The pseudouridine at position 43 in vertebrate U2 snRNA is one of the most conserved post-transcriptional modifications of spliceosomal snRNAs; the equivalent position is pseudouridylated in U2 snRNAs in different phyla including fungi, insects, and worms. Pseudouridine synthase Pus1p acts alone on U2 snRNA to form this pseudouridine in yeast  Saccharomyces cerevisiae  and mouse. Furthermore, in  S. cerevisiae , Pus1p is the only pseudouridine synthase for this position. Using an in vivo yeast cell system, we tested enzymatic activity of Pus1p from the fission yeast  Schizosaccharomyces pombe , the worm  Caenorhabditis elegans , the fruit fly  Drosophila melanogaster , and the frog  Xenopus tropicalis  We demonstrated that Pus1p from  C. elegans  has no enzymatic activity on U2 snRNA when expressed in yeast cells, whereas in similar experiments, position 44 in yeast U2 snRNA (equivalent to position 43 in vertebrates) is a genuine substrate for Pus1p from  S. cerevisiae ,  S. pombe ,  Drosophila ,  Xenopus , and mouse. However, when we analyzed U2 snRNAs from  Pus1  knockout mice and the  pus1Δ S. pombe  strain, we could not detect any changes in their modification patterns when compared to wild-type U2 snRNAs. In  S. pombe , we found a novel box H/ACA RNA encoded downstream from the  RPC10  gene and experimentally verified its guide RNA activity for positioning Ψ43 and Ψ44 in U2 snRNA. In vertebrates, we showed that SCARNA8 (also known as U92 scaRNA) is a guide for U2-Ψ43 in addition to its previously established targets U2-Ψ34/Ψ44.