The spatial-functional coupling of box C/D and C'/D' RNPs is an evolutionarily conserved feature of the eukaryotic box C/D snoRNP nucleotide modification complex

Abstract

Box C/D ribonucleoprotein particles guide the 2'-O-ribose methylation of target nucleotides in both archaeal and eukaryotic RNAs. These complexes contain two functional centers, assembled around the C/D and C'/D' motifs in the box C/D RNA. The C/D and C'/D' RNPs of the archaeal snoRNA-like RNP (sRNP) are spatially and functionally coupled. Here, we show that similar coupling also occurs in eukaryotic box C/D snoRNPs. The C/D RNP guided 2'-O-methylation when the C'/D' motif was either mutated or ablated. In contrast, the C'/D' RNP was inactive as an independent complex. Additional experiments demonstrated that the internal C'/D' RNP is spatially coupled to the terminal box C/D complex. Pulldown experiments also indicated that all four core proteins are independently recruited to the box C/D and C'/D' motifs. Therefore, the spatial-functional coupling of box C/D and C'/D' RNPs is an evolutionarily conserved feature of both archaeal and eukaryotic box C/D RNP complexes.

FIG. 1.

Sequence and structure of engineered human U24 snoRNA (hU24). (A) Terminal C and D and internal C′ and D′ box sequences are in bold letters (enclosed) and folded to form the box C/D and C′/D′ motifs, respectively. Human D and D′ guide regions were replaced with the indicated sequences complementary to yeast 18S rRNA to target methylation of novel nucleotides A534 and U547, respectively, designated by asterisks. (B) Schematic presentation of engineered human U24 snoRNA. Box C, D, C′, and D′ sequences are indicated as boxes with the respective box C/D (K-turn) and C′/D′ (K-loop) motifs designated. Intermotif spacer regions are shown with the guide sequences complementary to 18S rRNA in bold lines and associated spacer regions in thin lines. The nucleotide lengths of the guide and associated spacer regions are indicated.

FIG. 2.

The terminal box C/D RNP can guide nucleotide methylation independent of the internal C′/D′ RNP. (A) Box C/D RNP-guided nucleotide modification is independent of the C′/D′ RNP. Schematic presentations of hU24 and its mutant constructs are shown at the top of the appropriate gel lanes, with decreasing nucleotide concentrations in the primer extension assay indicated. Primer extension sequencing lanes are at the left, and the indicated primer extension control assays include cells transformed with no plasmid (WT) and empty plasmid (Blank). C′ and D′ mutations included alteration of the K-loop GA residues alone (indicated by a slash in the respective box) and in conjunction with loop mutations (indicated by an X in the respective box). The deleted C′/D′ motif is indicated by a dashed line. Termination sites due to box C/D RNP- and C′/D′ RNP-guided nucleotide modification are indicated on the left as D and D′, respectively. Altered methylation levels with respect to wild-type U24 are indicated below the respective gel lanes. A strong termination site seen between the hU24-targeted nucleotides is the naturally methylated A541 nucleotide. (B) The box C/D RNP requires a minimal size for box C/D RNP-guided nucleotide modification activity. Schematic presentations of full-length and shortened hU24 snoRNAs are shown at the top with targeted nucleotide methylation indicated on the left by D (A534) and D′ (U547). Altered methylation levels with respect to wild-type U24 are indicated below the respective gel lanes.

FIG. 3.

Internal box C/D RNP-guided nucleotide methylation requires the terminal box C/D RNP. (A) Schematic presentation of the chimeric hU24 (box C/D)-snR5 (box H/ACA) snoRNA. The bipartite structure of snR5 with conserved H and ACA boxes (enclosed) is shown. Box C/D snoRNA inserts are positioned between two NcoI restriction sites indicated with arrows. (B) Schematic presentations of the box C/D portion of the chimeric hU24-snR5 snoRNAs are shown above the respective primer extension gel lanes. WT and blank lanes correspond to yeast cells transformed with no plasmid and plasmid lacking the hU24-snR5 chimeric insert, respectively. Primer extension stop sites for the box C/D (D) and C′/D′ (D′) RNP-guided nucleotide methylations are indicated on the left. Altered methylation levels with respect to wild-type U24 are indicated below the respective gel lanes.

FIG. 4.

The terminal box C/D and internal C′/D′ RNPs are spatially coupled for snoRNA-guided nucleotide methylation. Schematic presentations of engineered U24 snoRNA constructs with lengthened or shortened D and D′ spacer regions are shown above the respective primer extension gel lanes. Blank gel lanes correspond to transformed yeast cells with empty plasmid. Primer extension stop sites for the box C/D (D) and C′/D′ (D′) RNP-guided nucleotide methylations are indicated at the side. Altered methylation levels with respect to wild-type U24 are indicated below the respective gel lanes. (A) Box C/D and C′/D′ RNPs exhibit a maximal guide/spacer region length for spatially coupled C′/D′ RNP-guided nucleotide methylation. (B) Box C/D and C′/D′ RNPs exhibit a minimal guide/spacer region length for spatially coupled C′/D′ RNP-guided nucleotide methylation.

FIG. 5.

Asymmetric alteration of the D and D′ guide/spacer regions demonstrates the independent function of the box C/D RNP. Schematic presentations of engineered U24 snoRNA constructs with asymmetrically altered D or D′ spacer regions are shown above the respective primer extension gel lanes. The blank gel lane corresponds to transformed yeast cells with empty plasmid. Primer extension stop sites for the box C/D (D) and C′/D′ (D′) RNP-guided nucleotide methylations are indicated on the left. Altered methylation levels with respect to wild-type U24 are indicated below the respective gel lanes.

FIG. 6.

Box C/D and C′/D′ RNPs are symmetric in core protein composition. Chimeric U24 box C/D-snR5 box H/ACA snoRNAs were coexpressed with epitope-tagged Snu13p, Nop56, Nop58, or fibrillarin core proteins. Yeast extracts were prepared, and individual tagged core proteins were affinity purified from the extract. Coprecipitated RNAs were isolated, resolved on polyacrylamide gels, and revealed by Northern blot analysis. Hybridization probes included U14 snoRNA (box C/D snoRNA positive control) and snR5 (endogenous box H/ACA negative control). The snR5 probe also recognized the chimeric U24-snR5 snoRNAs coprecipitating with the individual core proteins. The box C/D snoRNA region of the U24-snR5 chimeric construct is shown above the respective gel lanes. T and IP indicate total RNA in the yeast extract and affinity-isolated snoRNAs, respectively. Blank lanes represent yeast not expressing a U24-snR5 chimeric snoRNA. Migration positions of U14, snR5, and chimeric U24-snR5 snoRNAs are indicated at the side. (A) snoRNAs coprecipitated with 3×HA-tagged affinity-isolated Snu13p core protein. (B) snoRNAs coprecipitated with TAP-tagged and affinity-isolated fibrillarin core protein. (C) snoRNAs coprecipitated with TAP-tagged and affinity-isolated Nop58 core protein. (D) snoRNAs coprecipitated with TAP-tagged and affinity-isolated Nop56 core protein.

FIG. 7.

Yeast box C/D and C′/D′ RNPs exhibit a defined range of inter-RNP spatial positioning assisted by spacer region folding. (A) Box C/D and C′/D′ inter-RNP spacing of yeast box C/D snoRNAs. Distances between the C-D′ boxes (black bars) and the C′-D boxes (gray bars) were determined and plotted versus the number of yeast box C/D snoRNAs in the indicated nucleotide spacing range. Regions of each bar represented in white are that fraction of snoRNAs which exhibit potential hairpin structures in their C-D′ or C′-D spacer regions. (B) Yeast box C/D snoRNAs utilizing spacer region folding to facilitate coupling of the box C/D and C′/D′ RNPs. Box C/D and C′/D′ sequences are blocked in black with the D′ guide regions base paired to their complementary sequences in 25S rRNA. Evolutionarily conserved hairpin structures which shorten the inter-RNP distances are indicated.