Pseudouridines in U2 snRNA stimulate the ATPase activity of Prp5 during spliceosome assembly

Pseudouridine (Psi) is the most abundant internal modification identified in RNA, and yet little is understood of its effects on downstream reactions. Yeast U2 snRNA contains three conserved Psi s (Psi 35, Psi 42, and Psi 44) in the branch site recognition region (BSRR), which base pairs with the pre-mRNA branch site during splicing. Here, we show that blocks to pseudouridylation at these positions reduce the efficiency of pre-mRNA splicing, leading to growth-deficient phenotypes. Restoration of pseudouridylation at these positions using designer snoRNAs results in near complete rescue of splicing and cell growth. These Psi s interact genetically with Prp5, an RNA-dependent ATPase involved in monitoring the U2 BSRR-branch site base-pairing interaction. Biochemical analysis indicates that Prp5 has reduced affinity for U2 snRNA that lacks Psi 42 and Psi 44 and that Prp5 ATPase activity is reduced when stimulated by U2 lacking Psi 42 or Psi 44 relative to wild type, resulting in inefficient spliceosome assembly. Furthermore, in vivo DMS probing analysis reveals that pseudouridylated U2, compared to U2 lacking Psi 42 and Psi 44, adopts a slightly different structure in the branch site recognition region. Taken together, our results indicate that the Psi s in U2 snRNA contribute to pre-mRNA splicing by directly altering the binding/ATPase activity of Prp5.