Structurally Conserved Nop56/58 N-terminal Domain Facilitates Archaeal Box C/D Ribonucleoprotein-guided Methyltransferase Activity

• Published in: The Journal of biological chemistry 2012-06, Vol.287 (23), p.19418-19428
• Main Authors: Gagnon, Keith T., Biswas, Shyamasri, Zhang, Xinxin, Brown, Bernard A., Wollenzien, Paul, Mattos, Carla, Maxwell, E. Stuart
• Format: Article
• Language: English
• Subjects: Archaeal Proteins - chemistry; Archaeal Proteins - genetics; Archaeal Proteins - metabolism; Box C/D RNA; Chromosomal Proteins, Non-Histone - chemistry; Chromosomal Proteins, Non-Histone - genetics; Chromosomal Proteins, Non-Histone - metabolism; Fibrillin; Methanococcales - chemistry; Methanococcales - genetics; Methanococcales - metabolism; Methyltransferase; Methyltransferases - chemistry; Methyltransferases - genetics; Methyltransferases - metabolism; Nop56/58; Protein Binding; Protein Structure and Folding; Protein Structure, Tertiary; Protein-Protein Interactions; Ribonuclear Protein (RNP); Ribonucleoproteins - chemistry; Ribonucleoproteins - genetics; Ribonucleoproteins - metabolism; Ribosomal RNA (rRNA); RNA Methylation
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111.323253

Box C/D RNA-protein complexes (RNPs) guide the 2′-O-methylation of nucleotides in both archaeal and eukaryotic ribosomal RNAs. The archaeal box C/D and C′/D′ RNP subcomplexes are each assembled with three sRNP core proteins. The archaeal Nop56/58 core protein mediates crucial protein-protein interactions required for both sRNP assembly and the methyltransferase reaction by bridging the L7Ae and fibrillarin core proteins. The interaction of Methanocaldococcus jannaschii (Mj) Nop56/58 with the methyltransferase fibrillarin has been investigated using site-directed mutagenesis of specific amino acids in the N-terminal domain of Nop56/58 that interacts with fibrillarin. Extensive mutagenesis revealed an unusually strong Nop56/58-fibrillarin interaction. Only deletion of the NTD itself prevented dimerization with fibrillarin. The extreme stability of the Nop56/58-fibrillarin heterodimer was confirmed in both chemical and thermal denaturation analyses. However, mutations that did not affect Nop56/58 binding to fibrillarin or sRNP assembly nevertheless disrupted sRNP-guided nucleotide modification, revealing a role for Nop56/58 in methyltransferase activity. This conclusion was supported with the cross-linking of Nop56/58 to the target RNA substrate. The Mj Nop56/58 NTD was further characterized by solving its three-dimensional crystal structure to a resolution of 1.7 Å. Despite low primary sequence conservation among the archaeal Nop56/58 homologs, the overall structure of the archaeal NTD domain is very well conserved. In conclusion, the archaeal Nop56/58 NTD exhibits a conserved domain structure whose exceptionally stable interaction with fibrillarin plays a role in both RNP assembly and methyltransferase activity. Box C/D RNPs direct site-specific 2′-O-methylation of rRNA. The Nop56/58 and fibrillarin core proteins establish a very stable dimer with Nop56/58 contributing to methyltransferase activity. The Nop56/58 core protein plays a role not only in RNP assembly, but also methyltransferase activity. Our observations reveal a novel role for the Nop56/58 core protein in box C/D RNP function.