Research TopicsSpecies | MECHANISMS OF ENHANCER DEPENDENT SPLICE SITE ACTIVATIONSummaryPrincipal Investigator: Klemens J Hertel Abstract: DESCRIPTION (provided by applicant): With the completion of the human genome project, it has become clear that the number of genes cannot account for the complexity of the human proteome. This conclusion has lead to a dramatic increase in our appreciation of the abundance and importance of post-transcriptional mechanisms of gene regulation. Among several proposed mechanisms, alternative pre-mRNA splicing is considered to be one of the most efficient and wide spread avenues to generate multiple protein isoforms from individual genes. Current estimates indicate that over 90% of human genes undergo alternative splicing, thus greatly increasing the coding potential of our genome. This renewal application focuses on understanding the mechanisms of splice site selection with the long-term goal to generate a splicing code that permits alternative splicing predictions based on sequence analysis. The experiments outlined in this application build on the most exciting discoveries made during the previous funding period;charting highly significant correlations between alternative splicing and cis-acting RNA splicing elements, the discovery of a mechanism that describes position-dependent activities of splicing regulators, and the discovery that the retention of spliceosomal components along ligated exons ensures efficient processing. The proposal has three major goals: 1) To investigate experimentally and computationally the complexity of exon selection. A systems approach is proposed to examine methodically how the combination of cis-acting RNA splicing elements influences splice site selection with the objective to generate a splicing code that permits alternative splicing predictions based on sequence analysis. 2) To test the hypothesis that position dependent splicing activation or repression is due to unique interactions between splicing regulatory complexes and spliceosomal components. 3) To determine the mechanisms that ensure highly efficiency processing of multi-intron containing pre-mRNAs. Because defects in splicing lead to many human genetic diseases, including a number of genes that have been implicated in cancer, the proposed research will greatly enhance our understanding of regulated gene expression and human disease. In particular, the results from these studies will provide a quantitative framework for exon recognition and they will permit splicing predictions to classify exonic mutations, thus paving the way for the design of alternative therapeutic approaches to combat disease. Funding Period: 2001-02-01 - 2014-06-30 more information: NIH RePORT Top Publications
Research Grants
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Detail Information
Publications
Linking splicing to Pol II transcription stabilizes pre-mRNAs and influences splicing patternsMartin J Hicks
Department of Microbiology and Molecular Genetics, University of California Irvine, Irvine, California, USA
PLoS Biol 4:e147. 2006..We propose that the connection between transcription by Pol II and pre-mRNA splicing guarantees an extended half-life and proper processing of nascent pre-mRNAs...- G Run-mediated recognition of proteolipid protein and DM20 5' splice sites by U1 small nuclear RNA is regulated by context and proximity to the splice siteErming Wang
Department of Neurology, University of Kentucky, Lexington, Kentucky 40536, USA
J Biol Chem 286:4059-71. 2011..The data suggest that different mechanisms underlie G run-mediated recognition of 5' splice sites and that context and position play a critical role... - Complex and dynamic landscape of RNA polyadenylation revealed by PAS-SeqPeter J Shepard
Department of Microbiology and Molecular Genetics, University of California at Irvine, Irvine, California 92697, USA
RNA 17:761-72. 2011..Together, our PAS-Seq analyses revealed a complex landscape of RNA polyadenylation in mammalian cells and the dynamic regulation of APA during stem cell differentiation... - Efficient internal exon recognition depends on near equal contributions from the 3' and 5' splice sitesPeter J Shepard
Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, CA 92697 4025, USA
Nucleic Acids Res 39:8928-37. 2011.... - Evolution of SR protein and hnRNP splicing regulatory factorsAnke Busch
Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, CA 92697 4025, USA
Wiley Interdiscip Rev RNA 3:1-12. 2012.... - Extensive regulation of NAGNAG alternative splicing: new tricks for the spliceosome?Anke Busch
Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, Irvine, CA 92697 4025, USA
Genome Biol 13:143. 2012..A recent study using massive parallel sequencing demonstrates unequivocally that alternative tandem acceptor splicing is tissue-specifically regulated... - HEXEvent: a database of Human EXon splicing EventsAnke Busch
Department of Microbiology and Molecular Genetics, University of California, Irvine, CA 92697 4025, USA
Nucleic Acids Res 41:D118-24. 2013..Importantly, the generated data sets are downloadable for further analysis... - Position-dependent splicing activation and repression by SR and hnRNP proteins rely on common mechanismsSteffen Erkelenz
Institute of Virology, Heinrich Heine University, D 40225 Dusseldorf, Germany
RNA 19:96-102. 2013..Thus, SR and hnRNP splicing factors exploit similar mechanisms to positively or negatively influence splice site selection... 
Preparation of splicing competent nuclear extractsChiu Ho T Webb
Department of Microbiology and Molecular Genetics, University of California, Irvine, CA, USA
Methods Mol Biol 1126:117-21. 2014..Nuclear extracts derived from HeLa cells provide all of the obligatory components to carry out intron removal in vitro. This chapter describes the large-scale preparation of nuclear extract from HeLa cells. ..- In vitro assay of pre-mRNA splicing in mammalian nuclear extractMaliheh Movassat
Department of Microbiology and Molecular Genetics, University of California, Irvine, CA, USA
Methods Mol Biol 1126:151-60. 2014..Through the use of radiolabeled pre-mRNA and crude nuclear extract, spliced mRNAs can be purified and visualized by autoradiography for downstream analysis. .. - Retention of spliceosomal components along ligated exons ensures efficient removal of multiple intronsTara L Crabb
Department of Microbiology and Molecular Genetics, University of California at Irvine, Irvine, California 92697 4025, USA
RNA 16:1786-96. 2010..Thus, ligated exons do not require redefinition, providing an additional kinetic advantage for exon defined splice sites... - Competing upstream 5' splice sites enhance the rate of proximal splicingMartin J Hicks
Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, CA 92697 4025, USA
Mol Cell Biol 30:1878-86. 2010..These results reveal the discovery of a new splicing regulatory element, an upstream 5' splice site functioning as a splicing enhancer... - Restoration of full-length SMN promoted by adenoviral vectors expressing RNA antisense oligonucleotides embedded in U7 snRNAsTill Geib
Department of Microbiology and Molecular Genetics, School of Medicine, University of California Irvine, Irvine, California, United States of America
PLoS ONE 4:e8204. 2009..It is caused by deletions or mutations of the survival of motor neuron 1 gene (SMN1). The remaining copy of the gene, SMN2, generates only low levels of the SMN protein due to a mutation in SMN2 exon 7 that leads to exon skipping... - Genomic splice-site analysis reveals frequent alternative splicing close to the dominant splice siteYimeng Dou
Department of Information and Computer Sciences, University of California, Irvine, Irvine, California 92697 4025, USA
RNA 12:2047-56. 2006..This high probability of alternative 3'-splice-site activation in close proximity of the dominant 3'-splice site suggests that the second step of the splicing may be prone to violate splicing fidelity... - The splicing regulators Tra and Tra2 are unusually potent activators of pre-mRNA splicingKathryn S Sciabica
Department of Microbiology and Molecular Genetics, University of California, Irvine, CA 92697 4025, USA
Nucleic Acids Res 34:6612-20. 2006..Thus, the regulated assembly of the dsx enhancer complexes leads to the generation of an extended activation domain to guarantee the 'all or none' splicing switch that is required during Drosophila sexual differentiation... - Combinatorial control of exon recognitionKlemens J Hertel
Department of Microbiology and Molecular Genetics, University of California, Irvine, CA 92697 4025, USA
J Biol Chem 283:1211-5. 2008..The relative contributions of each of these parameters control how efficiently splice sites are recognized and flanking introns are removed... - Conserved RNA secondary structures promote alternative splicingPeter J Shepard
Department of Microbiology and Molecular Genetics, Institute for Genomics and Bioinformatics, University of California at Irvine, Irvine, California 92697 4025, USA
RNA 14:1463-9. 2008..Their frequent involvement with alternative splicing demonstrates that RNA structure formation is an important mechanism regulating gene expression and disease... - Spliceosome assembly pathways for different types of alternative splicing converge during commitment to splice site pairing in the A complexMatthew V Kotlajich
Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, California 92697 4025, USA
Mol Cell Biol 29:1072-82. 2009..These results identify the A complex as the spliceosomal assembly step dedicated to splice site pairing and suggest that ATP hydrolysis locks splice sites into a splicing pattern after stable U2 snRNP association to the branch point... - Splice-site pairing is an intrinsically high fidelity processKristi L Fox-Walsh
Department of Microbiology and Molecular Genetics, University of California, Irvine, CA 92697 4025, USA
Proc Natl Acad Sci U S A 106:1766-71. 2009..We conclude that all multi-intron-containing genes are alternatively spliced and that the reduction of SMN results in a general splicing defect that is mediated through alterations in the fidelity of splice-site pairing... - Spliceosomes walk the line: splicing errors and their impact on cellular functionShu Ning Hsu
Department of Microbiology and Molecular Genetics, University of California, Irvine, CA, USA
RNA Biol 6:526-30. 2009..Here we review recent progress made in understanding the extent of alternative splicing within the human genome with particular emphasis on splicing fidelity... - The SR protein familyPeter J Shepard
Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, CA 92697 4025, USA
Genome Biol 10:242. 2009..These activities point up the importance of SR proteins during the regulation of mRNA metabolism... - Identification of a novel nuclear localization signal and speckle-targeting sequence of tuftelin-interacting protein 11, a splicing factor involved in spliceosome disassemblySissada Tannukit
Center for Craniofacial Molecular Biology, University of Southern California, 2250 Alcazar Street, CSA Rm103, Los Angeles, CA 90033 1004, USA
Biochem Biophys Res Commun 390:1044-50. 2009..These data help define the mechanism of nuclear/nuclear speckle localization of the splicing factor TFIP11, with implications for it's function... - Kinetic analysis of in vitro pre-mRNA splicing in HeLa nuclear extractWilliam F Mueller
Department of Microbiology and Molecular Genetics, University of California, Irvine, CA, USA
Methods Mol Biol 1126:161-8. 2014..This chapter will describe the rationale and approach employed to use kinetic analysis to evaluate an in vitro splicing reaction using radiolabeled pre-mRNA incubated in splicing-competent HeLa nuclear extract (NE). ..
Research Grants
- Mechanisms of Signal-Induced Alternative Splicing: CD45Kristen W Lynch; Fiscal Year: 2013....
- Development and Evaluation of Dual Compartment Combination MicrobicidesROBERT WALTER BUCKHEIT; Fiscal Year: 2013..Through our industrial partnership with ImQuest Pharmaceuticals our methodology and products will have an immediate outlet to further clinical development. ..
- Spliceosome Mechanism Dissected at the Single Molecule LevelNils G Walter; Fiscal Year: 2013..Taken together, these advances will pave the way for, over the funding period, extensive mechanistic studies of yeast splicing and for studying alternative splicing in humans in the longer term. ..
- Regulation of Ovarian Cancer Multidrug Resistance by MAP KinasesScott T Eblen; Fiscal Year: 2013..Elucidation of this synergistic mechanism of drug resistance would provide important information for treating drug-resistant ovarian cancer. ..
- SMN Control of snRNP Biogenesis: Role in RNA Splicing and Motor Neuron SurvivalLivio Pellizzoni; Fiscal Year: 2013..These studies should provide insights into the basic mechanisms of RNA regulation and the molecular defects underlying SMA pathogenesis with the potential of identifying new candidate targets for therapeutic development. ..
- The Role of Caspase 9b Expression in NSCLC Survival/Chemotherapeutic SensitivityCharles E Chalfant; Fiscal Year: 2013..g. cisplatinum and paclitaxel). ..
- SPLICING OF MRNA PRECURSORSMichael R Green; Fiscal Year: 2013....
- BIOCHEMISTRY OF PRE-MRNA SPLICINGAdrian R Krainer; Fiscal Year: 2013..abstract_text> ..
- HORMONAL REGULATION OF BLOOD PRESSUREMichal Laniado Schwartzman; Fiscal Year: 2013..ular tone, in the pathophysiology of hypertension and cardiovascular disease. ..
- Alternative pre-mRNA Splicing in DrosophilaDonald C Rio; Fiscal Year: 2013....
- STEM CELL GENE THERAPY FOR HEMOGLOBINOPATHIESGeorge Stamatoyannopoulos; Fiscal Year: 2013..The focus of this Program Project, Gene Therapy, can provide a new paradigm for the treatment of these hemoglobinopathies as well as for other blood diseases. (End of Abstract) ..
- Vertebrate Non-Coding RNPs: Integrating function, biogenesis and survivalJoan A Steitz; Fiscal Year: 2013..Together, this understanding will contribute to the development of therapeutic approaches to combat multiple genetic diseases that derive from faulty RNA processing and surveillance, as well as cancer. ..
- Functional RNA elements in the human genomeEUGENE WEI MING YEO; Fiscal Year: 2013..This research has the potential to fundamentally change our view on splicing control and its contribution to human disease. ..
- Electron Microscopy of Biological MacromoleculesKenneth H Downing; Fiscal Year: 2013....