Comparative Analysis of the 15.5kD Box C/D snoRNP Core Protein in the Primitive Eukaryote Giardia lamblia Reveals Unique Structural and Functional Features

摘要

Box C/D ribonucleoproteins (RNP) guide then20n-O-methylation of targeted nucleotides in archaeal andneukaryotic rRNAs. The archaeal L7Ae and eukaryotic 15.5kDnbox C/D RNP core protein homologues initiate RNP assemblynby recognizing kink-turn (K-turn) motifs. The crystal structurenof the 15.5kD core protein fromthe primitive eukaryote Giardianlamblia is described here to a resolution of 1.8 Å. The Giardian15.5kD protein exhibits the typical Ru0002βu0002R sandwich foldnexhibited by both archaeal L7Ae and eukaryotic 15.5kD pro-nteins. Characteristic of eukaryotic homologues, the Giardian15.5kD protein binds the K-turn motif but not the variantnK-loop motif. The highly conserved residues of loop 9, criticalnfor RNA binding, also exhibit conformations similar to those ofnthe human 15.5kD protein when bound to the K-turn motif.nHowever, comparative sequence analysis indicated a distinctnevolutionary position between Archaea and Eukarya. Indeed, assessment of the Giardia 15.5kD protein in denaturing experimentsndemonstrated an intermediate stability in protein structure when compared with that of the eukaryotic mouse 15.5kD and archaealnMethanocaldococcus jannaschii L7Ae proteins. Most notable was the ability of the Giardia 15.5kD protein to assemble in vitro ancatalytically active chimeric box C/D RNP utilizing the archaeal M. jannaschii Nop56/58 and fibrillarin core proteins. In contrast, ancatalytically competent chimeric RNP could not be assembled using the mouse 15.5kD protein. Collectively, these analyses suggestnthat the G. lamblia 15.5kD protein occupies a unique position in the evolution of this box C/DRNP core protein retaining structuralnand functional features characteristic of both archaeal L7Ae and higher eukaryotic 15.5kD homologues