Ribosomal RNAs are covalently modified during biogenesis. Failure to properly modify rRNA can affect the efficiency of protein synthesis and may be involved in tumor-formation and disease pathogenesis. One of the most prevalent rRNA modification in both eukaryotes and archaea is the 2'-O-methylation of ribose 2'-hydroxyl groups. These modifications are directed by the conserved family of box C/D small nucleolar RNAs, which contain terminal box C/D and juxtaposed internal box C?O/D?O motifs. At present, archaeal box C'/D'small ribonucleoproteins (sRNPs) are the only RNA-guided nucleotide modification systems that can be reconstituted in vitro to obtain enzymatically active complexes. These systems are ripe for structural investigations that determine the mechanism of complex-assembly and its linkage to regulation, and assess how mutations affect methylation activity. The long-term goals of this research are to investigate the structural biology of the box C/D-guided 2'-O-methylation systems in archaeal organisms and compare them to eukaryotic counterparts. This investigation will specifically examine the structure <-->activity relationship of the Methanocaldococcus jannaschii and Aeropyrum pernix box C/D sRNPs. In these systems, the L7Ae core protein initiates sRNP assembly by binding box C/D RNAs and establishing interactions with the Nop56/58 and fibrillarin core proteins. The goals are to elucidate how protein-RNA and protein/protein interactions, binding, folding, and assembly regulate biological activity.
The specific aims are: 1. To compare and contrast the structures of the individual and juxtaposed archaeal box C/D and C'/D'sRNA motifs to those in isolated and juxtaposed L7Ae-box C/D and L7Ae-C'/D'sRNP complexes;and 2. To examine the archaeal core protein L7Ae's role in initiating sRNP assembly and the putative function of zinc in these interactions. The rationale for this investigation is to add complementary biophysical and structural data to support the mounting biochemical studies on archaeal box C/D RNA modification systems. These studies will provide insight to the evolution and development of ribonucleoprotein enzyme complexes, and illuminate the similarities and difference between the archaeal and eukaryotic sRNP systems.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM069699-04
Application #
7494458
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Preusch, Peter C
Project Start
2005-08-01
Project End
2011-03-31
Budget Start
2009-04-01
Budget End
2011-03-31
Support Year
4
Fiscal Year
2009
Total Cost
$230,377
Indirect Cost
Name
North Carolina State University Raleigh
Department
Biochemistry
Type
Schools of Earth Sciences/Natur
DUNS #
042092122
City
Raleigh
State
NC
Country
United States
Zip Code
27695
Bhuiya, Mohammad Wadud; Suryadi, Jimmy; Zhou, Zholi et al. (2013) Structure of the Aeropyrum pernix L7Ae multifunctional protein and insight into its extreme thermostability. Acta Crystallogr Sect F Struct Biol Cryst Commun 69:979-88
Gagnon, Keith T; Biswas, Shyamasri; Zhang, Xinxin et al. (2012) Structurally conserved Nop56/58 N-terminal domain facilitates archaeal box C/D ribonucleoprotein-guided methyltransferase activity. J Biol Chem 287:19418-28
de Silva, Udesh; Zhou, Zhaoli; Brown 2nd, Bernard A (2012) Structure of Aeropyrum pernix fibrillarin in complex with natively bound S-adenosyl-L-methionine at 1.7?Å resolution. Acta Crystallogr Sect F Struct Biol Cryst Commun 68:854-9
Gagnon, Keith T; Zhang, Xinxin; Qu, Guosheng et al. (2010) Signature amino acids enable the archaeal L7Ae box C/D RNP core protein to recognize and bind the K-loop RNA motif. RNA 16:79-90
Bleichert, Franziska; Gagnon, Keith T; Brown 2nd, Bernard A et al. (2009) A dimeric structure for archaeal box C/D small ribonucleoproteins. Science 325:1384-7
Zhang, Xinxin; Champion, Erica A; Tran, Elizabeth J et al. (2006) 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. RNA 12:1092-103