A novel approach to describe a U1 snRNA binding site.
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.
Freund
Marcel
M
Institut für Virologie, Heinrich-Heine-Universität Düsseldorf, Geb. 22.21, Universitätsstrasse 1, D-40225 Düsseldorf, Germany.
Asang
Corinna
C
Kammler
Susanne
S
Konermann
Carolin
C
Krummheuer
Jörg
J
Hipp
Marianne
M
Meyer
Imke
I
Gierling
Wolfram
W
Theiss
Stephan
S
Preuss
Thorsten
T
Schindler
Detlev
D
Kjems
Jørgen
J
Schaal
Heiner
H
D000465:Algorithms; D064007:Ataxia Telangiectasia Mutated Proteins; D001483:Base Sequence; D001665:Binding Sites; D018797:Cell Cycle Proteins; D002457:Cell Extracts; D004268:DNA-Binding Proteins; D005091:Exons / Q000235:genetics; D015686:Gene Products, env / Q000235:genetics; D015497:HIV-1 / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D006860:Hydrogen Bonding; D009154:Mutation / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D022821:RNA Splice Sites / Q000235:genetics*; D012342:RNA, Small Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000378:metabolism; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000378:metabolism; D025521:Tumor Suppressor Proteins
D018797:Cell Cycle Proteins; D002457:Cell Extracts; D004268:DNA-Binding Proteins; D015686:Gene Products, env; D022821:RNA Splice Sites; D012342:RNA, Small Nuclear; D017412:Ribonucleoprotein, U1 Small Nuclear; D017413:Ribonucleoprotein, U2 Small Nuclear; D025521:Tumor Suppressor Proteins; C405483:U1 small nuclear RNA; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases