Conserved spacing between the box C/D and C'/D' RNPs of the archaeal box C/D sRNP complex is required for efficient 2'-O-methylation of target RNAs

Abstract

RNA-guided nucleotide modification complexes direct the post-transcriptional nucleotide modification of both archaeal and eukaryotic RNAs. We have previously demonstrated that efficient 2'-O-methylation activity guided by an in vitro reconstituted archaeal box C/D sRNP requires juxtaposed box C/D and C'/D' RNP complexes. In these experiments, we investigate the importance of spatially positioning the box C/D and C'/D' RNPs within the sRNP complex for nucleotide modification. Initial sequence analysis of 245 archaeal box C/D sRNAs from both Eukyarchaeota and Crenarchaeota kingdoms revealed highly conserved spacing between the box C/D and C'/D' RNA motifs. Distances between boxes C to D' and C' to D (D' and D spacers, respectively) exhibit highly constrained lengths of 12 nucleotides (nt). Methanocaldococcus jannaschii sR8 sRNA, a model box C/D sRNA with D and D' spacers of 12 nt, was mutated to alter the distance between the two RNA motifs. sRNAs with longer or shorter spacer regions could still form sRNPs by associating with box C/D core proteins, L7, Nop56/58, and fibrillarin, comparable to wild-type sR8. However, these reconstituted box C/D sRNP complexes were severely deficient in methylation activity. Alteration of the D and D' spacer lengths disrupted the guided methylation activity of both the box C/D and C'/D' RNP complexes. When only one spacer region was altered, methylation activity of the corresponding RNP was lost. Collectively, these results demonstrate the importance of box C/D and C'/D' RNP positioning for preservation of critical inter-RNP interactions required for efficient box C/D sRNP-guided nucleotide methylation.

FIGURE 1.

Folded secondary structure of M. jannaschii sR8 box C/D RNA, a model archaeal box C/D sRNA. The spacing between the box C/D and C′/D′ motifs in archaeal box C/D sRNAs is conserved. Consensus sequence elements (boxes C and D of the terminal core motif and internal boxes C′ and D′) are indicated in bold lettering. The spacer regions between boxes C and D′ (D′ spacer) and boxes C′ and D (D spacer) are designated. These spacer regions include, but are not limited to, the sRNA guide sequence that base pairs with the respective target RNAs. Bold arrows indicate the sites of nucleotide insertions and deletions for the constructed sR8 sRNA mutants.

FIGURE 2.

The spacing between the box C/D and C′/D′ motifs in archaeal box C/D sRNAs is conserved. The length (nucleotides) of the D (black) and D′ (gray) spacer regions is plotted versus specific sRNA species of M. jannaschii (A) and S. acidocaldarius (B) box C/D sRNAs. The average spacer length of 12 nt is indicated by the dashed lines. Box C/D sRNA sequences were obtained from the Eddy snoRNA database (http://rna.wustl.edu/snoRNAdb/).

FIGURE 3.

Alteration of the spacing distance between the sR8 box C/D and C′/D′ motifs does not affect sRNP assembly. sR8 sRNA D and D′ spacer regions were shortened or lengthened in 2-nt increments by deleting or inserting nucleotides upstream and downstream of boxes C′ and C, respectively, as indicated in Figure 1 ▶. Spacing mutants lengthening the D and D′ spacers were typically created by inserting two uridine nucleotides unless otherwise indicated (spacing mutant 14/14 AG inserted the dinucleotide AG in place of UU). sR8 spacing mutants were either symmetric (A), where both the D and D′ spacers were shortened/lengthened, or asymmetric (B), where only one spacer region was altered. Altered spacing distances are indicated at the top (D spacer/D′ spacer). sR8 sRNPs were assembled in vitro and analyzed by electrophoretic mobility-shift analysis. The sequential addition of the box C/D sRNP core proteins to radiolabeled wild-type and mutant spacer sR8 RNAs is indicated at the top and the migration positions of the resulting RNP complexes are designated at the side. The slightly altered migration of the 14/14 (AG) RNPs is a result of slightly different electrophoretic conditions.

FIGURE 4.

Alteration of the distance between the sR8 box C/D and C′/D′ motifs disrupts 2′-O-methylation guided by the box C/D and C′/D RNP complexes. sR8 sRNAs possessing either shortened or lengthened D and D′ spacer regions were assembled in vitro with the three sRNP core proteins and incubated in the presence of ³H-S-adenosyl-methionine and target RNA oligonucleotides complementary to the D or D′ guide regions. 2′-O-methylation guided by the box C/D and C′/D′ RNPs was determined by measuring ³H-methyl incorporation into the respective D and D′ target RNAs as detailed in Materials and Methods. Target RNA oligonucleotides synthetically methylated at the target nucleotide prior to incubation served as controls to establish background levels of TCA-precipitable ³H counts as well as demonstrate site-specific nucleotide modification. Box C/D and C′/D′ RNP-guided methylation activities of symmetric (A) and asymmetric (B) mutant sR8 sRNAs are presented as the percentage of wild-type sR8 sRNP (12/12)-guided methylation activity minus the background of the synthetically methylated target RNA controls. The reported activities are the averages of three determinations. 14/14 (AG) is the sR8 mutant where the dinucleotide AG was inserted in place of UU.

FIGURE 5.

D and D′ spacer lengths of human and yeast box C/D snoRNAs. The length (nucleotides) of the D (black) and D′ (gray) spacer regions of selected box C/D snoRNAs of human and the yeast Saccharomyces cerevisiae is presented in A and B, respectively. These selected species from Kiss-Laszlo et al. (1998) possess known or putative C′ and D′ sequences. Asterisks indicate those box C/D snoRNAs known to guide 2′-O-methylation from both the terminal box C/D and internal C′/D′ RNPs.