The coiled-coil domain of the Nop56/58 core protein is dispensable for sRNP assembly but is critical for archaeal box C/D sRNP-guided nucleotide methylation

Abstract

Archaeal box C/D sRNAs guide the methylation of specific nucleotides in archaeal ribosomal and tRNAs. Three Methanocaldococcus jannaschii sRNP core proteins (ribosomal protein L7, Nop56/58, and fibrillarin) bind the box C/D sRNAs to assemble the sRNP complex, and these core proteins are essential for nucleotide methylation. A distinguishing feature of the Nop56/58 core protein is the coiled-coil domain, established by alpha-helices 4 and 5, that facilitates Nop56/58 self-dimerization in vitro. The function of this coiled-coil domain has been assessed for box C/D sRNP assembly, sRNP structure, and sRNP-guided nucleotide methylation by mutating or deleting this protein domain. Protein pull-down experiments demonstrated that Nop56/58 self-dimerization and Nop56/58 dimerization with the core protein fibrillarin are mutually exclusive protein:protein interactions. Disruption of Nop56/58 homodimerization by alteration of specific amino acids or deletion of the entire coiled-coil domain had no obvious effect upon core protein binding and sRNP assembly. Site-directed mutation of the Nop56/58 homodimerization domain also had no apparent effect upon either box C/D RNP- or C'/D' RNP-guided nucleotide modification. However, deletion of this domain disrupted guided methylation from both RNP complexes. Nuclease probing of the sRNP assembled with Nop56/58 proteins mutated in the coiled-coil domain indicated that while functional complexes were assembled, box C/D and C'/D' RNPs were altered in structure. Collectively, these experiments revealed that the self-dimerization of the Nop56/58 coiled-coil domain is not required for assembly of a functional sRNP, but the coiled-coil domain is important for the establishment of wild-type box C/D and C'/D' RNP structure essential for nucleotide methylation.

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

“…However, we have recently shown that this interaction is not required for sRNP assembly and is not likely to occur in the complete sRNP complex. 36 The observation that loss of the coiled-coil domain affects overall sRNP structure is, however, consistent with a structural role for Nop56/58 in establishing sRNA and sRNP conformations ultimately important for RNA-guided methylation.…”

“…We have previously reported the cooperative nature of L7 binding to sR8 and the cross-talk interactions important for efficient methylation activities guided by both box C/D and C′/D′ RNPs. 30,36 Therefore, it is reasonable to suggest that formation of inter-RNP interactions critical for methylation activity may well be dependent upon the inherent structural flexibility of the sRNA itself.…”

“…We have previously proposed that Nop56/58 and fibrillarin form a protein scaffold or "platform". 36 It is upon this platform that the sRNAtarget RNA duplex is formed and the base-paired target RNA for modification positioned. Recent work has suggested that in specific archaeal organisms, such as Archaeoglobus fulgidus, L7 may help position this platform on the target RNA via L7 proteinprotein interactions with Nop56/58.…”

“…Recent work has suggested that in specific archaeal organisms, such as Archaeoglobus fulgidus, L7 may help position this platform on the target RNA via L7 proteinprotein interactions with Nop56/58. 36 This core protein platform could establish an architectural bridge between both terminal and internal C/D motifs, providing inter-RNP spacing constraints necessary to position the fibrillarin active site for specific 2′-O-methylation of target RNA nucleotides according to the N+5 rule. 1,30,35 The need for sRNA structure to conform to this protein platform would also help explain the nature of Nop56/58 and fibrillarin-induced RNA remodeling.…”

“…Unfortunately, a complete understanding of box C/D RNP assembly is presently lacking and the nature of inter-RNP interactions is still undefined. 15,36 Mounting evidence demonstrating that the L7 core protein strongly stabilizes the box C/D K-turn by inducing RNA structural changes has suggested that sRNP assembly requires sRNA remodeling. [37][38][39][40][41] Crystal structures of both free and RNA-bound L7 from several archaeal organisms, as well as FRET analyses, have revealed a mechanism of induced-fit interaction for the RNA component with only minor changes in L7 protein structure upon RNA binding.…”