Pseudouridine 35 (psi35) in the branch site recognition region of yeast U2 small nuclear RNA is absolutely conserved in all eukaryotes examined. Pus7p catalyzes pseudouridylation at position 35 in Saccharomyces cerevisiae U2. The pus7 deletion strain, although viable in rich medium, is growth-disadvantaged under certain conditions. To clarify the function of U2 psi35 in yeast, we used this pus7 deletion strain to screen a collection of mutant U2 small nuclear RNAs, each containing a point mutation near the branch site recognition sequence, for a synthetic growth defect phenotype. The screen identified two U2 mutants, one containing a U40 --> G40 substitution (U40G) and another having a U40 deletion (U40Delta). Yeast strains carrying either of these U2 mutations grew as well as the wild-type strain in the selection medium, but they exhibited a temperature-sensitive growth defect phenotype when coupled with the pus7 deletion (pus7Delta). A subsequent temperature shift assay and a conditional pus7 depletion (via GAL promoter shutoff) in the U2-U40 mutant genetic background caused pre-mRNA accumulation, suggesting that psi35 is required for pre-mRNA splicing under certain conditions.