A novel Nop5-sRNA interaction that is required for efficient archaeal box C/D sRNP formation.
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 di-sRNPs. 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.
Abteilung Zelluläre Biochemie, Max-Planck-Institute for Biophysical Chemistry, D-37077 Goettingen, Germany.
Homa
H
Ghalei
He-Hsuan
HH
Hsiao
Henning
H
Urlaub
Markus C
MC
Wahl
Nicholas J
NJ
Watkins
D001483:Base Sequence; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D019281:Dimerization; D004526:Efficiency; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D020027:Pyrococcus furiosus / Q000235:genetics / Q000378:metabolism; D019642:RNA, Archaeal / Q000737:chemistry / Q000378:metabolism*; D020537:RNA, Small Nucleolar / Q000737:chemistry / Q000378:metabolism*; D017411:Ribonucleoproteins, Small Nuclear / Q000378:metabolism*; D058727:RNA, Small Untranslated
D002868:Chromosomal Proteins, Non-Histone; D003432:Cross-Linking Reagents; D019642:RNA, Archaeal; D020537:RNA, Small Nucleolar; D017411:Ribonucleoproteins, Small Nuclear; C047458:fibrillarin; D058727:RNA, Small Untranslated