Virtually all pre-mRNA introns begin with the sequence / GU and end with AG/ (where / indicates a border between an exon and an Intron). We have previously shown that the G residues at the first and last positions of the yeast act In Intron Interact during the second step of splicing. In this work, we ask If other highly conserved Intron nucleotides also take part In this / G - G/ interaction. Of special interest is the penultimate Intron nucleotlde (AGl), which Is important for the second step of splicing and is In proximity to other conserved intron nucleotides. Therefore, we tested interactions of the penultimate intron nucleotide with the second intron nucleotide (/GU) and with the branch site nucleotide. We also tested two models that predict Interactions between sets of three conserved Intron nucleotides. In addition, we used random mutagenesls and genetic selection to search for Interactions between nucleotides In the pre-mRNA. We find no evidence for other Interactions between intron nucleotides besides the interaction between the first and last intron nucleotides.
Bibliographical noteFunding Information:
We thank Cammie Lesser and Christine Guthrie for the U6 plasmids, and Guillaume Chanfreau, Pierre Legrain, and Alain Jacquier for sharing unpublished results. We are grateful to Kathleen Conklin, Hung-Ying Kao, Patricia Hilleren, and Mark Murphy for helpful comments on the manuscript. This work was supported by a grant from the NSF (MCB-9407272).