Nucleic Acids Research, 2003, Vol. 31, No. 23 6963-6975
© 2003 Oxford University Press
Article |
A novel approach to describe a U1 snRNA binding site
Institut für Virologie, Heinrich-Heine-Universität Düsseldorf, Geb. 22.21, Universitätsstraße 1, D-40225 Düsseldorf, Germany, 1 Result GmbH, Tempelsweg 16, D-47918 Tönisvorst, Germany, 2 thpr.net, Schützenstraße 23, D-72070 Tübingen, Germany, 3 Department of Human Genetics, University of Würzburg, Biozentrum, Würzburg, Germany and 4 Department of Molecular and Structural Biology, University of Aarhus, C.F. Mollers Alle, Building 130, DK-8000 Aarhus C, Denmark
*To whom correspondence should be addressed. Tel: +49 211 81 12393; Fax: +49 211 81 12227; Email: schaal{at}uni-duesseldorf.de
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, 
2-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.
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