Small nucleolar RNAs (snoRNAs) are localized in the nucleolus as small ribonucleoprotein particle (snoRNP) that are RNA-protein complexes and catalyze site specific modifications on the ribosomal RNA. When compared to many protein enzymes, the snoRNPs are unique in that they use the RNA component of the snoRNP for substrate recognition by base pairing through its antisense sequence and the protein component for catalyzing the modification. Hence, each snoRNP particle has the same catalytic unit but different substrate recognition unit. One member of snoRNP, called the “box C/D snoRNP” catalyzes 2’-O-ribose methylation in the most conserved and functionally important regions of the ribosome. The box C/D snoRNP consists of a bipartite box C/D snoRNA and four core proteins in eukaryotes (Fibrillarin, Nop56p, Nop58p, and Snu13p) and three core proteins in archaea (Fibrillarin, Nop56/58p and L7Ae). The box C/D RNA acts as a guide for substrate recognition by its antisense sequence to the substrate while the proteins distribute on the bipartite box C/D RNA to catalyze the 2’-O-ribose modification. In eukaryotes, the proteins are differentially distributed on the bipartite RNA with Snu13p, Nop58p and Fibrillarin on the C/D motif and Nop56p and fibrillarin on the C’/D’ motif. In archaea, a symmetric distribution of proteins on the C/D and C’/D’ motifs is observed with L7Ae, Nop56/58p and fibrillarin on both the motifs.
In the past decade a number of RNPs with RNA guided mechanisms for catalysis have been identified. With little information available, insights on any RNP assembly and catalysis would provide a global understanding of the protein-protein, protein-RNA and RNA-RNA interactions in a RNP particle. The present study focuses on understanding the box C/D snoRNP assembly and catalysis using biochemical and crystallographic techniques.
To understand the ability of Snu13p to initiate snoRNP assmebly and its differential specificity for the box C/D bipartite RNA, a crystal structure of free Snu13p was obtained. By comparing the structure of Snu13p with its eukaryal and archaeal homologs whose structures with RNA are available, similar structural features are observed thus confirming its ability to induce RNA conformational change. In addition the high resolution structure revealed a structural divergence between the eukaryotic and the archaeal homologs that may account for their different RNA specificities. Finally, structure of the core protein complex of an archaeal box C/D snoRNP, Nop56/58-Fibrillarin complex, has been obtained. We report two crystal structures of the Nop56/58-fibrillarin complex from Pyrococcus furiosus: one with full-length protein (3.6 Å) and the other in which the C-terminal KKE/D tail of Nop56/58 is truncated (2.7 Å). In both structures, a Nop56/58-fibrillarin homodimer is formed by interactions of the coiled-coil domains of Nop56/58, confirming the generality of this previously observed arrangement. However, the conformation of Nop56/58 in the new structures differs substantially from that in the earlier structure, resulting in repositioning of fibrillarin and the cognate catalytic sites within the complex. These studies provide an attractive model of box C/D snoRNP assembly that comprises “one complex, two active site” model. The structural features facilitating this model depend on protein-protein interactions as well as the plasticity in proteins and RNA. Finally, efforts to confirm this assembly model by X-ray crystallographic techniques are described.