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

  1. Xinxin Zhang1,
  2. Erica A. Champion2,
  3. Elizabeth J. Tran1,
  4. Bernard A. Brown II3,
  5. Susan J. Baserga2, and
  6. E. Stuart Maxwell1
  1. 1Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina 27695, USA
  2. 2Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA
  3. 3Department of Chemistry, Wake Forest University, Winston-Salem, North Carolina 27109, USA

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 α-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.

Keywords

Footnotes

  • Reprint requests to: E. Stuart Maxwell, Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695, USA; e-mail: stu_maxwell{at}ncsu.edu; fax: (919) 515-2047.

  • Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.2230106.

    • Received September 14, 2005.
    • Accepted February 16, 2006.
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  1. Published in Advance April 6, 2006, doi: 10.1261/rna.2230106 RNA 12: 1092-1103 Copyright © 2006 RNA Society

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