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In vitro transcription in the presence of a nucleoside 5'-O-(1-thiotriphosphate) has been used to prepare pre-mRNA analogues of the small intron of a rabbit beta-globin gene and flanking exon sequences. Incubation of transcripts prepared with adenosine 5'-O-(1-thiotriphosphate) in a HeLa cell nuclear extract showed that the presence of the thionucleotide in a transcript inhibited splicing, but a novel product was formed by cleavage three nucleotides upstream of the 3' splice site. This product was formed with the same kinetics as the intermediates of a normal splicing reaction, and its formation depended on the presence of intact small nuclear RNAs U1, U2, and U6. We conclude that activation of 3' splice site-proximal sequences need not be linked to exon ligation.

Type

Journal article

Journal

J Biol Chem

Publication Date

05/09/1988

Volume

263

Pages

12295 - 12304

Keywords

Adenosine Triphosphate, Affinity Labels, Animals, Base Sequence, DNA-Directed RNA Polymerases, Electrophoresis, Polyacrylamide Gel, Endoribonucleases, Exons, Exonucleases, Globins, HeLa Cells, Humans, Introns, Molecular Sequence Data, RNA Precursors, RNA Splicing, RNA, Messenger, RNA, Small Nuclear, Rabbits, T-Phages, Thionucleotides, Transcription, Genetic