INTERACTIONS BETWEEN THE TERMINAL BASES OF MAMMALIAN INTRONS ARE RETAINED IN INOSINE-CONTAINING PRE-MESSENGER-RNAS

Nuclear pre-mRNA splicing has a fundamentally similar two-step mechani sm to that employed by group II self-splicing introns. It is believed that nuclear pre-mRNA splicing involves a network of RNA-RNA interacti ons which form the catalytic core of the active spliceosome. We show h ere a non-Watson-Crick interaction between the first and last guanosin e residues of a mammalian intron. As in Saccharomyces cerevisiae, subs titution of the conserved guanosines at the 5' and 3' splice sites by A and C respectively, specifically suppresses step 2 splicing defects resulting from the individual mutations. No other combination of termi nal nucleotides was able to restore splicing. We additionally provide independent evidence for an indirect interaction between other nucleot ides of the consensus splice sites during step 2 of splicing. Substitu tion of the nucleotide in the +3 position of the 5' splice site affect s competition between closely spaced AG dinucleotides at the 3' splice site, although the interaction is not via direct differential base pa iring. Finally, we show that complete substitution of guanosine residu es by inosine in a pre-mRNA has only a modest effect upon step 2 of sp licing, although earlier spliceosome assembly steps are impaired. Pred ictions can thus be made about the precise configuration of the non-Wa tson-Crick interaction between the terminal residues.