A novel approach to describe a U1 snRNA binding site

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  • Marcel Freund, Institut für Virologie, Heinrich‐Heine‐Universität Düsseldorf, Germany
  • Corinna Asang, Institut für Virologie, Heinrich‐Heine‐Universität Düsseldorf, Germany
  • Susanne Kammler, Denmark
  • Carolin Konermann, Institut für Virologie, Heinrich‐Heine‐Universität Düsseldorf, Germany
  • Jörg Krummheuer, Denmark
  • Marianne Hipp, Institut für Virologie, Heinrich‐Heine‐Universität Düsseldorf, Germany
  • Imke Meyer, Institut für Virologie, Heinrich‐Heine‐Universität Düsseldorf, Germany
  • Wolfram Gierling, Institut für Virologie, Heinrich‐Heine‐Universität Düsseldorf, Germany
  • Stephan Theiss, Result GmbH, Tönisvorst, Germany
  • Thorsten Preuss, thpr.net, Tübingen, Germany
  • Detlev Schindler, Department of Human Genetics, University of Würzburg, Biozentrum, Würzburg, Germany
  • Jørgen Kjems
  • Heiner Schaal
RNA duplex formation between U1 snRNA and a splice donor (SD) site can protect pre-mRNA from degradation prior to splicing and initiates formation of the spliceosome. This process was monitored, using sub-genomic HIV-1 expression vectors, by expression analysis of the glycoprotein env, whose formation critically depends on functional SD4. We systematically derived a hydrogen bond model for the complementarity between the free 5' end of U1 snRNA and 5' splice sites and numerous mutations following transient transfection of HeLa-T4+ cells with 5' splice site mutated vectors. The resulting model takes into account number, interdependence and neighborhood relationships of predicted hydrogen bond formation in a region spanning the three most 3' base pairs of the exon (-3 to -1) and the eight most 5' base pairs of the intron (+1 to +8). The model is represented by an algorithm classifying U1 snRNA binding sites which can or cannot functionally substitute SD4 with respect to Rev-mediated env expression. In a data set of 5' splice site mutations of the human ATM gene we found a significant correlation between the algorithmic classification and exon skipping (P = 0.018, chi2-test), showing that the applicability of the proposed model reaches far beyond HIV-1 splicing. However, the algorithmic classification must not be taken as an absolute measure of SD usage as it may be modified by upstream sequence elements. Upstream to SD4 we identified a fragment supporting ASF/SF2 binding. Mutating GAR nucleotide repeats within this site decreased the SD4-dependent Rev-mediated env expression, which could be balanced simply by artificially increasing the complementarity of SD4.
Original languageEnglish
JournalNucleic Acids Research
Volume31
Issue23
Pages (from-to)6963-6975
Number of pages13
ISSN0305-1048
DOIs
Publication statusPublished - 1 Dec 2003

    Research areas

  • Algorithms, Base Sequence, Binding Sites, Cell Cycle Proteins, Cell Extracts, DNA-Binding Proteins, Exons, Gene Products, env, HIV-1, HeLa Cells, Humans, Hydrogen Bonding, Mutation, Protein-Serine-Threonine Kinases, RNA Splice Sites, RNA, Small Nuclear, Ribonucleoprotein, U1 Small Nuclear, Ribonucleoprotein, U2 Small Nuclear, Tumor Suppressor Proteins

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