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 nucleo lar RNAs (snoRNAs) that have been categorized into two major families based on evolutionarily conserved sequence elements. U14 snoRNA is a m ember of the larger, box C/D snoRNA family and possesses nucleotide bo x 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 proce ssing. These studies also revealed that nuclear proteins that recogniz e boxes C/D are required, We have now established an in vitro U14 snoR NP assembly system to characterize protein binding. Electrophoretic mo bility-shift analysis demonstrated that all the sequences and structur es of the box C/D cove motif required for U14 processing are also nece ssary for protein binding and snoRNP assembly. These required elements include a base paired 5',3' terminal stem and the phylogenetically co nserved nucleotides of boxes C and D, The ability of other box C/D sno RNAs to compete for protein binding demonstrated that the box C/D core motif-binding proteins are common to this family of snoRNAs. UV cross linking of nuclear proteins bound to the U14 core motif identified a 6 5-kDa mouse snoRNP protein that requires boxes C and D for binding. Tw o additional core motif proteins of 55 and 50 kDa were also identified by biochemical fractionation of the in vitro-assembled U14 snoRNP com plex. Thus, the U14 snoRNP core complex is a multiprotein particle who se assembly requires nucleotide boxes C and D.