An RNA Switch at the 59 Splice Site Requires ATP and the DEAD Box Protein Prp28p

site cleavage, the spliceosome rearranges to permit exon ligation (Umen and Guthrie, 1995b), the mRNA and lariat products are released (Company et al. Pre-mRNA splicing requires dramatic RNA rearrange-Since this pathway is conserved from yeast to mammals, ments hypothesized to be catalyzed by ATP-depen-it likely reflects fundamental requirements for intron rec-dent RNA unwindases of the DExD/H box family. In a ognition and catalysis. rearrangement critical for the fidelity of 5؅ splice site At the molecular level, the assembly pathway corre-recognition, a base-pairing interaction between the sponds to rearrangements of RNA–RNA interactions, 5؅ splice site and U1 snRNA must be switched for a many of which must be disrupted to allow formation mutually exclusive interaction between the 5؅ splice site and U6 snRNA. By lengthening the U1:5؅ splice example, after the 5Ј splice site is recognized by U1 site duplex, we impeded this switch in a temperature-through base pairing (Zhuang and Weiner, 1986; Sé r-dependent manner and prevented formation of the aphin et al., 1988; Siliciano and Guthrie, 1988), this inter-spliceosome's catalytic core. Using genetics, we iden-action must be switched for a mutually exclusive interac-tified the DExD/H box protein Prp28p as a potential isomerization of RNA at the 5؅ splice site and promotes 1993b). Similarly, a base-pairing interaction between U4 fidelity in splicing. and U6 must be disrupted to allow for a mutually exclusive U2/U6 interaction that comprises a major compo-Introduction nent of the spliceosome's catalytic core (Madhani and Guthrie, 1992). The mutually exclusive design of RNA In pre-mRNA splicing, the spliceosome must remove interactions may ensure coordinated and regulated as-introns with single-nucleotide precision to avoid cata-sembly of the spliceosome. Intriguingly, the assembly steps that require ATP also strophic errors. Introns are defined, however, by only require " DExD/H box " proteins (Staley and Guthrie, minimal sequences at the 5Ј splice site, 3Ј splice site, 1998; Burge et al., 1999). This protein family belongs to and branch point (Burge et al., 1999). Consequently, the a superfamily of ATPases that include RNA unwindases spliceosome must discriminate against erroneous yet (Gorbalenya and Koonin, 1993). Thus, it has been hy-similar splice sites. Sequential inspection of potential pothesized that the spliceosomal DExD/H box proteins splice sites plays a role in ensuring accuracy (Kandels-catalyze RNA rearrangements in splicing by using ATP To examine the molecular basis for fidelity in splicing, Challenging this hypothesis, none of the DExD/H box we have investigated an RNA rearrangement that allows …