A novel Nop5-sRNA interaction that is required for efficient archaeal box C/D sRNP formation

Abstract

Archaeal and eukaryotic box C/D RNPs catalyze the 2′-O-methylation of ribosomal RNA, a modification that is essential for the correct folding and function of the ribosome. Each archaeal RNP contains three core proteins - L7Ae, Nop5, and fibrillarin (methyltransferase) - and a box C/D sRNA. Base-pairing between the sRNA guide region and the rRNA directs target site selection with the C/D and related C′/D′ motifs functioning as protein binding sites. Recent structural analysis of in vitro assembled archaeal complexes has produced two divergent models of box C/D sRNP structure. In one model, the complex is proposed to be monomeric, while the other suggests a dimeric sRNP. The position of the RNA in the RNP is significantly different in each model. We have used UV-cross-linking to characterize protein - RNA contacts in the in vitro assembled Pyrococcus furiosus box C/D sRNP. The P. furiosus sRNP components assemble into complexes that are the expected size of disRNPs. Analysis of UV-induced protein - RNA cross-links revealed a novel interaction between the ALFR motif, in the Nop domain of Nop5, and the guide/spacer regions of the sRNA. We show that the ALFR motif and the spacer sequence adjacent to box C or C′ are important for box C/D sRNP assembly in vitro. These data therefore reveal new RNA - protein contacts in the box C/D sRNP and suggest a role for Nop5 in substrate binding and/or release. Published by Cold Spring Harbor Laboratory Press. Copyright © 2010 RNA Society.