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. 1998 May;4(5):582–593. doi: 10.1017/s1355838298980128

In vitro assembly of the mouse U14 snoRNP core complex and identification of a 65-kDa box C/D-binding protein.

N J Watkins 1, D R Newman 1, J F Kuhn 1, E S Maxwell 1
PMCID: PMC1369641  PMID: 9582099

Abstract

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.

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Selected References

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