Site-specific cross-linking analyses reveal an asymmetric protein distribution for a box C/D snoRNP.
Methylation of the ribose 2'-hydroxyl, the most widespread modification of ribosomal and splicesomal RNAs, is guided by the box C/D class of small nucleolar RNAs (snoRNAs). Box C/D small nucleolar ribonucleoproteins (snoRNPs) contain four core proteins: fibrillarin, Nop56, Nop58 and 15.5 kDa. We constructed U25 snoRNAs containing a single photoactivatable 4-thiouridine at each U position within the conserved box C/D and C'/D' motifs. Proteins assembled on the snoRNA after injection into Xenopus oocyte nuclei were identified by cross-linking, and reconstituted particles characterized by functional rescue and mutational analyses. Our data argue that box C/D snoRNPs are asymmetric, with the C' box contacting Nop56 and fibrillarin, the C box interacting with Nop58, and the D and D' boxes contacting fibrillarin. No cross-link to 15.5 kDa was detected; its binding is disrupted by 4-thiouridine substitution in position 1 of the C box. Repositioning the guide sequence of U25 upstream of box D instead of D' revealed that both C/D motifs have the potential to function as guide centers, but, surprisingly, there was no alteration in protein cross-linking.
Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06536, USA.
Niamh M
NM
Cahill
Kyle
K
Friend
Wayne
W
Speckmann
Zhu-Hong
ZH
Li
Rebecca M
RM
Terns
Michael P
MP
Terns
Joan A
JA
Steitz
D000818:Animals; D001483:Base Sequence; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D008745:Methylation; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D016601:RNA-Binding Proteins; D020718:Ribonucleoproteins, Small Nucleolar / Q000235:genetics / Q000378:metabolism*; D014981:Xenopus; D029867:Xenopus Proteins
D002868:Chromosomal Proteins, Non-Histone; C486598:Nop56 protein, Xenopus; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D016601:RNA-Binding Proteins; D020718:Ribonucleoproteins, Small Nucleolar; D029867:Xenopus Proteins; C047458:fibrillarin