Structure of a functional ribonucleoprotein pseudouridine synthase bound to a substrate RNA

Abstract

Box H/ACA small nucleolar and Cajal body ribonucleoprotein particles comprise the most complex pseudouridine synthases and are essential for ribosome and spliceosome maturation. The multistep and multicomponent-mediated enzyme mechanism remains only partially understood. Here we report a crystal structure at 2.35 Å of a substrate-bound functional archaeal enzyme containing three of the four proteins, Cbf5, Nop10 and L7Ae, and a box H/ACA RNA that reveals detailed information about the protein-only active site. The substrate RNA, containing 5-fluorouridine at the modification position, is fully docked and catalytically rearranged by the enzyme in a manner similar to that seen in two stand-alone pseudouridine synthases. Structural analysis provides a mechanism for plasticity in the diversity of guide RNA sequences used and identifies a substrate-anchoring loop of Cbf5 that also interacts with Gar1 in unliganded structures. Activity analyses of mutated proteins and RNAs support the structural findings and further suggest a role of the Cbf5 loop in regulation of enzyme activity.

Pseudouridines in spliceosomal snRNAs

“…To address this question, a great deal of efforts have been expended to solve the crystal structures of archaeal box H/ACA snRNP complexes. As a result, the structures of various forms, from the initial complex of three core proteins to a complete, substrate-bound box H/ACA RNP, have been solved (Li and Ye, 2006;Manival et al, 2006;Rashid et al, 2006;Liang et al, 2007;Duan et al, 2009;Liang et al, 2009). A detailed picture of how this most complex pseudouridylase modifies its substrate has now become available.…”

Reconstitution and structural analysis of the yeast box H/ACA RNA-guided pseudouridine synthase

“…In the substrate-loaded archaeal H/ACA RNP structures, it adopts a closed conformation, pinching the substrate at the active site (Duan et al 2009;Liang et al 2009), while in the substrate-free state or an inactive substrate complex, the thumb is partially disordered and docks at Gar1 in an open conformation ( Fig. 5C; Li and Ye 2006;Liang et al 2007).…”

“…1F). In the structure of substrate-loaded archaeal H/ACA RNPs (Duan et al 2009;Liang et al 2009), the upper stem coaxially stacks with the substrate-guide duplex formed between the 39 arm of the substrate and the 59 guide. Proper anchoring of the upper stem by L7Ae would be important to correctly place the substrate at the active site.…”

“…The crystal structure of Pyrococcus furiosus (Pf) H/ACA RNP assembled with a single-hairpin H/ACA RNA demonstrates that the upper stem of the guide RNA is bound sequentially by L7Ae, Nop10, and the catalytic domain of Cbf5, and the lower stem and the ACA motif are anchored at the PUA domain of Cbf5 (Li and Ye 2006). The substrate is recruited through base-pairing with the bipartite guides and extensive protein interactions, precisely placing the target uridine at the active site (Duan et al 2009;Liang et al 2009). …”

“…Catalytically active archaeal H/ACA RNPs have been reconstituted with recombinant proteins (Baker et al 2005;Charpentier et al 2005) and are amenable for crystallographic analysis (Hamma et al 2005;Li and Ye 2006;Manival et al 2006;Rashid et al 2006;Liang et al 2007Liang et al , 2009Ye 2007;Duan et al 2009). The crystal structure of Pyrococcus furiosus (Pf) H/ACA RNP assembled with a single-hairpin H/ACA RNA demonstrates that the upper stem of the guide RNA is bound sequentially by L7Ae, Nop10, and the catalytic domain of Cbf5, and the lower stem and the ACA motif are anchored at the PUA domain of Cbf5 (Li and Ye 2006).…”