Dissecting the role of conserved box C/D sRNA sequences in di-sRNP assembly and function.
In all three kingdoms of life, nucleotides in ribosomal RNA (rRNA) are post-transcriptionally modified. One type of chemical modification is 2'-O-ribose methylation, which is, in eukaryotes and archaea, performed by box C/D small ribonucleoproteins (box C/D sRNPs in archaea) and box C/D small nucleolar ribonucleoproteins (box C/D snoRNPs in eukaryotes), respectively. Recently, the first structure of any catalytically active box C/D s(no)RNP determined by electron microscopy and single particle analysis surprisingly demonstrated that they are dimeric RNPs. Mutational analyses of the Nop5 protein interface suggested that di-sRNP formation is also required for the in vitro catalytic activity. We have now analyzed the functional relevance of the second interface, the sRNA interface, within the box C/D di-sRNP. Mutations in conserved sequence elements of the sRNA, which allow sRNP assembly but which severely interfere with the catalytic activity of box C/D sRNPs, prevent formation of the di-sRNP. In addition, we can observe the dimeric box C/D sRNP architecture with a different box C/D sRNP, suggesting that this architecture is conserved. Together, these results provide further support for the functional relevance of the di-sRNP architecture and also provide a structural explanation for the observed defects in catalysis of 2'-O-ribose methylation.
Bleichert
Franziska
F
Department of Genetics, Yale University, New Haven, CT 06520, USA.
Baserga
Susan J
SJ
D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D017124:Conserved Sequence; D019281:Dimerization; D017015:Methanococcales / Q000235:genetics; D009154:Mutation; D019642:RNA, Archaeal / Q000737:chemistry* / Q000378:metabolism; D058727:RNA, Small Untranslated / Q000737:chemistry* / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000378:metabolism
D019843:Archaeal Proteins; D019642:RNA, Archaeal; D058727:RNA, Small Untranslated; D012261:Ribonucleoproteins