Substitution of pre-mRNA with phosphorothioate linkages reveals a new splicing-related reaction

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.