The guide snoRNAs and their target RNAs. The class (A) C/D and (B) H/ACA snoRNAs (grey) contain conserved and class-specific sequence motifs ‘boxes’ (blue) and unique guide regions that define their respective target RNA site(s) (magenta). The sites targeted for nucleotide modification are marked with a star.

The conserved C/D methyltransferase fibrillarin. Crystal structure of (A) the archaeal fibrillarin (1FBN) and (B) of the double-stranded DNA methyltransferase HhaI (1MHT). The catalytic domains are colored according to their secondary structural elements (red: α-helices; violet: β-strand; grey: variable regions). S-adenosylmethionine (AdoMet) binds at a conserved site (yellow star) of these related enzymes. Structural details of fibrillarin interaction with the sno(s)RNA-target RNA complex are unknown. HhaI flips out the nucleobase to be modified in dsDNA (blue). (C) Fibrillarin catalyzes the ribose-2′-O-ribose methylation of its RNA substrates by converting AdoMet to S-adenosylhomocycteine (AdoHcy).

The conserved H/ACA Ψ synthase Cbf5. Crystal structures of (A) the archaeal H/ACA Ψ synthase Cbf5 (2APO) and (B) the E. coli tRNA Ψ synthase TruB (1K8W). The catalytic domains are colored according to their conserved secondary structural elements (yellow: α-helices; green: β-strand; grey: PUA domain and variable regions). The site of uridine isomerization (red star) is surrounded by highly conserved residues in Cbf5 and TruB. The TruB induces a base flipping of its tRNA substrate (blue). Structural differences between Cbf5 and TruB reflect differences in RNA substrate specificity; for example, a characteristic TruB peptide sequence (coral) is absent from Cbf5 homologs. (C) The isomerization of uridine to Ψ requires an overall 120° rotation of the uracil base.

K-turn recognition by L7Ae. (A) Sequence of a canonical K-turn motif with conserved C/D nucleotides in blue. (B) Crystal structure of the archaea L7Ae protein (gold) bound to the canonical K-turn motif of C/D sRNAs (1RLG). RNA elements are colored as in (A). The K-turn adopts a characteristic ∼60° bend in the phosphate backbone and L7Ae makes several interactions with conserved structural features of this motif.

Crystal structure of the archaeal fibrillarin-Nop5 tetrameric complex (1NT2). Nop5 (blue) interacts with fibrillarin (Fib; red) through its N- and C-terminal domains. The C-terminal domain of Nop5 binds to the guide RNA and is positioned near the catalytic site (yellow star) of fibrillarin. The N- and C-terminal domains of Nop5 are connected by a coiled–coiled motif that facilitates dimerization of the Fib-Nop5 complex.

Class C/D sno(s)RNP architecture. (A) The archaeal C/D sRNP assembles with a copy of each core C/D proteins (colored as in Figures 4 and 5) at both the C/D and C′/D′ RNA motifs (blue) of the sRNA (grey), and guides methylation of a RNA nucleotide (pink) at each site. The observed quaternary structure of the fibrillarin-Nop5 complex would position two fibrillarin catalytic sites (yellow star) ∼80 Å apart. In contradiction to this observation, the distance between catalytic C/D and C′/D′ sites of the sRNAs is highly conserved and only ∼25–35 Å, suggesting the coiled–coiled interactions (transparent coloring) of Nop5 may be reorganized and/or disrupted in the fully assembled sRNP. (B) The C/D snoRNP is proposed to assemble into a pseudo-symmetric architecture. In contrast to the archaeal L7Ae protein, the Snu13/15.5 kDa protein appears to bind solely at the C/D site of the snoRNA. The Nop5 paralogs, Nop56 and Nop58, recognize the C and C′ Box elements, respectively, while a copy of fibrillarin (Fib) interacts with each D and D′ Box, consistent with its role in the catalytic center (yellow star).

Crystal structure of the archaeal Cbf5-Gar1-Nop10 heterotrimer (2EY4). The catalytic domain of Cbf5 (yellow) is surrounded by the Cbf5 PUA domain (green), Nop10 (blue) and Gar1 (purple) proteins. The Cbf5 PUA domain contributes to the extended RNA-binding surface of this protein. Nop10 packs against the back-side of the catalytic site (red star) and contributes to the organization and stabilization of the active site itself. Gar1 contacts Cbf5 through conserved hydrophobic interactions that may modulate substrate RNA loading and release.

Class H/ACA sno(s)RNP architecture. (A) The crystal structure of the archaeal H/ACA core proteins with a guide sRNA (2HVY). (B) The H/ACA sRNA secondary structure and color scheme (base-paired stems and apical loop: grey; conserved ACA Box and K-loop nucleotides: blue; pseudouridylation pocket: red). The sRNA is recognized by Cbf5, L7Ae and Nop10 (colored as in Figures 4 and 7). The ACA Box and proximal stem are bound by the Cbf5 PUA domain, while the apical stem and K-loop are bound by Nop10 and L7Ae, respectively. These interactions act as conserved molecular clamps that precisely position the sRNA pseudouridylation pocket at the catalytic site of Cbf5 (red star). (C) The H/ACA snoRNP is proposed to adopt a bipartite structure with a copy of the core H/ACA proteins assembled at each hairpin motif of the snoRNA. The core H/ACA interactions are expected to be similar between eukaryotic and archaeal sRNPs, but some interactions with the snoRNA are expected to be unique to eukaryotes. For example, the Nhp2-snoRNA interaction differs from the L7Ae-sRNA K-turn (or K-loop) interaction.