In vitro assembly of the mouse U14 snoRNP core complex and identification of a 65-kDa box C/D-binding protein.
The eukaryotic nucleolus contains a diverse population of small nucleolar RNAs (snoRNAs) that have been categorized into two major families based on evolutionarily conserved sequence elements. U14 snoRNA is a member of the larger, box C/D snoRNA family and possesses nucleotide box C and D consensus sequences. In previous studies, we have defined a U14 box C/D core motif that is essential for intronic U14 snoRNA processing. These studies also revealed that nuclear proteins that recognize boxes C/D are required. We have now established an in vitro U14 snoRNP assembly system to characterize protein binding. Electrophoretic mobility-shift analysis demonstrated that all the sequences and structures of the box C/D core motif required for U14 processing are also necessary for protein binding and snoRNP assembly. These required elements include a base paired 5',3' terminal stem and the phylogenetically conserved nucleotides of boxes C and D. The ability of other box C/D snoRNAs to compete for protein binding demonstrated that the box C/D core motif-binding proteins are common to this family of snoRNAs. UV crosslinking of nuclear proteins bound to the U14 core motif identified a 65-kDa mouse snoRNP protein that requires boxes C and D for binding. Two additional core motif proteins of 55 and 50 kDa were also identified by biochemical fractionation of the in vitro-assembled U14 snoRNP complex. Thus, the U14 snoRNP core complex is a multiprotein particle whose assembly requires nucleotide boxes C and D.
Watkins
N J
NJ
Department of Biochemistry, North Carolina State University, Raleigh 27695-7622, USA.
Newman
D R
DR
Kuhn
J F
JF
Maxwell
E S
ES
D000818:Animals; D001202:Ascitic Fluid / Q000166:cytology; D002457:Cell Extracts; D003432:Cross-Linking Reagents; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D009865:Oocytes; D011485:Protein Binding; D012342:RNA, Small Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017411:Ribonucleoproteins, Small Nuclear / Q000096:biosynthesis* / Q000737:chemistry; D014466:Ultraviolet Rays; D014981:Xenopus
D002457:Cell Extracts; D003432:Cross-Linking Reagents; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; D017411:Ribonucleoproteins, Small Nuclear