Exclusive interaction of the 15.5 kD protein with the terminal box C/D motif of a methylation guide snoRNP.
Box C/D small nucleolar RNAs (snoRNAs) direct site-specific methylation of ribose 2'-hydroxyls in ribosomal and spliceosomal RNAs. To identify snoRNA functional groups contributing to assembly of an active box C/D snoRNP in Xenopus oocytes, we developed an in vivo nucleotide analog interference mapping procedure. Deleterious substitutions consistent with requirements for binding the 15.5 kD protein clustered within the terminal box C/D motif only. In vitro analyses confirmed a single interaction site for recombinant 15.5 kD protein and identified the exocyclic amine of A89 in box D as essential for binding. Our results argue that the 15.5 kD protein interacts asymmetrically with the two sets of conserved box C/D elements and that its binding is primarily responsible for the stability of box C/D snoRNAs in vivo.
Szewczak
Lara B Weinstein
LB
Department of Molecular Biophysics and Biochemistry, New Haven, CT 06536, USA.
DeGregorio
Suzanne J
SJ
Strobel
Scott A
SA
Steitz
Joan A
JA
D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D017124:Conserved Sequence; D008745:Methylation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D009865:Oocytes; D011485:Protein Binding; D020537:RNA, Small Nucleolar / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000737:chemistry / Q000378:metabolism*; D013873:Thionucleotides / Q000737:chemistry / Q000378:metabolism; D014981:Xenopus / Q000378:metabolism
D020537:RNA, Small Nucleolar; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D017411:Ribonucleoproteins, Small Nuclear; D013873:Thionucleotides; D000241:Adenosine