Mechanistic characterization of pseudouridine formation by eukaryotic H/ACA snoRNPs

The H/ACA snoRNP complex directs the formation of pseudouridines in ribosomal RNA (rRNA); however, the mechanisms by which the eukaryotic two-hairpin structured H/ACA snoRNP mediates pseudouridylation in rRNA are poorly understood. This study investigates the function of the conserved two-hairpin structure of eukaryotic H/ACA snoRNA while modifying large, structured rRNA. The biochemical analysis reveals that the two hairpins of the snoRNP complex can independently convert two uridines to two pseudouridines in long rRNA fragments. Moreover, the H/ACA snoRNP can likely unfold secondary structures in rRNA without the help of additional factors. Further, the GAR domains of Gar1 are required for pseudouridine formation, but the KKE/D extensions of Cbf5 play no role in pseudouridylation of rRNA. In summary, this study addresses the mechanism of the H/ACA snoRNP complex in modifying rRNA during ribosome biogenesis.