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Single nucleotide changes to the sequence between two alternative 5' splice sites, separated by 25 nucleotides in a beta-globin gene derivative, caused substantial shifts in pre-mRNA splicing preferences, both in vivo and in vitro. An activating sequence for splicing was located. Models for the recognition by U1 small nuclear ribonucleoproteins (snRNPs) of competing 5' splice sites were tested by altering the distance separating the two sites. Use of the upstream splice site declined sharply when it was separated from the downstream (natural) site by distances of 40 nucleotides or more. This effect was reversed in vivo, but not in vitro, by altering the upstream sequence to that of a consensus 5' splice site sequence. Dilution of an extract used for splicing in vitro shifted preferences when the sites were close towards the downstream site. We conclude that the mechanism of selection depends on the distance apart of the potential splice sites and that with close sites steric interference between factors bound to both sites may impede splicing and affect splicing preferences.

Type

Journal article

Journal

J Mol Biol

Publication Date

20/01/1991

Volume

217

Pages

265 - 281

Keywords

Animals, Base Sequence, Cloning, Molecular, DNA Mutational Analysis, Globins, In Vitro Techniques, Molecular Sequence Data, RNA Splicing, RNA, Messenger, Rabbits, Ribonucleoproteins, Ribonucleoproteins, Small Nuclear, Structure-Activity Relationship