Pseudouridylation of yeast U2 snRNA is catalyzed by either an RNA-guided or RNA-independent mechanism

Yeast U2 small nuclear RNA ( snRNA) contains three pseudouridines (Psi 35, Psi 42, and Psi 44). Pus7p and Pus1p catalyze the formation of Psi 35 and Psi 44, respectively, but the mechanism of Psi 42 formation remains unclear. Using a U2 substrate containing a single P-32 radiolabel at position 42, we screened a GST-ORF library for pseudouridylase activity. Surprisingly, we found a Psi 42-specific pseudouridylase activity that coincided with Nhp2p, a protein component of a Box H/ACA sno/scaRNP ( small nucleolar/Cajal body-specific ribonucleoprotein). When isolated by tandem affinity purification ( TAP), the other protein components of the H/ACA sno/scaRNP also copurified with the pseudouridylase activity. Micrococcal nuclease-treated TAP preparations were devoid of pseudouridylase activity; however, activity was restored upon addition of RNAs from TAP preparations. Pseudouridylation reconstitution using RNAs from a Box H/ACA RNA library identified snR81, a snoRNA known to guide rRNA pseudouridylation, as the Psi 42-specific guide RNA. Using the snR81-deletion strain, Nhp2p- or Cbf5p-conditional depletion strain, and a cbf5 mutation strain, we further demonstrated that the pseudouridylase activity is dependent on snR81 snoRNP in vivo. Our data indicate that snRNA pseudouridylation can be catalyzed by both RNA-dependent and RNA-independent mechanisms.