Evidence for a Prp24 binding site in U6 snRNA and in a putative intermediate in the annealing of U6 and U4 snRNAs.

A mutation (U4‐G14C) that destabilizes the base‐pairing interaction between U4 and U6 snRNAs causes the accumulation of a novel complex containing U4, U6 and Prp24, a protein with RNA binding motifs. An analysis of suppressors of this cold‐sensitive mutant led to the hypothesis that this complex is normally a transient intermediate in the annealing of U4 and U6. It was proposed that Prp24 must be released to form a fully base‐paired U4/U6 snRNP. By using a chemical probing method we have tested the prediction that nucleotides A40‐C43 in U6 mediate the binding of Prp24. Consistent with the location of recessive suppressors in U6, we find that residues A40‐C43 are protected from chemical modification in U4/U6 complexes from the U4‐G14C mutant but not from the wild‐type or suppressor strains carrying mutations in U6 or PRP24. Furthermore, we find that base‐pairing is substantially disrupted in the mutant complexes. Notably, the base‐paired structure is restored in recessive suppressors despite the presence of a mismatched base‐pair at the U4‐G14C site. Our results support the model that Prp24 binds to U6 to promote its association with U4, but must dissociate to allow complete annealing.