Crystal structure of a Cbf5-Nop10-Gar1 complex from Saccharomyces cerevisiae
Classification:
ISOMERASE/PROTEIN BINDING
Technique:
X-Ray Diffraction
Resolution:
1.9
R value free:
0.233
R value observed:
0.205
R value work:
0.204
Abstract of the PDB Structure's related Publication:
Box H/ACA ribonucleoprotein particles (RNPs) mediate pseudouridine synthesis, ribosome formation, and telomere maintenance. The structure of eukaryotic H/ACA RNPs remains poorly understood. We reconstituted functional Saccharomyces cerevisiae H/ACA RNPs with recombinant proteins Cbf5, Nop10, Gar1, and Nhp2 and a two-hairpin H/ACA RNA; determined the crystal structure of a Cbf5, Nop10, and Gar1 ternary complex at 1.9 Å resolution; and analyzed the structure-function relationship of the yeast complex. Although eukaryotic H/ACA RNAs have a conserved two-hairpin structure, isolated single-hairpin RNAs are also active in guiding pseudouridylation. Nhp2, unlike its archaeal counterpart, is largely dispensable for the activity, reflecting a functional adaptation of eukaryotic H/ACA RNPs to the variable RNA structure that Nhp2 binds. The N-terminal extension of Cbf5, a hot spot for dyskeratosis congenita mutation, forms an extra structural layer on the PUA domain. Gar1 is distinguished from the assembly factor Naf1 by containing a C-terminal extension that controls substrate turnover and the Gar1-Naf1 exchange during H/ACA RNP maturation. Our results reveal significant novel features of eukaryotic H/ACA RNPs.
Pseudouridine (Ψ) is retained to stabilize the conformation of rRNAs and plays a key role in ribosomal RNA processing, being exploited in different modification positions during pre-RNAs biogenesis (Zebarjadian et al. 1999). Cbf5 is a low-affinity binding protein (Jiang et al. 1993) and the catalytic subunit of H/ACA small nuclear ribonucleoprotein (H/ACA snoRNP) complex that catalyzes the pseudouridylation of rRNA (Zebarjadian et al. 1999). A fifth protein (Shq1) is required for H/ACA RNP biogenenesis. H/ACA snoRNAs consist minimally of a helix (P1) and a hairpin (P2) flanking bulge, with a single stranded region including the conserved ACA sequence from the helix at the 3' side (Hamma et al. 2005). H/ACA and substrate RNAs form a four-helix junction that binds to Cbf5 (NAP57 in rat and dyskerin in human), Gor1, L7Ae (Nhp2 in Eukarya) and Nop10. All four proteins determine the viability in yeast. Cbf5, Nhp2, and Nop10 are required for in vivo stability of H/ACA RNAs. Cbf5 contains a repeating KKD/E sequence domain near the C-terminus that allows for microtubules binding (Jiang et al. 1993) . Catalyzes the pseudouridylation of rRNA (including 5S and 5.8S rRNA) and small RNAs (snRNAs, snoRNAs). In Eukarya, H/ACA RNPs are essential for three fundamental cellular processes: protein synthesis, mRNA splicing, and maintenance of genome integrity. For detailed information on modified positions see the (snoRNA) table.