Self-splicing of Tetrahymena rRNA can proceed with phosphorothioate substitution at the splice sites

The self-excision of a 413-base intervening sequence of the 26S rRNA of Tetrahymena thermophila has been investigated using phosphorothioate-substituted RNA. Transcripts containing this intron were prepared by T7 RNA polymerase-catalyzed polymerisation using a M13 mICE10 vector in the presence of various nucleoside alpha-thiotriphosphate analogues. Wild-type transcripts incorporating phosphorothioates 5' to adenosine or uridine were inactive, whereas incorporation 5' to cytidine or guanosine allowed splicing. The first two substitutions place phosphorothioates inter alia at the 5' and 3' splice sites respectively. Mutagenesis at either site allowed phosphorothioate substitution 5' to guanosine at each splice site. This did not block splicing, suggesting that substitution at internal sites within the intron has more effect.