Purified box C/D snoRNPs are able to reproduce site-specific 2'-O-methylation of target RNA in vitro.
Small nucleolar RNAs (snoRNAs) are associated in ribonucleoprotein particles localized to the nucleolus (snoRNPs). Most of the members of the box C/D family function in directing site-specific 2'-O-methylation of substrate RNAs. Although the selection of the target nucleotide requires the antisense element and the conserved box D or D' of the snoRNA, the methyltransferase activity is supposed to reside in one of the protein components. Through protein tagging of a snoRNP-specific factor, we purified to homogeneity box C/D snoRNPs from the yeast Saccharomyces cerevisiae. Mass spectrometric analysis demonstrated the presence of Nop1p, Nop58p, Nop56p, and Snu13p as integral components of the particle. We show that purified snoRNPs are able to reproduce the site-specific methylation pattern on target RNA and that the predicted S-adenosyl-L-methionine-binding region of Nop1p is responsible for the catalytic activity.
Galardi
Silvia
S
Department of Genetics and Molecular Biology, Cenci-Bolognetti Foundation, Institute Pasteur, University of Rome La Sapienza, Italy.
Fatica
Alessandro
A
Bachi
Angela
A
Scaloni
Andrea
A
Presutti
Carlo
C
Bozzoni
Irene
I
D001665:Binding Sites; D017124:Conserved Sequence; D005656:Fungal Proteins / Q000737:chemistry; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D008745:Methylation; D008780:Methyltransferases / Q000737:chemistry* / Q000302:isolation & purification; D009687:Nuclear Proteins / Q000737:chemistry; D009843:Oligoribonucleotides / Q000737:chemistry; D012331:RNA, Fungal / Q000737:chemistry*; D012335:RNA, Ribosomal / Q000737:chemistry; D017411:Ribonucleoproteins, Small Nuclear / Q000737:chemistry; D020718:Ribonucleoproteins, Small Nucleolar / Q000737:chemistry* / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D013379:Substrate Specificity
D005656:Fungal Proteins; D046911:Macromolecular Substances; C064793:NOP1 protein, S cerevisiae; C430407:NOP56 protein, S cerevisiae; C438199:NOP58 protein, S cerevisiae; D009687:Nuclear Proteins; D009843:Oligoribonucleotides; D012331:RNA, Fungal; D012335:RNA, Ribosomal; D017411:Ribonucleoproteins, Small Nuclear; D020718:Ribonucleoproteins, Small Nucleolar; D029701:Saccharomyces cerevisiae Proteins; C440569:Snu13 protein, S cerevisiae; C065039:RNA, ribosomal, 25S; D008780:Methyltransferases