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Determinants of the inherent strength of human 5' splice sites.
RNA. 2005 May; 11(5):683-98.RNA

Abstract

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.

Authors+Show Affiliations

Cold Spring Harbor Laboratory, PO Box 100, Cold Spring Harbor, NY 11724, USA.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

15840817

Citation

Roca, Xavier, et al. "Determinants of the Inherent Strength of Human 5' Splice Sites." RNA (New York, N.Y.), vol. 11, no. 5, 2005, pp. 683-98.
Roca X, Sachidanandam R, Krainer AR. Determinants of the inherent strength of human 5' splice sites. RNA. 2005;11(5):683-98.
Roca, X., Sachidanandam, R., & Krainer, A. R. (2005). Determinants of the inherent strength of human 5' splice sites. RNA (New York, N.Y.), 11(5), 683-98.
Roca X, Sachidanandam R, Krainer AR. Determinants of the Inherent Strength of Human 5' Splice Sites. RNA. 2005;11(5):683-98. PubMed PMID: 15840817.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Determinants of the inherent strength of human 5' splice sites. AU - Roca,Xavier, AU - Sachidanandam,Ravi, AU - Krainer,Adrian R, PY - 2005/4/21/pubmed PY - 2005/5/25/medline PY - 2005/4/21/entrez SP - 683 EP - 98 JF - RNA (New York, N.Y.) JO - RNA VL - 11 IS - 5 N2 - 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. SN - 1355-8382 UR - https://www.unboundmedicine.com/medline/citation/15840817/Determinants_of_the_inherent_strength_of_human_5'_splice_sites_ L2 - http://www.rnajournal.org/cgi/pmidlookup?view=long&pmid=15840817 DB - PRIME DP - Unbound Medicine ER -