Determinants of the inherent strength of human 5' splice sites.
We previously showed that the authentic 5' splice site (5'ss) of the first exon in the human beta-globin gene is intrinsically stronger than a cryptic 5'ss located 16 nucleotides upstream. Here we examined by mutational analysis the contribution of individual 5'ss nucleotides to discrimination between these two 5'ss. Based on the in vitro splicing efficiencies of a panel of 26 wild-type and mutant substrates in two separate 5'ss competition assays, we established a hierarchy of 5'ss and grouped them into three functional subclasses: strong, intermediate, and weak. Competition between two 5'ss from different subclasses always resulted in selection of the 5'ss that belongs to the stronger subclass. Moreover, each subclass has different characteristic features. Strong and intermediate 5'ss can be distinguished by their predicted free energy of base-pairing to the U1 snRNA 5' terminus (DeltaG). Whereas the extent of splicing via the strong 5'ss correlates well with the DeltaG, this is not the case for competition between intermediate 5'ss. Weak 5'ss were used only when the competing authentic 5'ss was inactivated by mutation. These results indicate that extensive complementarity to U1 snRNA exerts a dominant effect for 5'ss selection, but in the case of competing 5'ss with similarly modest complementarity to U1, the role of other 5'ss features is more prominent. This study reveals the importance of additional submotifs present in certain 5'ss sequences, whose characterization will be critical for understanding 5'ss selection in human genes.
Roca
Xavier
X
Cold Spring Harbor Laboratory, PO Box 100, Cold Spring Harbor, NY 11724, USA.
Sachidanandam
Ravi
R
Krainer
Adrian R
AR
D017398:Alternative Splicing / Q000235:genetics; D020029:Base Pairing; D001483:Base Sequence; D019295:Computational Biology; D005091:Exons / Q000235:genetics; D005914:Globins / Q000235:genetics; D006801:Humans; D007438:Introns / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D022821:RNA Splice Sites / Q000235:genetics*; D012326:RNA Splicing / Q000235:genetics*; D012342:RNA, Small Nuclear / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics
D012322:RNA Precursors; D022821:RNA Splice Sites; D012342:RNA, Small Nuclear; C405483:U1 small nuclear RNA; D005914:Globins