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

RNA. 2010 Dec;16(12):2341-8. doi: 10.1261/rna.2380410. Epub 2010 Oct 20.

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 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Chromosomal Proteins, Non-Histone / metabolism
  • Cross-Linking Reagents / pharmacology
  • Dimerization
  • Efficiency
  • Models, Biological
  • Models, Molecular
  • Molecular Sequence Data
  • Nucleic Acid Conformation
  • Protein Binding
  • Pyrococcus furiosus / genetics
  • Pyrococcus furiosus / metabolism
  • RNA, Archaeal / chemistry
  • RNA, Archaeal / metabolism*
  • RNA, Guide / chemistry
  • RNA, Guide / metabolism
  • RNA, Small Nucleolar / chemistry
  • RNA, Small Nucleolar / metabolism*
  • Ribonucleoproteins, Small Nuclear / metabolism*

Substances

  • Chromosomal Proteins, Non-Histone
  • Cross-Linking Reagents
  • RNA, Archaeal
  • RNA, Guide
  • RNA, Small Nucleolar
  • Ribonucleoproteins, Small Nuclear
  • fibrillarin