The Interaction of Prp2 with a Defined Region of the Intron Is Required for the First Splicing Reaction

Abstract

In Saccharomyces cerevisiae, the 3' splice site is not required for the first catalytic reaction of splicing. We have previously reported that at least 24 nucleotides downstream of the branch point is required for the first reaction to take place, but the precatalytic spliceosome forms efficiently on the truncated pre-mRNA with only 5 nucleotides retained downstream of the branch point. The factors that mediate this length-dependent control of the first catalytic step are not known. We show here that Prp2 can be recruited to the spliceosome without interacting with pre-mRNA when the 3' tail is short. Prp2 interacts with the intron when the 3' tail is extended, which results in destabilization of Prp2 and, consequently, progression of the first reaction. An RNA segment at 23 to 33 nucleotides downstream of the branch point is necessary and sufficient for the ATP-dependent action of Prp2. We also show that Prp2 directly interacts with the carboxyl-terminal fragment of Brr2 by pulldown assays. We propose that Prp2 is recruited to the spliceosome via interaction with Brr2 and is spatially positioned to interact with this specific region of the pre-mRNA, which stimulates the ATPase activity of Prp2 to promote the progression of the first catalytic step.