Determinants of the inherent strength of human 5' splice sites

Roca, Xavier, Sachidanandam, Ravi, Krainer, Adrian R. (May 2005) Determinants of the inherent strength of human 5' splice sites. Rna, 11 (5). pp. 683-98. ISSN 1355-8382 (Print)

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DOI: 10.1261/rna.2040605


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.

Item Type: Paper
Uncontrolled Keywords: Alternative Splicing genetics Base Pairing Base Sequence Computational Biology Exons genetics Globins genetics Humans Introns genetics Molecular Sequence Data Mutation genetics RNA Precursors chemistry genetics metabolism RNA Splice Sites genetics RNA Splicing genetics RNA Small Nuclear genetics metabolism Substrate Specificity Thermodynamics 5'ss
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > sRNA
CSHL Authors:
Communities: CSHL labs > Krainer lab
CSHL labs > Sachidanandam lab
Depositing User: CSHL Librarian
Date: May 2005
Date Deposited: 06 Jan 2012 16:10
Last Modified: 09 Apr 2014 15:51
PMCID: PMC1370755
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