BIOCHEMISTRY OF PRE-MRNA SPLICING

Summary

Principal Investigator: ADRIAN KRAINER
Abstract: DESCRIPTION (provided by applicant): Pre-mRNA splicing is an essential step in gene expression. It involves precise excision of introns and joining of exons from primary transcripts in the nucleus to generate mature mRNA, the template for protein synthesis in the cytoplasm. Many genes express multiple mRNA and protein isoforms by alternative splicing of their primary transcripts. The catalyst for splicing is the spliceosome, a macromolecular machine composed of five small RNAs and numerous proteins, which undergo dynamic assembly with each exon-intron-exon substrate. Prior to translation, the mature mRNA is subject to a quality-control pathway, known as nonsense- mediated decay (NMD). NMD degrades mature mRNAs with a premature termination codon (PTC). In mammals, the difference between premature and normal PTCs is established in relation to their position and distance from the last exon-exon junction. This reflects splicing-dependent loading of an exon-junction protein complex (EJC). PTCs arise by nonsense, frameshift, or splicing mutations, as well as being present in some normal alternatively spliced mRNA isoforms. The overall goal of the proposed research is to study selected aspects of mammalian pre-mRNA splicing, with an emphasis on the recognition of the splice sites by selected human splicing factors. Specific aspects of splicing-dependent mRNA surveillance will also be investigated. The specific aims are: 1. To study the mechanism of 5'-splice-site selection;2. To study RNA-binding proteins involved in 3'-splice-site recognition;3. To study the links between pre-mRNA splicing and NMD;4. To study exon-recognition defects in disease-susceptibility genes. These objectives will be achieved through a combination of biochemical approaches using cell-free systems, purified components, and recombinant proteins together with molecular and reverse-genetic approaches in cell culture, bioinformatics analysis of the genome, and methods to modulate gene expression. Aberrant splicing due to mutations in intron-containing genes is a frequent cause of many human genetic diseases. Information gained from these basic studies already informs genetic counseling and phenotypic-risk assessment in the clinic, yet there is much room for improvement in predicting genotype-phenotype correlations that involve aberrant splicing or NMD. Mutations in splicing factors are also associated with inherited diseases, such as retinitis pigmentosa, and with cancer. For example, the gene encoding the splicing factor SF2/ASF is a proto-oncogene that can be activated by gene amplification in breast cancer. Drugs are being developed to correct aberrant splicing, e.g., through antisense methods, small molecules, or bifunctional modulators. Some of these approaches are already being tested in clinical trials for muscular dystrophy and spinal muscular atrophy, while others are still in the pre-clinical stages. The proposed studies, which are aimed at understanding basic cellular mechanisms of gene expression, may provide the basis for future development of new or improved diagnostic tools and therapeutic agents. PROJECT NARRATIVE - Pre-mRNA splicing and mRNA quality-control are key steps in gene expression. Mutations that affect these processes in specific genes, or that alter the properties or levels of the factors involved in these pathways, underlie many genetic diseases and cancer predispositions. The proposed studies are aimed at understanding basic cellular mechanisms of gene expression, and may provide the basis for developing or improving diagnostic tools and therapeutic agents for these diseases.
Funding Period: ----------------1989 - ---------------2011-
more information: NIH RePORT

Top Publications

  1. ncbi An increased specificity score matrix for the prediction of SF2/ASF-specific exonic splicing enhancers
    Philip J Smith
    Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA
    Hum Mol Genet 15:2490-508. 2006
  2. pmc Peripheral SMN restoration is essential for long-term rescue of a severe spinal muscular atrophy mouse model
    Yimin Hua
    Cold Spring Harbor Laboratory, PO Box 100, Cold Spring Harbor, New York 11724, USA
    Nature 478:123-6. 2011
  3. pmc Splicing therapeutics in SMN2 and APOB
    Bernard Khoo
    University College London Medical School, Department of Endocrinology, Hampstead Campus, London, UK
    Curr Opin Mol Ther 11:108-15. 2009
  4. pmc Identification of synaptic targets of Drosophila pumilio
    Gengxin Chen
    Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA
    PLoS Comput Biol 4:e1000026. 2008
  5. pmc Antisense masking of an hnRNP A1/A2 intronic splicing silencer corrects SMN2 splicing in transgenic mice
    Yimin Hua
    Cold Spring Harbor Laboratory, PO Box 100, Cold Spring Harbor, NY 11724, USA
    Am J Hum Genet 82:834-48. 2008
  6. pmc Dual-specificity splice sites function alternatively as 5' and 3' splice sites
    Chaolin Zhang
    Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA
    Proc Natl Acad Sci U S A 104:15028-33. 2007
  7. pmc Deletion of the N-terminus of SF2/ASF permits RS-domain-independent pre-mRNA splicing
    Stephanie D Shaw
    Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, United States of America
    PLoS ONE 2:e854. 2007
  8. ncbi Evolutionary impact of limited splicing fidelity in mammalian genes
    Chaolin Zhang
    Cold Spring Harbor Laboratory, NY 11724, USA
    Trends Genet 23:484-8. 2007
  9. pmc Splicing remodels messenger ribonucleoprotein architecture via eIF4A3-dependent and -independent recruitment of exon junction complex components
    Zuo Zhang
    Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA
    Proc Natl Acad Sci U S A 104:11574-9. 2007
  10. ncbi SR proteins function in coupling RNAP II transcription to pre-mRNA splicing
    Rita Das
    Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA
    Mol Cell 26:867-81. 2007

Scientific Experts

  • ADRIAN KRAINER
  • Thomas W Prior
  • Yimin Hua
  • Michelle L Hastings
  • Xavier Roca
  • Chaolin Zhang
  • Zuo Zhang
  • Bernard Khoo
  • Michael Q Zhang
  • Eric Allemand
  • C Frank Bennett
  • Shuying Sun
  • Suhyung Cho
  • Tomoya Kubota
  • Mafalda Martins de Araujo
  • Gourisankar Ghosh
  • Gengxin Chen
  • Timothy A Vickers
  • Emanuele Buratti
  • Karsten Bork Nielsen
  • Stephanie D Shaw
  • Rita Das
  • Michael P Myers
  • Shern L Chew
  • Hiroshi Kimura
  • Philip J Smith
  • Nihar Sheth
  • Oliver Fregoso
  • Xiang Dong Fu
  • Takashi Kimura
  • Amy Hoang
  • Saburo Sakoda
  • Masanori P Takahashi
  • Yosuke Kokunai
  • Ichizo Nishino
  • Xiang Yang Zhong
  • Frank Rigo
  • Gene Hung
  • Rahul Sinha
  • Kentaro Sahashi
  • Guy Horev
  • Juan Valcarcel
  • Sophie Bonnal
  • Nishi Sinha
  • Jody Barditch
  • Michael Regulski
  • Tim Tully
  • Josh Dubnau
  • Qing Shuo Zhang
  • Hazeem L Okunola
  • Wanhe Li
  • Brage Storstein Andresen
  • Michael V Murray
  • Robin Reed
  • Niels Gregersen
  • Martin Chivers
  • Line Sinnathamby Reinert
  • Jana Kralovicova
  • Sutapa Chakrabarti
  • Luca Cartegni
  • Suzette Sørensen
  • Thomas Juhl Corydon
  • Jiong Yu
  • Thomas Koed Doktor
  • Jørgen Kjems
  • Lisbeth Dahl Schroeder
  • Steven P Gygi
  • Melanie P Gygi
  • Orly Elpeleg
  • Marco Baralle
  • Igor Vorechovsky
  • Brenda F Baker
  • Maurizio Romano
  • Dominik M Duelli
  • Nanako Takizawa
  • Jinhua Wang
  • Ted Roeder
  • Francisco J Iborra
  • Ravi Sachidanandam
  • Tatsuo Fukagawa
  • Michiko Muraki
  • Naohito Nozaki
  • Tetsuya Hori
  • Katsuya Okawa
  • Masatoshi Hagiwara

Detail Information

Publications24

  1. ncbi An increased specificity score matrix for the prediction of SF2/ASF-specific exonic splicing enhancers
    Philip J Smith
    Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA
    Hum Mol Genet 15:2490-508. 2006
    ..Finally, we have derived an increased specificity score matrix that incorporates information from both of our SF2/ASF-specific matrices and that accurately predicts the exon-skipping phenotypes of deleterious point mutations...
  2. pmc Peripheral SMN restoration is essential for long-term rescue of a severe spinal muscular atrophy mouse model
    Yimin Hua
    Cold Spring Harbor Laboratory, PO Box 100, Cold Spring Harbor, New York 11724, USA
    Nature 478:123-6. 2011
    ..These results suggest that the liver is important in SMA pathogenesis, underscoring the importance of SMN in peripheral tissues, and demonstrate the efficacy of a promising drug candidate...
  3. pmc Splicing therapeutics in SMN2 and APOB
    Bernard Khoo
    University College London Medical School, Department of Endocrinology, Hampstead Campus, London, UK
    Curr Opin Mol Ther 11:108-15. 2009
    ....
  4. pmc Identification of synaptic targets of Drosophila pumilio
    Gengxin Chen
    Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA
    PLoS Comput Biol 4:e1000026. 2008
    ..Finally, we show that the endogenous dlg1 mRNA can be regulated by Pumilio in a neuronal context, the adult mushroom bodies (MB), which is an anatomical site of memory storage...
  5. pmc Antisense masking of an hnRNP A1/A2 intronic splicing silencer corrects SMN2 splicing in transgenic mice
    Yimin Hua
    Cold Spring Harbor Laboratory, PO Box 100, Cold Spring Harbor, NY 11724, USA
    Am J Hum Genet 82:834-48. 2008
    ..Most importantly, our results highlight the therapeutic potential of some of these ASOs in the context of SMA...
  6. pmc Dual-specificity splice sites function alternatively as 5' and 3' splice sites
    Chaolin Zhang
    Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA
    Proc Natl Acad Sci U S A 104:15028-33. 2007
    ..We discuss the implications of this unusual splicing pattern for the diverse mechanisms of exon recognition and for gene evolution...
  7. pmc Deletion of the N-terminus of SF2/ASF permits RS-domain-independent pre-mRNA splicing
    Stephanie D Shaw
    Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, United States of America
    PLoS ONE 2:e854. 2007
    ..Our data provide additional evidence that the SF2/ASF RS domain is not strictly required for constitutive splicing in vitro, contrary to prevailing models for how the domains of SR proteins function to promote splicing...
  8. ncbi Evolutionary impact of limited splicing fidelity in mammalian genes
    Chaolin Zhang
    Cold Spring Harbor Laboratory, NY 11724, USA
    Trends Genet 23:484-8. 2007
    ..These results suggest AS events introducing mild changes are generally favored during evolution and explain the excess of shorter, frame-preserving cassette exons in present mammalian genomes...
  9. pmc Splicing remodels messenger ribonucleoprotein architecture via eIF4A3-dependent and -independent recruitment of exon junction complex components
    Zuo Zhang
    Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA
    Proc Natl Acad Sci U S A 104:11574-9. 2007
    ....
  10. ncbi SR proteins function in coupling RNAP II transcription to pre-mRNA splicing
    Rita Das
    Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA
    Mol Cell 26:867-81. 2007
    ..Thus, these factors readily out-compete inhibitory hnRNP proteins, resulting in efficient spliceosome assembly on nascent RNAP II transcripts...
  11. pmc Control of pre-mRNA splicing by the general splicing factors PUF60 and U2AF(65)
    Michelle L Hastings
    Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, United States of America
    PLoS ONE 2:e538. 2007
    ..Our results indicate that recognition of 3' splice sites involves different U2AF-like molecules, and that modulation of these general splicing factors can have profound effects on splicing...
  12. pmc A novel histone exchange factor, protein phosphatase 2Cgamma, mediates the exchange and dephosphorylation of H2A-H2B
    Hiroshi Kimura
    Nuclear Function and Dynamics Unit, Horizontal Medical Research Organization, Graduate School of Medicine, Kyoto University, Sakyo ku, Kyoto 606 8501, Japan
    J Cell Biol 175:389-400. 2006
    ....
  13. pmc Comprehensive splice-site analysis using comparative genomics
    Nihar Sheth
    Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA
    Nucleic Acids Res 34:3955-67. 2006
    ..Our study proves that the identification of broad patterns in naturally-occurring splice sites, through the analysis of genomic datasets, provides mechanistic and evolutionary insights into pre-mRNA splicing...
  14. pmc Antisense oligonucleotide-induced alternative splicing of the APOB mRNA generates a novel isoform of APOB
    Bernard Khoo
    Endocrinology, William Harvey Research Institute, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK
    BMC Mol Biol 8:3. 2007
    ..Down-regulation of APOB100 is a potential therapy to lower circulating LDL and cholesterol levels...
  15. pmc Seemingly neutral polymorphic variants may confer immunity to splicing-inactivating mutations: a synonymous SNP in exon 5 of MCAD protects from deleterious mutations in a flanking exonic splicing enhancer
    Karsten Bork Nielsen
    Research Unit for Molecular Medicine, Aarhus University Hospital and Faculty of Health Science, Skejby Sygehus, Denmark
    Am J Hum Genet 80:416-32. 2007
    ....
  16. pmc Enhancement of SMN2 exon 7 inclusion by antisense oligonucleotides targeting the exon
    Yimin Hua
    Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, United States of America
    PLoS Biol 5:e73. 2007
    ..Some of the ASOs we identified are sufficiently active to proceed with experiments in SMA mouse models...
  17. ncbi Alternative splicing regulation by interaction of phosphatase PP2Cgamma with nucleic acid-binding protein YB-1
    Eric Allemand
    Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA
    Nat Struct Mol Biol 14:630-8. 2007
    ..We propose that PP2Cgamma modulates alternative splicing of specific pre-mRNAs coregulated by YB-1...
  18. pmc Aberrant 5' splice sites in human disease genes: mutation pattern, nucleotide structure and comparison of computational tools that predict their utilization
    Emanuele Buratti
    International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34012 Trieste, Italy
    Nucleic Acids Res 35:4250-63. 2007
    ..The development of an online database of aberrant 5'ss will be useful for studying basic mechanisms of splice-site selection, identifying splicing mutations and optimizing splice-site prediction algorithms...
  19. pmc Interaction between the RNA binding domains of Ser-Arg splicing factor 1 and U1-70K snRNP protein determines early spliceosome assembly
    Suhyung Cho
    Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA 92093, USA
    Proc Natl Acad Sci U S A 108:8233-8. 2011
    ....
  20. pmc A mutation in a rare type of intron in a sodium-channel gene results in aberrant splicing and causes myotonia
    Tomoya Kubota
    Department of Neurology, Osaka University Graduate School of Medicine, 2 2 Yamadaoaka, Suita, Osaka, Japan
    Hum Mutat 32:773-82. 2011
    ..This is the first report of a disease-associated mutation in an AT-AC type II intron, and also the first intronic mutation in a voltage-gated ion channel gene showing a gain-of-function defect...
  21. pmc A positive modifier of spinal muscular atrophy in the SMN2 gene
    Thomas W Prior
    Department of Pathology, Ohio State University, Columbus, OH 43210, USA
    Am J Hum Genet 85:408-13. 2009
    ..We have shown not only that the SMA phenotype is modified by the number of SMN2 genes but that SMN2 sequence variations can also affect the disease severity...
  22. pmc Differential 3' splice site recognition of SMN1 and SMN2 transcripts by U2AF and U2 snRNP
    Mafalda Martins de Araujo
    Centre de Regulacio Genomica, Barcelona, Spain
    RNA 15:515-23. 2009
    ..Collectively, the results suggest that both U2AF binding and other steps of U2 snRNP recruitment can be control points in SMN splicing regulation...
  23. pmc Recognition of atypical 5' splice sites by shifted base-pairing to U1 snRNA
    Xavier Roca
    Cold Spring Harbor Laboratory, PO Box 100, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA
    Nat Struct Mol Biol 16:176-82. 2009
    ..The unexpected flexibility in 5' ss-U1 base-pairing challenges an established paradigm and has broad implications for splice-site prediction algorithms and gene-annotation efforts in genome projects...
  24. pmc Mechanisms of activation and repression by the alternative splicing factors RBFOX1/2
    Shuying Sun
    Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA
    RNA 18:274-83. 2012
    ..RNA interference experiments showed that hnRNP H1 and TFG modulate the splicing activity of RBFOX1/2, whereas RALY had no effect. However, TFG is localized in the cytoplasm, and likely modulates alternative splicing indirectly...