In vitro reconstitution and activity of a C/D box methylation guide ribonucleoprotein complex

Abstract

The genomes of hyperthermophilic Archaea encode dozens of methylation guide, C͞D box small RNAs that guide 2 -O-methylation of ribose to specific sites in rRNA and various tRNAs. The genes encoding the Sulfolobus homologues of eukaryotic proteins that are known to be present in C͞D box small nucleolar ribonucleoprotein (snoRNP) complexes were cloned, and the proteins (aFIB, aNOP56, and aL7a) were expressed and purified. The purified proteins along with an in vitro transcript of the Sulfolobus sR1 small RNA were reconstituted in vitro, into an RNP complex. The order of assembly of the three proteins onto the RNA was aL7a, aNOP56, and aFIB. The complex was active in targeting S-adenosyl methionine (SAM)-dependent, site-specific 2 -O-methylation of ribose to a short fragment of ribosomal RNA (rRNA) that was complementary to the D box guide region of the sR1 small RNA. The presence of aFIB was essential for methylation; mutant proteins having amino acid replacements in the SAM-binding motif of aFIB were able to assemble into an RNP complex, but the resulting complexes were defective in methylation activity. These experiments define the minimal number of components and the conditions required to achieve in vitro RNA guide-directed 2 -O-methylation of ribose in a target RNA.T he eukaryotic nucleolus is a highly specialized organelle where rRNA is transcribed, processed, folded, and assembled along with ribosomal proteins into small and large ribosomal subunits (1-5). During this process, up to a hundred or more nucleotide modifications are introduced into the ribosomal RNA (rRNA) by two distinct families of small nucleolar ribonucleoprotein (snoRNP) complexes. The snoRNAs in these RNP complexes contain short antisense guide elements that are used to target modifications to specific locations within the rRNAs. One guide family, the C͞D box snoRNPs, targets site-specific 2Ј-O-methylation of ribose (6-9), and the other guide family, the H͞ACA snoRNPs, targets site-specific conversion of uridine to pseudouridine (10).The C͞D box snoRNAs are characterized by a bipartite structure with conserved C box (RUGAUGA) and D box (CUGA) motifs near their respective 5Ј and 3Ј ends and related CЈ (UGAUGA) and DЈ (CUGA) motifs near the center of the molecule. The antisense elements are located upstream of the D or DЈ motifs and are generally 10 or more nucleotides (nt) in length. Methylation is directed to the rRNA nucleotide that participates in a Watson-Crick base pair five nucleotides upstream from the start of the D or DЈ box; this is the N plus five rule (10-12). Although the general mechanism used by these RNP complexes in mediating modification has been deduced from in vivo biochemical and genetic observations, isolation and characterization of the structure and the in vitro activity of these guide complexes have not been described.The human C͞D box snoRNAs associate with several essential proteins, including fibrillarin, NOP56, and NOP58 (paralogous proteins derived from a gene duplication event), and a 15.5-kDa protein (8,(12)...

Kink turn sRNA folding upon L7Ae binding using molecular dynamics simulations

“…[11][12][13][14] L7Ae protein binds directly to box C/D elements, 8,14 Nop5p binds to L7Ae, and fibrillarin serves as the core catalytic component. [15][16][17] This conserved structure is homologous to the box C/D snoRNP in eukaryotes, 16 which contains a K-turn snoRNA and four protein components. 18,19 Both sRNA and protein components are required in the methylation process.…”

Structural and Thermodynamic Evidence for a Stabilizing Role of Nop5p in S-Adenosyl-L-methionine Binding to Fibrillarin

“…For instance, nop1.2, nop1.3, and nop1.7 mutations occurred in motif III, motif I, and motifs II and IV, respectively (9). Furthermore, by using an in vitro reconstituted archaeal sRNP enzyme, Omer et al (13) demonstrated that substitutions of two amino acids within motifs I and III of Sulfolobus solfataricus fibrillarin (A85V and P129V) resulted in completely or partially abolished methylation activities, whereas sRNP assembly was unaffected. These data suggest that fibrillarin has a function similar to other previously known AdoMet-dependent MTases.…”

Circular box C/D RNAs in Pyrococcus furiosus

“…The modification is catalyzed by fibrillarin, one of three protein components of the box C͞D RNP in archaea (2,4,8). The other two box C͞D RNP proteins are L7Ae (a multifunctional RNA binding protein that is also a component of the ribosome and of box H͞ACA pseudouridylation guide RNPs) and Nop56͞58 (2,4,8,9). More than 50 box C͞D RNAs are encoded in the genomes of humans and Pyrococcus furiosus (1,2,4).…”

“…The RNAs are characterized by the presence of one or two sets of the short, conserved sequence elements termed box C and box D. Base-pairing of the guide region of a box C͞D RNA to the target RNA establishes a site of 2Ј-O-methylation of the target RNA. The modification is catalyzed by fibrillarin, one of three protein components of the box C͞D RNP in archaea (2,4,8). The other two box C͞D RNP proteins are L7Ae (a multifunctional RNA binding protein that is also a component of the ribosome and of box H͞ACA pseudouridylation guide RNPs) and Nop56͞58 (2,4,8,9).…”