A TERTIARY INTERACTION IN THE TETRAHYMENA INTRON CONTRIBUTES TO SELECTION OF THE 5' SPLICE-SITE

The utilization of cryptic splice sites has been observed in a number of RNA splicing reactions. In the self-splicing group I intron of Tetrahymena thermophila, point mutations of either A(57) or A(95) promote cleavage at two sites other than the normal 5' splice site, suggesting that these nucleotides are involved in a common tertiary interaction. These results are unusual since A(57) and A(95) are neither at nor near the 5' splice site in the sequence or secondary structure. Cleavage at the alternative sites appears to occur by intron cyclization, a reaction with well-established structural and mechanistic similarities to the first step of RNA self-splicing. Alternative docking of P1 (the helix containing the 5' splice site paired to the internal guide sequence of the intron) into the catalytic core accounts for cleavage at the cryptic reaction sites. We propose that the A(57)/A(95) interaction, along with an element implicated previously (J1/2), provide structural connectivity from the reaction site in p1 to the catalytic core of the Tetrahymena intron. It seems likely that RNA splicing in general will require such tertiary interactions to position RNA helices.