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.
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.
Zhang
Xinxin
X
Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, 27695, USA.
Champion
Erica A
EA
Tran
Elizabeth J
EJ
Brown
Bernard A
BA
Baserga
Susan J
SJ
Maxwell
E Stuart
ES
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D019281:Dimerization; D008745:Methylation; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D019642:RNA, Archaeal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020537:RNA, Small Nucleolar / Q000737:chemistry / Q000378:metabolism*; D017411:Ribonucleoproteins, Small Nuclear / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
D019843:Archaeal Proteins; D002868:Chromosomal Proteins, Non-Histone; D009711:Nucleotides; D019642:RNA, Archaeal; D020537:RNA, Small Nucleolar; D017411:Ribonucleoproteins, Small Nuclear; C047458:fibrillarin