Interaction of the yeast DExH-box RNA helicase Prp22p with the 3 ' splice site during the second step of nuclear pre-mRNA splicing

Using site-specific incorporation of the photochemical cross-linking reagen t 4-thiouridine, we demonstrate the previously unknown association of two p roteins with yeast 3' splice sites. One of these is an unidentified similar to 122 kDa protein that cross-links to 3' splice sites during formation of the prespliceosome. The other factor is the DExH-box RNA helicase, Prp22p. With substrates functional in the second step of splicing, only very weak cross-linking of Prp22p to intron sequences at the 3' splice site is observ ed. In contrast, substrates blocked at the second step exhibit strong cross -linking of Prp22 to intron sequences at the 3' splice site, but not to adj acent exon sequences. In vitro reconstitution experiments also show that th e association of Prp22p with intron sequences at the 3' splice site is depe ndent on Prp16p and does not persist when release of mature mRNA from the s pliceosome is blocked. Taken together, these results suggest that the 3' sp lice site of yeast introns is contacted much earlier than previously envisi oned by a protein of similar to 120 kDa, and that a transient association o f Prp22p with the 3' splice site occurs between the first and second cataly tic steps.