Site-specific cross-linking analyses reveal an asymmetric protein distribution for a box C/D snoRNP

Abstract

Methylation of the ribose 2¢-hydroxyl, the most widespread modi®cation 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: ®brillarin, 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 identi®ed 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 ®brillarin, the C box interacting with Nop58, and the D and D¢ boxes contacting ®bril-larin. 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.

Pol I Transcription and Pre-rRNA Processing Are Coordinated in a Transcription-dependent Manner in Mammalian Cells

“…Nop58 was chosen because immunoprecipitation studies in yeast show that fibrillarin binds to box C/D RNA independent of Nop58 (Gautier et al, 1997; Tollervey, 1999, 2000) and additional site-specific cross-linking analyses confirm and map the independent association of Nop58 and fibrillarin with their respective box C/D elements (Cahill et al, 2002). Therefore, fibrillarin can only be coprecipitated with Nop58 through being a part of snoRNPs.…”

Assembly of the Archaeal Box C/D sRNP Can Occur via Alternative Pathways and Requires Temperature-facilitated sRNA Remodeling

“…12,21 Two highly related nucleolar proteins, Nop58 and Nop56, differentially bind the box C/D and C′/ D′ motifs, respectively, while the methyltransferase enzyme fibrillarin interacts with both motifs. [22][23][24][25][26] The eukaryotic snoRNP is said to be asymmetric with 15.5 kDa, Nop58, and fibrillarin binding to the terminal box C/D motif and core proteins Nop56 and fibrillarin binding the internal C′/D′ motif. 26,27 Box C/D snoRNA-like RNAs, or sRNAs, are abundant in archaeal organisms and guide nucleotide methylation of rRNAs and tRNAs.…”

“…[22][23][24][25][26] The eukaryotic snoRNP is said to be asymmetric with 15.5 kDa, Nop58, and fibrillarin binding to the terminal box C/D motif and core proteins Nop56 and fibrillarin binding the internal C′/D′ motif. 26,27 Box C/D snoRNA-like RNAs, or sRNAs, are abundant in archaeal organisms and guide nucleotide methylation of rRNAs and tRNAs. 14,28 In vitro assembly of catalytically active archaeal box C/D sRNPs has recently been accomplished and used to structurally and functionally characterize sRNP complexes.…”

Conserved spacing between the box C/D and C′/D′ RNPs of the archaeal box C/D sRNP complex is required for efficient 2′-O-methylation of target RNAs

“…In eukaryotes, biochemical and genetic experiments have defined four box C/D snoRNP core proteins; the 15.5kD protein (Snu13p in yeast), nucleolar proteins Nop56 and Nop58, and the methylase fibrillarin (Gauthier et al 1997;Lafontaine andTollervey 1999, 2000;Newman et al 2000;Watkins et al 2000). In vivo crosslinking of the core proteins has indicated a differential distribution on the terminal box C/D core and internal CЈ/DЈ motif (Cahill et al 2002;Szewczak et al 2002). The 15.5kD protein, which initiates snoRNP assembly, binds only the terminal core motif, whereas the Nop56 and Nop58 proteins crosslink to the CЈ/DЈ and core C/D motifs, respectively.…”