Among the four known mechanisms of intron removal, three are reputedly catalyzed by RNA molecules. In the fourth mechanism, a protein endonuclease removes introns from nuclear tRNA and all archaeal RNAs. Three strictly conserved residues of the splicing endonuclease, a histidine, a lysine, and a tyrosine, were predicted to catalyze the intron cleavage reaction in a manner similar to that of the catalytic triad of ribonuclease A. Single-turnover kinetic parameters were obtained for the wild-type enzyme and two triad mutants. Mutation of histidine to alanine produced an at least ∼28-fold reduction; mutation of tyrosine to phenylalanine produced an at least ∼7-fold reduction in activity, while a histidine and tyrosine double mutation abolished cleavage. The single mutation of lysine to glutamic acid abolished RNA cleavage activity in the absence of a divalent metal but maintained a substantial level of activity in the presence of specific divalent metals. These data support important functional roles already proposed for the catalytic triad and suggest an intriguing hypothesis in which the splicing endonuclease is an intermediate in the transition from the RNA to the RNP world.

Nop56p and Nop58p are two core proteins of the box C/D snoRNPs that interact concurrently with fibrillarin and snoRNAs to function in enzyme assembly and catalysis. Here we report the 2.9 Å resolution co-crystal structure of an archaeal homolog of Nop56p/Nop58p, Nop5p, in complex with fibrillarin from Archaeoglobus fulgidus (AF) and the methyl donor S-adenosyl-L-methionine. The N-terminal domain of Nop5p forms a complementary surface to fibrillarin that serves to anchor the catalytic subunit and to stabilize cofactor binding. A coiled coil in Nop5p mediates dimerization of two fibrillarin−Nop5p heterodimers for optimal interactions with bipartite box C/D RNAs. Structural analysis and complementary biochemical data demonstrate that the conserved C-terminal domain of Nop5p harbors RNA-binding sites. A model of box C/D snoRNP assembly is proposed based on the presented structural and biochemical data.