1a Gene Controls Potato Plant Architecture.

    PubMed

    Nicolas, Michael; Rodríguez-Buey, María Luisa; Franco-Zorrilla, José Manuel; Cubas, Pilar

    2015-07-20

    Amplification and diversification of transcriptional regulators that control development is a driving force of morphological evolution. A major source of protein diversity is alternative splicing, which leads to the generation of different isoforms from a single gene. The mechanisms and timing of intron evolution nonetheless remain unclear, and the functions of alternative splicing-generated protein isoforms are rarely studied. In Solanum tuberosum, the BRANCHED1a (BRC1a) gene encodes a TCP transcription factor that controls lateral shoot outgrowth. Here, we report the recent evolution in Solanum of an alternative splice site in BRC1a that leads to the generation of two BRC1a protein isoforms with distinct C-terminal regions, BRC1a(Long) and BRC1a(Short), encoded by unspliced and spliced mRNA, respectively. The BRC1a(Long) C-terminal region has a strong activation domain, whereas that of BRC1a(S) lacks an activation domain and is predicted to form an amphipathic helix, the H domain, which prevents protein nuclear targeting. BRC1a(Short) is thus mainly cytoplasmic, while BRC1a(Long) is mainly nuclear. BRC1a(Long) functions as a transcriptional activator, whereas BRC1a(Short) appears to have no transcriptional activity. Moreover, BRC1a(Short) can heterodimerize with BRC1a(Long) and act as a dominant-negative factor; it increases BRC1a(Long) concentration in cytoplasm and reduces its transcriptional activity. This alternative splicing mechanism is regulated by hormones and external stimuli that control branching. The evolution of a new alternative splicing site and a novel protein domain in Solanum BRC1a led to a multi-level mechanism of post-transcriptional and post-translational BRC1a regulation that effectively modulates its branch suppressing activity in response to environmental and endogenous cues. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Genome-wide identification and characterization of tissue-specific RNA editing events in D. melanogaster and their potential role in regulating alternative splicing.

    PubMed

    Mazloomian, Alborz; Meyer, Irmtraud M

    2015-01-01

    RNA editing is a widespread mechanism that plays a crucial role in diversifying gene products. Its abundance and importance in regulating cellular processes were revealed using new sequencing technologies. The majority of these editing events, however, cannot be associated with regulatory mechanisms. We use tissue-specific high-throughput libraries of D. melanogaster to study RNA editing. We introduce an analysis pipeline that utilises large input data and explicitly captures ADAR's requirement for double-stranded regions. It combines probabilistic and deterministic filters and can identify RNA editing events with a low estimated false positive rate. Analyzing ten different tissue types, we predict 2879 editing sites and provide their detailed characterization. Our analysis pipeline accurately distinguishes genuine editing sites from SNPs and sequencing and mapping artifacts. Our editing sites are 3 times more likely to occur in exons with multiple splicing acceptor/donor sites than in exons with unique splice sites (p-value < 2.10(-15)). Furthermore, we identify 244 edited regions where RNA editing and alternative splicing are likely to influence each other. For 96 out of these 244 regions, we find evolutionary evidence for conserved RNA secondary-structures near splice sites suggesting a potential regulatory mechanism where RNA editing may alter splicing patterns via changes in local RNA structure.

  9. Phosphorylation inhibits DNA-binding of alternatively spliced aryl hydrocarbon receptor nuclear translocator

    SciTech Connect

    Kewley, Robyn J. . E-mail: rkewley@csu.edu.au; Whitelaw, Murray L.

    2005-12-09

    The basic helix-loop-helix/PER-ARNT-SIM homology (bHLH/PAS) transcription factor ARNT (aryl hydrocarbon receptor nuclear translocator) is a key component of various pathways which induce the transcription of cytochrome P450 and hypoxia response genes. ARNT can be alternatively spliced to express Alt ARNT, containing an additional 15 amino acids immediately N-terminal to the DNA-binding basic region. Here, we show that ARNT and Alt ARNT proteins are differentially phosphorylated by protein kinase CKII in vitro. Phosphorylation had an inhibitory effect on DNA-binding to an E-box probe by Alt ARNT, but not ARNT, homodimers. This inhibitory phosphorylation occurs through Ser77. Moreover, a point mutant, Alt ARNT S77A, shows increased activity on an E-box reporter gene, consistent with Ser77 being a regulatory site in vivo. In contrast, DNA binding by an Alt ARNT/dioxin receptor heterodimer to the xenobiotic response element is not inhibited by phosphorylation with CKII, nor does Alt ARNT S77A behave differently from wild type Alt ARNT in the context of a dioxin receptor heterodimer.

  10. Subcellular RNA profiling links splicing and nuclear DICER1 to alternative cleavage and polyadenylation

    PubMed Central

    Neve, Jonathan; Burger, Kaspar; Li, Wencheng; Hoque, Mainul; Patel, Radhika; Tian, Bin; Gullerova, Monika; Furger, Andre

    2016-01-01

    Alternative cleavage and polyadenylation (APA) plays a crucial role in the regulation of gene expression across eukaryotes. Although APA is extensively studied, its regulation within cellular compartments and its physiological impact remains largely enigmatic. Here, we used a rigorous subcellular fractionation approach to compare APA profiles of cytoplasmic and nuclear RNA fractions from human cell lines. This approach allowed us to extract APA isoforms that are subjected to differential regulation and provided us with a platform to interrogate the molecular regulatory pathways that shape APA profiles in different subcellular locations. Here, we show that APA isoforms with shorter 3′ UTRs tend to be overrepresented in the cytoplasm and appear to be cell-type–specific events. Nuclear retention of longer APA isoforms occurs and is partly a result of incomplete splicing contributing to the observed cytoplasmic bias of transcripts with shorter 3′ UTRs. We demonstrate that the endoribonuclease III, DICER1, contributes to the establishment of subcellular APA profiles not only by expected cytoplasmic miRNA-mediated destabilization of APA mRNA isoforms, but also by affecting polyadenylation site choice. PMID:26546131

  11. Alternative splicing of anciently exonized 5S rRNA regulates plant transcription factor TFIIIA.

    PubMed

    Fu, Yan; Bannach, Oliver; Chen, Hao; Teune, Jan-Hendrik; Schmitz, Axel; Steger, Gerhard; Xiong, Liming; Barbazuk, W Brad

    2009-05-01

    Identifying conserved alternative splicing (AS) events among evolutionarily distant species can prioritize AS events for functional characterization and help uncover relevant cis- and trans-regulatory factors. A genome-wide search for conserved cassette exon AS events in higher plants revealed the exonization of 5S ribosomal RNA (5S rRNA) within the gene of its own transcription regulator, TFIIIA (transcription factor for polymerase III A). The 5S rRNA-derived exon in TFIIIA gene exists in all representative land plant species but not in green algae and nonplant species, suggesting it is specific to land plants. TFIIIA is essential for RNA polymerase III-based transcription of 5S rRNA in eukaryotes. Integrating comparative genomics and molecular biology revealed that the conserved cassette exon derived from 5S rRNA is coupled with nonsense-mediated mRNA decay. Utilizing multiple independent Arabidopsis overexpressing TFIIIA transgenic lines under osmotic and salt stress, strong accordance between phenotypic and molecular evidence reveals the biological relevance of AS of the exonized 5S rRNA in quantitative autoregulation of TFIIIA homeostasis. Most significantly, this study provides the first evidence of ancient exaptation of 5S rRNA in plants, suggesting a novel gene regulation model mediated by the AS of an anciently exonized noncoding element.

  12. Defective control of pre-messenger RNA splicing in human disease.

    PubMed

    Chabot, Benoit; Shkreta, Lulzim

    2016-01-04

    Examples of associations between human disease and defects in pre-messenger RNA splicing/alternative splicing are accumulating. Although many alterations are caused by mutations in splicing signals or regulatory sequence elements, recent studies have noted the disruptive impact of mutated generic spliceosome components and splicing regulatory proteins. This review highlights recent progress in our understanding of how the altered splicing function of RNA-binding proteins contributes to myelodysplastic syndromes, cancer, and neuropathologies.

  13. Defective control of pre–messenger RNA splicing in human disease

    PubMed Central

    Shkreta, Lulzim

    2016-01-01

    Examples of associations between human disease and defects in pre–messenger RNA splicing/alternative splicing are accumulating. Although many alterations are caused by mutations in splicing signals or regulatory sequence elements, recent studies have noted the disruptive impact of mutated generic spliceosome components and splicing regulatory proteins. This review highlights recent progress in our understanding of how the altered splicing function of RNA-binding proteins contributes to myelodysplastic syndromes, cancer, and neuropathologies. PMID:26728853

  14. Alternative splicing of MALT1 controls signalling and activation of CD4(+) T cells.

    PubMed

    Meininger, Isabel; Griesbach, Richard A; Hu, Desheng; Gehring, Torben; Seeholzer, Thomas; Bertossi, Arianna; Kranich, Jan; Oeckinghaus, Andrea; Eitelhuber, Andrea C; Greczmiel, Ute; Gewies, Andreas; Schmidt-Supprian, Marc; Ruland, Jürgen; Brocker, Thomas; Heissmeyer, Vigo; Heyd, Florian; Krappmann, Daniel

    2016-04-12

    MALT1 channels proximal T-cell receptor (TCR) signalling to downstream signalling pathways. With MALT1A and MALT1B two conserved splice variants exist and we demonstrate here that MALT1 alternative splicing supports optimal T-cell activation. Inclusion of exon7 in MALT1A facilitates the recruitment of TRAF6, which augments MALT1 scaffolding function, but not protease activity. Naive CD4(+) T cells express almost exclusively MALT1B and MALT1A expression is induced by TCR stimulation. We identify hnRNP U as a suppressor of exon7 inclusion. Whereas selective depletion of MALT1A impairs T-cell signalling and activation, downregulation of hnRNP U enhances MALT1A expression and T-cell activation. Thus, TCR-induced alternative splicing augments MALT1 scaffolding to enhance downstream signalling and to promote optimal T-cell activation.

  15. Alternative splicing of MALT1 controls signalling and activation of CD4+ T cells

    PubMed Central

    Meininger, Isabel; Griesbach, Richard A.; Hu, Desheng; Gehring, Torben; Seeholzer, Thomas; Bertossi, Arianna; Kranich, Jan; Oeckinghaus, Andrea; Eitelhuber, Andrea C.; Greczmiel, Ute; Gewies, Andreas; Schmidt-Supprian, Marc; Ruland, Jürgen; Brocker, Thomas; Heissmeyer, Vigo; Heyd, Florian; Krappmann, Daniel

    2016-01-01

    MALT1 channels proximal T-cell receptor (TCR) signalling to downstream signalling pathways. With MALT1A and MALT1B two conserved splice variants exist and we demonstrate here that MALT1 alternative splicing supports optimal T-cell activation. Inclusion of exon7 in MALT1A facilitates the recruitment of TRAF6, which augments MALT1 scaffolding function, but not protease activity. Naive CD4+ T cells express almost exclusively MALT1B and MALT1A expression is induced by TCR stimulation. We identify hnRNP U as a suppressor of exon7 inclusion. Whereas selective depletion of MALT1A impairs T-cell signalling and activation, downregulation of hnRNP U enhances MALT1A expression and T-cell activation. Thus, TCR-induced alternative splicing augments MALT1 scaffolding to enhance downstream signalling and to promote optimal T-cell activation. PMID:27068814

  16. Alternative Splicing in Plant Genes: A Means of Regulating the Environmental Fitness of Plants.

    PubMed

    Shang, Xudong; Cao, Ying; Ma, Ligeng

    2017-02-20

    Gene expression can be regulated through transcriptional and post-transcriptional mechanisms. Transcription in eukaryotes produces pre-mRNA molecules, which are processed and spliced post-transcriptionally to create translatable mRNAs. More than one mRNA may be produced from a single pre-mRNA by alternative splicing (AS); thus, AS serves to diversify an organism's transcriptome and proteome. Previous studies of gene expression in plants have focused on the role of transcriptional regulation in response to environmental changes. However, recent data suggest that post-transcriptional regulation, especially AS, is necessary for plants to adapt to a changing environment. In this review, we summarize recent advances in our understanding of AS during plant development in response to environmental changes. We suggest that alternative gene splicing is a novel means of regulating the environmental fitness of plants.

  17. Alternative Splicing in Plant Genes: A Means of Regulating the Environmental Fitness of Plants

    PubMed Central

    Shang, Xudong; Cao, Ying; Ma, Ligeng

    2017-01-01

    Gene expression can be regulated through transcriptional and post-transcriptional mechanisms. Transcription in eukaryotes produces pre-mRNA molecules, which are processed and spliced post-transcriptionally to create translatable mRNAs. More than one mRNA may be produced from a single pre-mRNA by alternative splicing (AS); thus, AS serves to diversify an organism’s transcriptome and proteome. Previous studies of gene expression in plants have focused on the role of transcriptional regulation in response to environmental changes. However, recent data suggest that post-transcriptional regulation, especially AS, is necessary for plants to adapt to a changing environment. In this review, we summarize recent advances in our understanding of AS during plant development in response to environmental changes. We suggest that alternative gene splicing is a novel means of regulating the environmental fitness of plants. PMID:28230724

  18. RBM5/Luca-15/H37 regulates Fas alternative splice site pairing after exon definition.

    PubMed

    Bonnal, Sophie; Martínez, Concepción; Förch, Patrik; Bachi, Angela; Wilm, Matthias; Valcárcel, Juan

    2008-10-10

    RBM5/Luca-15/H37 is a gene frequently inactivated in lung cancers and overexpressed in breast tumors. Its protein product has been detected in prespliceosomal complexes and modulates cell proliferation and Fas-mediated apoptosis. We report that RBM5 is a component of complexes involved in 3' splice site recognition and regulates alternative splicing of apoptosis-related genes, including the Fas receptor, switching between isoforms with antagonistic functions in programmed cell death. In contrast with classical mechanisms of splicing regulation, RBM5 does not affect early events of splice site recognition that lead to Fas exon 6 definition. Instead, RBM5 inhibits the transition between prespliceosomal complexes assembled around exon 6 to mature spliceosomes assembled on the flanking introns and promotes sequence-specific pairing of the distal splice sites. An OCRE domain important for RBM5 function contacts components of the U4/5/6 tri-snRNP, consistent with the idea that RBM5 modulates splice site pairing after prespliceosome assembly and exon definition.

  19. The transcription factor FBI-1 inhibits SAM68-mediated BCL-X alternative splicing and apoptosis.

    PubMed

    Bielli, Pamela; Busà, Roberta; Di Stasi, Savino M; Munoz, Manuel J; Botti, Flavia; Kornblihtt, Alberto R; Sette, Claudio

    2014-04-01

    Alternative splicing (AS) is tightly coupled to transcription for the majority of human genes. However, how these two processes are linked is not well understood. Here, we unveil a direct role for the transcription factor FBI-1 in the regulation of AS. FBI-1 interacts with the splicing factor SAM68 and reduces its binding to BCL-X mRNA. This, in turn, results in the selection of the proximal 5' splice site in BCL-X exon 2, thereby favoring the anti-apoptotic BCL-XL variant and counteracting SAM68-mediated apoptosis. Conversely, depletion of FBI-1, or expression of a SAM68 mutant lacking the FBI-1 binding region, restores the ability of SAM68 to induce BCL-XS splicing and apoptosis. FBI-1's role in splicing requires the activity of histone deacetylases, whose pharmacological inhibition recapitulates the effects of FBI-1 knockdown. Our study reveals an unexpected function for FBI-1 in splicing modulation with a direct impact on cell survival.

  20. Identification of genetic variants associated with alternative splicing using sQTLseekeR

    PubMed Central

    Monlong, Jean; Calvo, Miquel; Ferreira, Pedro G.; Guigó, Roderic

    2014-01-01

    Identification of genetic variants affecting splicing in RNA sequencing population studies is still in its infancy. Splicing phenotype is more complex than gene expression and ought to be treated as a multivariate phenotype to be recapitulated completely. Here we represent the splicing pattern of a gene as the distribution of the relative abundances of a gene’s alternative transcript isoforms. We develop a statistical framework that uses a distance-based approach to compute the variability of splicing ratios across observations, and a non-parametric analogue to multivariate analysis of variance. We implement this approach in the R package sQTLseekeR and use it to analyze RNA-Seq data from the Geuvadis project in 465 individuals. We identify hundreds of single nucleotide polymorphisms (SNPs) as splicing QTLs (sQTLs), including some falling in genome-wide association study SNPs. By developing the appropriate metrics, we show that sQTLseekeR compares favorably with existing methods that rely on univariate approaches, predicting variants that behave as expected from mutations affecting splicing. PMID:25140736